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Sr. Presidente Enrique Peña Nieto, President of the Republic of Mexico
Sr. Alberto Elías Beltrán, Interim Attorney General of Mexico
January 23, 2018
Dear President Peña Nieto and Interim Attorney General Beltrán:
We are deeply disturbed by the crackdown on indigenous communities in the Acapluco district of Guerrero by state and federal police following a violent incident precipitated by unknown armed men on January 7. The incident left 11 dead and at least 32 detained, including 30 members of CRAC-PC (Community Police of the Regional Coordination of Community Authorities) and environmental defenders Marco Antonio Suástegi Muñoz and his brother Vicente Suástegi Muñoz, spokespersons for CECOP (Council of Communal Lands and Communities Opposing the La Parota Dam). This is a clear attack against communities organizing to defend indigenous and environmental rights.
The incident begain on January 7 when the residents of the village of La Concepción were celebrating a local festival. Unknown armed men entered the village and attacked the community police (CRAC-PC), killing two of their members. Six of the attackers were also killed.
La Concepción is one of 47 villages in the indigenous communal landholdings of Cacahuatepec. CRAC-PC and CECOP have been spearheading resistance since 2006 to a proposed hydroelectric power plant to be set up on the Papagayo River. The communities are also advocating for reforms to the Political Constitution of Guerrero on Matters of Indigenous and Afro-Mexican Rights, specifically, reforms to Law 701 of Recognition, Rights and Culture of Indigenous Communities. They are also outspoken in their opposition to the Law of Interior Security (Ley de Seguridad Interior) and the installation of Special Economic Zones (Zonas Económicas Especiales).
Following the January 7 attack, a large number of state and federal police, as well as members of the army, mobilized in and around La Concepción. State armed forces spread throughout the surrounding villages and illegally raided the houses of CECOP members without search warrants. Later, state armed forces launched a second round of violent attacks against residents of La Concepción, killing another three members of CRAC-PC. That's when police arrested 30 members of CRAC-PC and the two CECOP spokespersons named above.
Because of our serious concern about the continuing violence and threats towards members of CRAC-PC and CECOP, we strongly urge you to:
- provide an impartial investigation into the attacks on CRAC-PC and CECOP, the killing of the CRAC-PC members, publish the results, and bring those responsible to justice;
- provide individual and collective protection measures which guarantee the life and physical integrity of leaders and human rights defenders of CRAC-PC and CECOP, in consultation with them and in accordance with their wishes;
- immediately release from detention CECOP spokespersons Marco Antonio Suástegi Muñoz and Vincente Suástegi Muñoz and the 30 members of CRAC-PC.
Christine Stonebraker-Martínez and Brian J. Stefan Szittai
Gerónimo Gutiérrez, Mexican Ambassador to the US ~ via email
Roberta Jackson, US Ambassador to Mexico ~ via fax: 011-52-55-5080-2834
Elizabeth MacDonald and Linda Neilan, Mexico Desk, US State Dept ~ via email
Esmeralda Arosemena de Troitiño, Rapporteur for Mexico, Inter-American Commission on Human Rights ~ via email
Antonia Urrejola, Rapporteur on the Rights of Indigenous Peoples, Inter-American Commission on Human Rights ~ via email
US Senators Brown & Portman ~ via email
US Representatives Beatty, Fudge, Gibbs, Johnson, Jordan, Joyce, Kaptur, Latta, Renacci, Ryan ~ via email


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Historic Wallace, Idaho
Wallace, Idaho is home to a rich and colorful history which has earned the entire downtown of Wallace a listing on The National Register of Historic Places. In 1884, Colonel W. R. Wallace was drawn to the area's rich deposits of silver and gold. He purchased and developed 80 acres of land, which eventually became the site of the City of Wallace. By 1887, downtown businesses were booming and mines were flourishing in the surrounding mountains. Railroads brought in prospectors and miners, took out ore and put the Wild West town of Wallace on the map.
Fire-related tragedy struck the first time in 1890 when an out-of-control blaze burned down the majority of downtown's wooden buildings. But, the independent-minded townspeople persevered and rebuilt the business district with brick, stone and masonry structures, many of which still stand today. Fire once again hit Wallace two decades later, during the largest US forest fire in recorded history. The "Big Burn" swept through Wallace, taking most of the business district with it, and making a hero of "Big Ed" Pulaski who saved 40 fire fighters by guiding them into a mine tunnel south of town.
The town and surrounding region have long been famous as the "Silver Capital of the World" with over 1.2 billion ounces of silver having been produced since 1884. Active, hard rock silver mining in the area continues to this day.
Much of Historic Wallace has been preserved in its buildings, lore, and numerous museums. Today Wallace is a popular tourist destination. In 2004, our City was declared "Center of the Universe" by mayoral proclamation. The reasoning - if something can't be disproven, then it must be true. A manhole cover at the corner of Bank and Sixth Streets commemorates the exact Center of the Universe.
The Silver Valley and Wallace's Place in American History
The Silver Valley and Wallace have played pivotal roles in major chapters of American History over the last 125 years. This would be a remarkable achievement for a large city or a populous industrial state. However, it is nothing short of astounding that a narrow valley in a sparsely populated state, and its tiny flagship town, should have contributed so much to so many truly important American historical developments.
American Labor History:
The Mineowners, the Wobblies and the American Labor Movement
The early days of mining in the West helped build great fortunes for the mine owners – and long hours, grueling work conditions and punishing practices for the miners. The enormously rich veins of silver and other metals in the Coeur d'Alene Mining District created wealth and jobs. This also led to a feverish and growing labor movement and, at times, protracted battles between mine owners and labor unions. The Silver Valley became fertile ground for the I.W.W., the "Wobblies" of American Labor lore. Labor strife in the area attracted headlines around the globe, as the struggles of local miners became a national symbol of working class activism in the face of ascendant American Capitalism.
At a time of heightened tensions, an explosion at the Bunker Hill Mine in nearby Kellogg set off a stiff response by law enforcement. Governor Frank Steunenberg declared martial law and federal troops soon filled the town. Suspected perpetrators, who included many immigrant mineworkers, were rounded-up and placed in a makeshift "bullpen." Subsequent trials, convictions and incarceration stirred local fervor and national debate.
Union activity was later implicated again in the shocking assassination of then former Governor, Frank Steunenberg – as detailed in J. Anthony Lukas's big book, Big Trouble. This era of Labor and Capital counter-punches cemented the Silver Valley's place, and notoriety, in the early history of the American West.
American Environmental History:
"The Big Burn": The Great Fire of 1910 and the Establishment of the U.S. Forest Service and the Environmental Movement
Wallace and the Silver Valley rebuilt following the damage endured in the Great Fire of 1890. However, rebuilders could not have foreseen what was to come in 1910.
The largest fire in American history – ultimately spanning 3 million acres, an area comparable to the entire state of Connecticut – ripped through western Montana, the Panhandle of North Idaho, and eastern Washington in August, 1910. The havoc wreaked by the fire is recounted in Pulitzer Prize-winning author Timothy Egan's 2010 book, The Big Burn.
Egan also details his interpretation that this was "the fire that saved America." He theorizes that Teddy Roosevelt and Gifford Pinchot had ambitious plans for the U.S. Forest Service, but that the Forest Service was woefully under-manned in the field and politically weak in Washington, D.C. The Service might easily have been marginalized and relegated to bureaucratic irrelevancy had the Great Fire of 1910 not so vividly demonstrated the critical need for a forest management and firefighting agency. Thus, the basis for new American environmental and land management policies was established.
Visitors to Wallace can take a short, pleasant hike to the Pulaski Tunnel, where the legendary U.S. Forest Ranger, Ed Pulaski, bravely assembled his crew in his brilliant strategy to escape the fire's carnage.
American Historic Preservation:
Harry F. Magnuson and the Battle to Save the City of Wallace from the Federal Highway Administration and the Wrecking Ball
More recent is the trail-blazing role that Wallace and one of its leading citizens played in charting the course of the American Historic Preservation Movement.
In 1970, the City of Wallace was thrust into this role in a fight for its survival. The Federal Highway Administration and the Idaho Transportation Department planned to route Interstate 90 directly through the center of downtown Wallace. Blocks of Queen Anne buildings, including the iconic Northern Pacific Railroad Depot, were at risk of imminent destruction.
Historic preservation in the 1970's was a little-known concept and, indeed, wrecking crews and bulldozers had already razed part of downtown Wallace. Local businessman and community activist, Harry F. Magnuson, resolved to fight these plans in the courts, taking on the Federal government by turning one of its own instruments on itself.
Congress had recently passed the National Environmental Policy Act of 1970 which required filing environmental impact statements in advance of major projects. Harry Magnuson filed suit, and won an injunction against the Federal Highway Administration stopping bulldozers in their tracks.
A protracted legal battle ensued, with Magnuson tying up the FHA in the courts for 17 years. In 1979, he secured placement of the entire downtown of Wallace on the National Register of Historic Places. A compromise was finally struck and a new overhead freeway was designed to bypass Wallace. In 1985, the Depot was moved across the South Fork of the Coeur d'Alene River to its permanent home on 6th Street.
Coincidentally, Harry Magnuson went on to channel new efforts into constructing a first-class hotel in Wallace as a resource for the community, and as a lovely place for visitors to stay and absorb the abundant history and natural beauty of Wallace. This resulted in the Wallace Inn of today, proudly and continuously overseen by Mr. Magnuson's family.
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- Route of the Hiawatha trail and shuttle passes for two
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- Special package rate $209.
- Special package rate $199.
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To book please call 1-208-752-1252.
Upgrades and extra nights available at a discounted rate. Package rates not available for walk-ins. Rates may increase during festival or holiday periods.
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Wallace Inn News
August 03, 2020 | THE TOP 5 ADVENTURES IN (AND NEAR) WALLACE, IDAHO
Discovering new places that are off the beaten path is our jam. It's 90% of the reason why we do what we do! (10% is for the food, obvi!) As we road tripped around Northern Idaho, we found some real hidden gems – many of which are in and around the small mining town of Wallace! It's…Read Full Story »
March 25, 2019 | Passage Into the Panhandle – Part 2
COMMUNITY Passage Into the Panhandle – Part 2 A Tale of Two Towns Gail Newbold | Photos by Kevin Kiernan Mar 15, 2019 Facebook22TwitterLinkedInSMSEmail Part 1 of this article, published in our January/February 2019 issue, focused on Priest Lake, the Roosevelt Grove of Ancient Cedars, Sandpoint,…Read Full Story »

subset: commoncrawl


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Buy a Franchise Instead of Starting Your Own Business
For running a successful business, then very few people have the natural ability or expertise to be efficient in all aspects. That is where the franchisor's experience comes into play.
Franchise organizations offer a structure for launching, operating and growing a business. Franchisors usually create comprehensive operations manuals and training programs for their franchise owners that cover marketing, operations, accounting, technology and other areas that are specific to the particular business model. These efficiencies are designed to enable franchise owners to earn more and spend less time and effort than otherwise would be required to open and operate a similar business on their own. Here comes the first:
The ability is offered by the franchise organization model to the franchise, to grow under a common brand and share in the benefits of a larger group of business owners. All franchisees share in the collaborative benefits of the organization through the support and oversight of the franchisor including:
- Group advertising resources not typically available to small, independent business owners
- The ability to sell products and services to markets that company-owned outlets have difficulty serving because of higher operational costs and lower motivation of employees in company-owned outlets
- Lower risk of failure and/or loss of investments than if you were to start your own business from scratch
- An opportunity to enhance your management abilities within an established business model that you couldn't experience in most employment situations
This collaboration from the franchisor's perspective:
- Offers the franchisor a method of rapid expansion
- Allows the franchise owner community to grow due to a duplicable system and support
- Features increased buying power for goods and services due to higher volume with suppliers
- Provides steady cash flow to the franchisor to facilitate overall growth of the system
- Can fund the brand recognition effort to grow nationally and globally
Franchising offers you for a better chance to succeed
Research by Government over the years has indicated that the success rate for franchise-owned endeavors is significantly better than the rate for non-franchise-owned small businesses. franchising makes up a significant part of the national economy and presents a statistically better chance for success than other business options, which is good news.
Freedom Factor
Most individuals seek three common elements when choosing a franchised business:
- Flexibility
- Money
- Status
Franchise owners are happy and making more money
There's a lot of truth to this statement, "It's been said that if you love what you do, you can't help but succeed." If the franchisor understands that its franchisees are the heart and soul of their success and understand a very basic premise, if the franchisees are happy then they'll generate more revenue, then it will build on that reputation and financial model.
Be sure to investigate their commitment to their franchise owners as you evaluate franchise organizations. as well as their future development plans to enable their franchisees enjoy continued growth and success.


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Raise your game to win tenders fight
GPs must have a unique selling proposition if they want to beat off the challenge of private firms in PCT tender processes, new guidance is advising.
The guidance from Surrey and Sussex LMCs says GPs need to decide how they are going to compete and what sort of organisation they will be to set themselves apart.
Strategies practices need to consider include whether they are going to compete by differentiating the type of service they will offer, focusing on a limited field of activity or concentrating solely on price.
Successful GPs will be outward looking, quickly engage with ideas, and adapt working conventions in response to patient needs, the guidance states.
Dr James Gillgrass, chief executive of Surrey and Sussex LMCs, said it drew up the advice after it became obvious GP bids were often inferior to the private sector. He said: 'After two or three tenders, doctors involved were saying the quality of tenders by the private sector was way above what GPs were providing. There was no comment that GPs' efforts were slapdash but the quality was different.'
Nigel Grinstead, managing director of consultancy Healthskills, which helped draw up the guidance, said he had seen some GP tender documents written in biro. He said: 'You need substance of course but the reality is there were a lot of very professional, well-presented approaches.'
How to win PCT tenders
• Be more outward looking
• Engage quickly with new
• Show how you react to changing needs of PCTs and the public
• Develop enhanced marketing, planning and finance skills
• Identify your competitive advantage
• Present your ideas professionally and coherently on paper – not in biro!
• Be prepared to think on your feet in oral questioning, and to cater for lay interviewers


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Whitlock - Breedon
(Wedding ~ 05/15/03)
SIKESTON -- Barbara Joyce Whitlock and Jeffrey Wayne Breedon were married in a 2 p.m. ceremony Nov. 30, 2002, at the First United Methodist Church in Sikeston. The Rev. Sandra Schaller, associate pastor, performed the ceremony. The bride is the daughter of Mr. and Mrs. William Whitlock of Sikeston and the bridegroom's parents are Mr. and Mrs. Larry Clayton of Rector, Ark., and the late Alfred Breedon...
Stricker - Christian
(Engagement ~ 05/15/03)
CHARLESTON -- Carl and Bernadette Stricker have announced the engagement of their daughter, Angela Denise, to Paul David Christian, son of Gary and Patricia Christian of Dexter and Robert and Regena Barrett of Wappapello. The future bride is the granddaughter of Margaret R. ...
Beggs - Barnett
(Engagement ~ 05/15/03)
BENTON -- Sheila Beggs and Mr. and Mrs. Cameron Beggs of Benton have announced the engagement of their daughter, Ashley Brooks, to Keith Alan Barnett, son of Bill Barnett of Cape Girardeau and Cathy O'Dell of Scott City. A graduate of Thomas W. Kelly High School, the future bride received a degree in social work in December 2002 from Southeast Missouri State University in Cape Girardeau. She is employed by Big Brothers Big Sisters of Eastern Missouri...
Rhodes one-hits New Madrid County
(High School Sports ~ 05/15/03)
NEW MADRID - The Sikeston Bulldogs baseball team made it a clean sweep in the SEMO Conference with a 12-0, six inning win against New Madrid County Central on Wednesday in New Madrid. The Bulldogs, who had already clinched the regular season championship, ran the table in the conference for the first time in several years with a 9-0 record...
Mid-America lands East Prairie guard
(High School Sports ~ 05/15/03)
OKLAHOMA CITY -- East Prairie's Matt Boyer signed a letter of intent to play basketball for Mid-America Bible College on Wednesday. Mid-America won the NCAA Division II National Championship this season and is consistently ranked among the top Division II schools in the nation...
Southeast signs 6-10 transfer from St. Louis
(High School Sports ~ 05/15/03)
CAPE GIRARDEAU - Southeast Missouri State University basketball coach Gary Garner has announced that Randy Holbrook, 6-10, 240, from St. Louis has signed a national letter of intent to attend Southeast in the fall of 2003. Holbrook was ranked as one of the top 100 high school players in the nation while a senior at Kirkwood High School in 2000. He spent two seasons at Odessa Community College in Texas and will have two years eligibility remaining at Southeast...
Hearing delayed for man charged in rape, robbery
(Local News ~ 05/15/03)
BENTON -- The preliminary hearing for the Sikeston man accused of raping and robbing an elderly woman has been moved to June 4. Donald Wayne Triblett, 18, was slated to appear Wednesday in front of Judge David Mann in Associate Court for the preliminary hearing on felony charges of forcible rape, forcible sodomy, first degree assault, armed criminal action, first degree burglary and first degree elderly abuse. His bond remains at $1 million...
James Eakens
(Obituary ~ 05/15/03)
MALDEN - James William "Tater" Eakens, 69, died May 12, 2003, at Three Rivers Healthcare North Campus in Poplar Bluff. Born March 20, 1934, in Tallapoosa, son of the late Lathie and Nona Kent Eakens, he was a member of Steelworkers Union Local 7686 and the First Southern Baptist Church in Risco. He was a ginner for the Campbell-Kirksey Cotton Gin for 21 years and also worked for Noranda Aluminum in Marston before retiring in 1996...
Zelpha Garrett
(Obituary ~ 05/15/03)
DEXTER - Zelpha Garrett, 88, died May 14, 2003, at Cypress Point Healthcare. Born Nov. 12, 1914, in Indiana, daughter of the late Henry and Ada Hayes Harris, she was a member of the Second General Baptist Church in Dexter and had been a clerk for Stovall's Department Store in Dexter...
Bobby Prayor
(Obituary ~ 05/15/03)
LaCENTER, Ky. -- Bobby Joe Prayor, 69, died at 1:10 p.m. May 14, 2003, at Lourdes Hospital in Paducah. Born Nov. 21, 1933, in Wyatt, Mo., son of the late Donald Eugene and Kathleen O'Connor Prayor, he had lived in Granite City, Ill., most of his life, where he was employed as a chef with Hullings Cafeteria. In 1993, he moved to Paducah and had lived at the Life Care Center in LaCenter for the past year. He was of the Catholic belief...
Lottie Byrd
(Obituary ~ 05/15/03)
SIKESTON - Lottie Lora Byrd, 84, died May 14, 2003, at St. Francis Medical Center in Cape Girardeau, of injuries sustained in a fall at her home. Born Dec. 4, 1918, in Fornfelt, daughter of the late Harrison and Allie Taylor Spradlin, she was a member of the First United Methodist Church in Sikeston. She was a self-employed beautician and had also been employed by the International Shoe Factory in Sikeston before retiring...
SpeakOut 5/15
(Opinion ~ 05/15/03)
I agree with Mike Jensen about Kmart. They had the rudest people wait on you whom I have ever seen. They were nasty to the customers. Good riddance to Kmart. It's a real shame that our institute of higher learning north of Sikeston has to call in each year and ask them to mow their lawn. ...
Gephardt doesn't represent majority
(Column ~ 05/15/03)
Dick Gephardt's presidential bid is sputtering. Missouri's favorite son is trying desperately to carve a position for himself among nine or so Democratic presidential hopefuls but thus far, Gephardt's name is usually found around the middle of the crowded field. He is hanging much of his hopes on a strong showing in the early Iowa caucus process since he has fairly strong support in our neighboring state...
Looking Back in Time at May 15
(History ~ 05/15/03)
SIKESTON - In the second War Loan Drive, Scott County purchased, $576,474 in present cash value of bonds, according to incomplete returns received by Stephen A. Barton, county chairman. The total is for cash value, not maturity value, he said. GIDEON - An election has been called for June 11 to vote on the construction of a sewer system for the city of Gideon...
Districts prepare to tighten belts
(Local News ~ 05/15/03)
SIKESTON -- From making teacher cuts to falling back on reserves to increasing class sizes, Missouri school district officials have a lot on their minds lately, and one of the hardest parts for Scott Downing is keeping his staff's morale up at this difficult time...
Noranda bill heads to governor's desk
(Local News ~ 05/15/03)
JEFFERSON CITY -- The bill that would let Noranda Aluminum Inc. negotiate for power contracts not under the Missouri Public Service Commission's control is on its way to the governor. The revised measure cleared both chambers without opposition -- 32-0 in the Senate and 129-0 in the House of Representatives. It is sponsored by Senate President Pro Tem Peter Kinder, R-Cape Girardeau, and state Rep. Lanie Black, R-Charleston...
Margaret Martin
(Obituary ~ 05/15/03)
CONRAN - Margaret Martin, 70, died May 14, 2003, at Missouri Delta Medical Center in Sikeston. Arrangements are incomplete at DeLisle Funeral Home in Portageville.
Stories from Thursday, May 15, 2003
Browse other days


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Signed in as:
Weathering the Storm and Staying Prepared
With the self-quarantine measures occurring around the country, I know most if not all conferences and events have been cancelled or postponed. Many workers are now working from home, perhaps struggling with productivity as they balance the needs of their job, their kids, and all the other home distractions.
"All storms, no matter how bad they are, will always pass, and the blue sky will always prevail".
Given the novelty of the current pandemic business environment, I'm sure your company or organization has many questions about how to move forward. How much longer do we have to be in this? How should we approach the new week, or month, or year for what lies ahead?
As a veteran of 4 Everest expeditions weathering frequent brutal storms, as well as on other 8000m peaks, I have learned many lessons about not only surviving through isolation and uncertainty, but thriving.
Please check out the the abstract for my webinar, "Weathering Storms and Staying Prepared". This 45-55 minute talk is truly uplifting and inspiring, and will leave your organization with daily actionable items to help you thrive.
As a life-long wilderness enthusiast, Dr. Jon Kedrowski is one of the world's leading high altitude mountaineer experts. He has studied (Ph.D. in Environmental Geography, Weather, and Climate) and survived on peaks around the world, learning valuable lessons and elevating his mindset. These are the lessons he wishes to share with and inspire others through his speaking engagements. Watch the video to learn more:
Small and big mountains - we've all tried to climb them. Where do you start?
By Everesting - elevating your mindset and your life!
In this inspirational keynote, Dr. Jon distills his mountain-tested techniques for reaching the BIG goals you have in life and business. He shares practical methods of how to:
Get to the Top in Life and Business!
Dr. Jon will take your audience on an entertaining and awe-inspiring journey through the adversity he has faced on Mt. Everest, continually facing failure from outside forces and the qualities we all need to climb the Everest's in our lives. This includes:
What does it take to endure the storms in our lives and business environment?
After decades of climbing and skiing off of high peaks around the world, as well being an entrepreneur, Dr. Jon knows what it takes to endure life's storms. In this motivating talk, he shares his stories and insights on how to:
Dr. Jon's authenticity is real, and his message will motivate...he was strong in front of the group, but the real treat was his commitment to some one on one interaction...our people really appreciated that time.
Joe Carlson
Financial Advisor
General Partner
Edward Jones
Jon was an incredible speaker and brought our group a brand new perspective on what it means to go through challenges and rise above them when it counts! I took a picture with him afterwards and noted that this was the closest I'll ever get to the top of Mount Everest. He was an inspiration and I would strongly recommend him to any organization looking to make an impact on their staff or clients.
Senior Account Executive
Minnesota Timberwolves / Lynx
Our community can't get enough of Jon's stories and lectures about his adventures on Everest. His multi-media presentation is as entertaining as it is educational and we welcome him to our stage any time.
K. Sabel
Executive Director
Vail Symposium


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Gee, why did it take so long to find that out?
Visits to Pakistan, wife beating and child abuse, will it ever end for France's notorious polygamist, w/ the burqa driving wife
Islamization Watch
Of course this all started, with the wife / mistress being asked to pay a ticket for wearing a burqa while driving. If only they have agreed to pay the 22 euro penalty….
Along with the new allegations of wife beating and child neglect, he was actually on the radar for Muslim extremists after he made several visits to Pakistan and dangerously enough Londonistan. He enquired about opening a mosque near where he lives in France and was planning to open an …. Islamic bookstore.
It might be that the family's -Â extended lots of mistresses/ Muslim wives – refusal to pay the fine ~ was a part of pushing back against French secular society's encroachment on their Islamic way of life.
All while ~ they and their 12 children were living off the French state.
In fresh allegations, the French Muslim man suspected of polygamy and welfare fraud has been accused of beating at least one of his women companions and keeping his children "locked away".
Lies Hebbadj, a French Muslim butcher suspected of polygamy, has been accused of domestic violence and mistreating his children, the French interior minister, Brice Hortefeux, told French lawmakers on Wednesday.
On Wednesday, police sources said Hebbadj's father-in-law filed a complaint Monday, saying his daughter was "beaten" by her husband and their children were kept "locked away". He also reported having "little or almost no contact" with his daughter.
Hebbadj, an Algerian-born 35-year-old butcher in France's north-western city of Nantes, has been at the centre of a political storm over alleged polygamy and welfare fraud.
According to Hortefeux, Hebbadj lives with four women who have borne him 12 children, and each wife receives "single parent" welfare benefits.
The Algerian-born butcher, now 37, came to France at the age of two. He did not acquire French citizenship until 1999 when he married Anne, a Frenchwoman who converted to Islam.
The well-regarded businessman recently opened a halal meat market and gocery store. He is also planning a bookstore.
Mr. Hebbadj is an adherent of a group known as Tablighi, a proselytizing movement that aims to bring Muslims closer to the practices of the Prophet Muhammad. It has about 100,000 members in France.
He has visited Pakistan several times. His trips, which include one to London, have brought him to the attention of various foreign security services.
The French government has said it has information Mr. Hebbadj is married to four women with 12 children and accuses them of fraudulently claiming single-parent benefits. NP
Hortefeux last Saturday called for Hebbadj's French passport to be removed, saying polygamy is "not welcome in France".
The controversy began last week, when one of Hebbadj's companions complained that she had been fined 22 euros for driving while wearing her niqab full-face veil, in Nantes.
"I'd go so far as to say that polygamists here (in France) are breeding for cash"
Flashback: The Imam of Vénissieux
As well as in service of a supremacist agenda. "Polygamists 'breeding for cash,'" fromAP, May 2 (thanks to Jihad Watch, which has excellent comments on the subject):
The burqa, or face-covering veil, is getting all the attention in the debate over Muslim immigrants in France. But another controversial tradition among some immigrants is less noticed and far more widespread: Polygamy.The issue resurfaced last week after a woman received a traffic citation in the western city of Nantes for driving with a veil over her face. Officials then accused her husband of having at least three other wives, and said he may be profiting from them financially while the state pays the bill.
Polygamy is one of several issues, like forced marriage or genital mutilation, that France and other European nations face, as immigrants arrive with customs that conflict with the law of the land. But experts say polygamy in France can also be linked to fraud, where husbands hijack a generous social welfare system to line their pockets with state funds from each of their wives.
"They practice polygamy just for that," said Jean-Marie Ballo, founder of an association that helps women escape from polygamous situations, Nouveaux Pas, or New Steps. "I'd go so far as to say that polygamists here (in France) are breeding for cash."
Ballo said he's even aware of cases where a legal wife's papers are used for hospital care for a second – a health risk as medical records intermingle.
It's hard to count how many polygamous families live in France because of the secrecy of the practice. But the National Consultative Commission on Human Rights in a 2006 report made a minimal estimate of 16,000 to 20,000 polygamous families in France, or some 180,000 people, including children. That compares to fewer than 2,000 women who are thought to wear burqa-style garments.
For decades, polygamy was legal in France for immigrants arriving from any of about 50 countries where it is legally recognised. Historically, taking numerous wives was either a social and economic necessity in poor countries with high death rates, or a sign of external wealth or male domination.
France banned polygamy in 1993. At the same time, it launched a process of "decohabitation" to help multiple wives trapped in small apartments with numerous children to move into their own homes. Experts say that system has been largely successful.
But abuses thrive. Especially vulnerable are women who arrived in France after 1993 – often here illegally and, therefore, with limited means to extricate themselves….
Chantal Brunel, a lawmaker from the governing conservative UMP party, called last weekend for a region-by-region examination of the family subsidies program to stop corruption by men profiting from state aid to illegal wives. Brunel, who has written a book about violence against women, said she has polygamous families in her district east of Paris "and since 2004-2005 I have asked that the state stop closing its eyes."…
Other countries in Europe also struggle with polygamy. Fines and prison sentences, in some cases up to seven years, are the norm for those convicted of polygamy in Europe. An exception is Norway. In France, marriage to more than one person is punishable by a year in prison and a 45,000 (almost $60,000) fine.
However, the law is being challenged in Ireland. And in Cyprus, with a 5-year prison term, the court can take into account arguments that the accused's culture or religion permits polygamy.
Carina Hagg, a Swedish lawmaker for the opposition Social Democrats, warns against mixing notions of polygamy and culture.
"You have to be careful not to make it an issue about ethnicity," she said. "Fundamentally it's about women's rights."…
Ballo, whose Malian father and grandfather were both polygamous, said he helped "decohabit" 12 households with 26 wives and 145 children in Les Ulis, south of Paris, where his group is based.
The human rights commission report notes that "there is, of course, no question of generalising and considering all polygamous men as executioners."
Ballo is more cynical: "There are always people in life who defend hell."
How very true!


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Create paintings from photos Did you ever wanted to be a great artist? But unfortunately you are really bad at it? Well that's ok because now with Psykopaint you can be a great artist with no skills. But how does it work? Open a photo you like and paint it using a variety of tools like brush, spraycan, ribbon, knife, Psykopaint will choose the colors automatically for you So you don't need to worry about it and focus on a style you like. Photography 101: Know Your DSLR Dial You know all those little symbols on that wheel at the top of your DSLR camera? They can elevate your photos to a more professional level. We've broke the important ones down for you symbol-by-symbol so you can make the most of your camera. Auto ('A' or camera symbol, sometimes it's colored green) This mode is for camera newbies since it sets the exposure and shutter speed automatically for you. All you need to do is push the button and snap a photo, which can be helpful when someone who is not experienced is using your camera to take a photo of you.
50 essential photography tips Whether photography is a hobby or a profession, you'll get a whole lot more out of it if you understand how it works. With a firm grasp of aperture, shutter speed, sensitivity and focal length, the ratio of truly great to merely mediocre shots you download at the end of an expedition is all but guaranteed to climb. Here we present CNET UK's 50 essential shooters' tips. Don't uncap your lens without them. Aperture
70 Excellent Photoshop Resources By Kawsar Ali Adobe Photoshop is the tool of choice for many designers and artists. Photoshop can be used for almost every little task that involves graphic design. It is one of the finest pieces of software available on the market when it comes to digital image editing. A great attribute of Photoshop is that you can extend it with additional brushes, patterns, shapes, and more. 20 Free PSD Templates to Mockup Your Poster Designs Creating an artistic poster is one of the most fun projects a designer can work on. It's especially satisfying when you have your hard work professionally printed into a physical poster, but this can be quite costly. You don't have to spend all that money to see how your design would look freshly printed and mounted though, there's a range of PSD mockup templates that simulate your design being presented as a framed print, wrapped canvas or even an outdoor billboard.
72 Creative And Smart Typographic Logo Inspiration If we are starting to go crazy in typography style, then let's continue with typographic and smart logo designs, shall we? Here you'll find 72 beautiful logos created by talented designers. This showcase just proves the point how much can one/two words, icons tell using correctly white, negative, positive spacing, colors, accents creating interesting associations. Explore and get creative as I did! 1. Killed Productions


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Special Offers
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The municipal register in Rojales currently stands at 17,649 residents, of which 35% are from the United Kingdom.
According to the figures compiled by the town hall about 12300 are immigrants from other countries, 7 out of 10 come from overseas while the other three come from Spain.
This means that the municipality, which currently has residents belonging to 85 different nationalities, is one of the towns in Spain with the highest percentage of foreign residents, a great melting pot of cultures where the inhabitants coexist perfectly integrated in life daily of the town.
Most inhabitants come from the United Kingdom, with more than 35%, followed by Spaniards, which are around 30%, while Norwegian and German residents make up approximately 4.4%.
In recent years Rojales has continued to increase the number of its Spanish classes, provided totally free of charge, to attract citizens who do not speak the language. The town has also created a program of language exchanges, which allows different nationalities to get to know each other and have the opportunity to practice the languages that interest them, while promoting coexistence and integration.


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Alice in Wonderland1999
Alice in Wonderland (1999)
Alice in Wonderland Photos
Movie Info
Watch it now
as Alice
as Mad Hatter
as Cheshire Cat
as Mock Turtle
as White Knight
as Queen of Hearts
as Major Caterpillar
as Ned Tweedledum
as Fred Tweedledee
as Walrus
as Carpenter
as King of Hearts
as Knave of Hearts
as White Rabbit
as Cook
as Duchess
as Pat the Gardener
as Bill the Gardener
as Chief Executioner
as Mr. Mouse
Critic Reviews for Alice in Wonderland
All Critics (6) | Fresh (2) | Rotten (4) | DVD (7)
The Flop that tried
Dismal: Stick with the Disney version instead
. Her crazy adventure teaches Alice about change, the multiple uses of words, curiosity, nonsense, and the great mysteries of life
Audience Reviews for Alice in Wonderland
Shrill, flat filming of a classic tale. Many stars are wasted here, the voice Miranda Richardson affects for the Red Queen may be intended to be funny but is incredibly grating instead.
Super Reviewer
Beautiful High-budget CGI realization of Wonderland! Nice fantasy creatures and characters; some computerized and some Henson-created. The assorted settings are magnificent to behold. The All-Star cast is impressive. The pacing is somewhat bogged down by insignificant long scenes (eg. the Griffon and Mock Turtle, the Library and the Greenhouse Gardeners). I suggest fast-forwarding through those scenes to improve flow. Martin Short is good as the Mad Hatter. Whoopi Goldberg makes a good Cheshire Cat. The story has a nice theme of Alice's Wonderland encounters helping her gain confidence to perform a a song in at her parents' tea party.
Super Reviewer
I feel the all star cast in this film was a little overkill. Granted, Louis Carrol?s ?Alice in Wonderland? is a classic and deserves for the film to tell it well and really capture the adventure and imagination and all this was certainly the case, unfortunately, this is one film that I feel only benefited a younger audience as such, the famous actors could be quite lost on them.
Super Reviewer
Alice in Wonderland Quotes
There are no approved quotes yet for this movie.


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Before I say anything about Affiliate Marketing or ow to Increase your affiliate marketing sales, kindly take a good look at the screenshot below.
Before I talk about how to Increase your affiliate marketing sales, I highly recommend you to read my article on If you are new to this business. I'm not here to speculate, rather I am here to give you legit strategies used by successful affiliate marketers from all walks of life. I know affiliate marketing seems formidable, yes. That was how I felt It when I started newly. I have realized my mistakes now and there Is need to Improve on them.
tips to Increase your affiliate marketing sales
Find High Commission Affiliate Programs-
Let us do little mathematics here.
For Instance, If your target Is to hit around $1000 this month, what would be your best approach to this ?..
Remember To Brand Yourself-
One of the best ways to Increase your affiliate sales, Is to brand youself.
- You should start a blog or run a Youtube channel. This platforms will Increase your audience trust.
- Show your earning history so far as an affiliate
- Try your possible best to always stay consistent and active on social media.
- Place an attractive photo of yourself.
- Relate your cintent with your own life experiences.
- Solve the problems of your audience.
- Add your blog or channel URL to your social media bios and profile.
Start Building Relationship-
Building relationships Is the most essential part of affiliate marketing business.
You should build relationships If you really what to Increase your affiliate sales by far.
The relationship foundation should be solid because It's going to last for a longer period.
Place Affiliate Banners On Your Webpages-
You whitespace on your blog or website.
Place the affiliate marketing banner of the product you want to promote.
Write A Detailed Review Or Tutorial-
Experts has proven that a detailed review or tutorial brings more sales.
Write an In-depth review or tutorial about the product you are using.
In your review, Include the beneficial aspect.
Tell your audience how the product(s) will help them In their daily life.
There are absolutely two (2) ways by which you can achieve this. If you have to sell a product worth $10, It means you must have 100 buyers. But If you have a product worth $100, you only need to sell to ten (10) people to hit your $1000.
Obviously, It Is very easy to find ten (10) people who would purchase a product rather than hundred (100). The possibility of getting ten people Is far higher than your chances of getting a hundred.
For this reason, you should find an affiliate program that pays high commission rates for products sold. There are hundreds and thousands of affiliate programs that are ready to give out commissions to an affiliate. The best places to head over to, Is or .
This two (2) companies have thousands of products, plus merchants are given to affiliates at 30% - 50%. This websites have products for any blog niche you can ever think of. In fact, ClickBank wins here because they paid as high as 75% commission for each sale.
Both ClickBank and ShareAsale are capable of Increasing your affiliate sales. The bad side of ClickBank Affiliate Is that ClickBank Is only available to some countries. That Is very unfortunate.
What does this mean ?..
The chances of getting more affiliate sales will highly Increase If people can see you as a real person. Branding Is one of the primary key to every successful affiliate marketing business.
For Instance, someone who you haven't meet before tells you to buy a product, maybe using a Link privided, this looks like a scam. You would be curious to know who the person really Is.
Ins't It ?..
So, to give your audience a good Impression, you must show yourself and an authority or a brand. I have prepared some legit branding tips that would help you:
Learn more about branding with the video tutorial below !.
Having known all this branding tips, soon you will begin to build trust between your audience and this automatically fetch you more sales.
Descover your audience problems. By using Email list, you can achieve this. Find out the best solutions that can help to solve their problems. You could offer them free E-Books, resources or abything that would add value to the life of your subscriber's.
Try to build }relationships with other affiliate marketers. You both can share marketing tips and tactics. Approach other affiliate marketers as a source of learning and not as a competition.
A research says that visual Images leaves great Impact on the mind of your audience. Once you place attractive banners, they will surely want to find out more about the product.
I highly recommend you to write a detailed review of the product If you have not done that. Tell your audience how the product has helped you to solve your problems, by doing this, you are gradually Increasing your readers attachment to the product.
Wrapping Up: how to Increase your affiliate sales
With all the tips have provided above, you should have no excuse. This are the best tactics In the affiliate market. If you have tried all this and things aren't working as expected, I'll advice you to seek for help from an expert In affiliate marketing field. Perhaps, you may find It very difficult to find them because they are always busy with their business and thousands of newbies/beginners approach them every single day.
So, what Is the best approach to this !..
The best way to approach this Is to buy their affiliate marketing courses or anything they can offer. I'm not trying to say you should spend hundreds or thousands of dollars on their courses. I did not purchase any course, but one (1) surefire thing Is that you have offered yourself a biggest favour. Their course will be detailed enough to see you throughout your markering journey because they have been doing this business for many years and they know In and out of affiliate marketing. I just hope my article on how to Increase your affiliate sales Is detailed enough to help you Increase your affiliate marketing Income rapidly.
Having known all this, I'll love to show you some affiliate program product to promote even without a website or blog. You can promote them on forum websites but be sure that such forum allows affiliate link sharing so that on the long run, you won't get suspended or banned by the forum site.
You can promote your affiliate products on social media websites. Pinterest Is a good example.
Top Affiliate Program Products To Promote
- CashSuperStar.Com If you are very active on Pinterest and you have gathered some huge about of followers, you can start pay per lead (PPL) Income program. Cash Super Star Is a platform that will pay you about $3.0 for every unique signup you get from countries like Canada, USA, UK, New Zealand and Australia..
- ShareASale.Com This Is one of the best affiliate companies you shoud try. They pay very high commission and no matter your blog niche, you surely will find product to promote. It could also promote your products on Pinterest.
- TailWind.Com Tailwind will automatically Increase your affiliate sales.through Pinterest. If you haven't heard of tailwind, then you should try It out now. Tailwind Is a must and best scheduler for people using Pinterest.
- UniqueRewards.Com Unique Rewards Is an Internet marketing website that will pay you when you watch videos and read Emails.
- UniquePaid.Com This website Is similar to Unique Rewards. You get paid when you shop online, when you read emas and when you visit other websites.
You can start a 30 days free trial then schedule your first 400 Pins. After you create your PIN, add your tailwind affiliate link then start pinning to the most relevant Pinterest boards. Anytime a person signup using the free trial In the PIN, you will be rewarded handsomely with $0.50, and when they ungrade their plan, you get about 15% affiliate commission.
The Best Survey And Cash Back Sites To Make Money.By using this sites, you can earn In two sure ways. You can take part In paid surveys and you can also promote your affiliate links on pinterest.
Start your affiliate marketing journey with any of the websites above. I hope you achieve your dreams soon.. Have a nice day and kindly share this post using the share buttons below.


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The call for the 2017-18 European Astro Pi challenge is open! For the third year running, we are inviting teams of European school students up to 19 years old to enter the competition and have their code run on the International Space Station (ISS).
ESA astronauts Paolo Nespoli and Alexander Gerst are our proud ambassadors this year. Paolo Nespoli is already onboard the ISS, and he has prepared a special video message inviting you to accept the challenge!
Paolo Nespoli launches the European Astro Pi Challenge
ESA astronaut Paolo Nespoli launches the European Astro Pi Challenge for the school year 2017 – 2018. On board the ISS there are two Astro Pi computers – nicknamed Ed and Izzy – that are available to run students experiments which make use of computer coding and computer sensors to be executed.
An version in Italian is available here.
Find out more and choose your mission on the home page.


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Community briefs
Association of Learning disabilities conference
STAMFORD -- The Connecticut Association for Children and Adults with Learning Disabilities will present its' 39th annual fall conference from 8 a.m. to 4 p.m. Saturday at the Stamford Marriott Hotel, 243 Tresser Blvd. Rick Lavoie, an expert and author on learning disabilities and special education, will speak. Registration fees range from $75 to $135. A limited number of full and partial scholarships are available for those who otherwise could not attend the conference.
For more information, visit www.cacld.org or call 203-838-5010.
Family fun at Yerwood Center
STAMFORD -- Yerwood Center will host its Family Fun Carnival Night from 5 to 9 p.m. Saturday at 90 Fairfield Ave. The event will feature rides, food, games, open pool and a movie. Cost is $10 for rides only, $5 for movie and $12 for the movie and rides.
Call 203-327-2790 to purchase tickets.
Glenbrook Neighborhood Association cleanup
STAMFORD -- The Glenbrook Neighborhood Association will hold Make a Difference Day from 9 a.m. to noon Saturday at the Glenbrook train station, 502 Glenbrook Road. Collect trash, clear vegetation and more. Gloves and trash bags will be provided. Bring garden tools. There will also be a Beverley Ecker Plaque Dedication.
For more information, call Christie Fountain at 203-964-0078 or Anne Goslin at 203-969-0696.


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February 14 2019
Sheriff Koutoujian Selected VP of Major County Sheriffs of America
By: BNEWS
Middlesex County's sheriff will be second in command of a country-wide organization of sheriffs.
Middlesex Sheriff Peter J. Koutoujian has been selected by his colleagues from across the country to serve as vice president of the Major County Sheriffs of America (MCSA), a release from his office states.
MCSA is a professional law enforcement association comprised of sheriffs of the nation's most populous counties and parishes and its members represent over 100 million people across more than 30 states.
"I am honored to be selected for this leadership position to help showcase the innovative work being done by sheriffs from across the country," said Sheriff Koutoujian. "I look forward to working with President Grady Judd, the executive board and membership to promote the incredible work our colleagues do on a daily basis to increase safety in our communities."
Koutoujian, who had been tapped to serve in an acting capacity in mid-December by President Judd, was formally voted to the post on a permanent basis by his colleagues at the MCSA's annual winter conference on Thursday, February 14.
"I am honored and excited to work shoulder to shoulder with Sheriff Koutoujian in the coming year," said President Judd. "I believe Peter is a visionary with the strong leadership and fresh perspective that will propel the MCSA to next level."
A longtime state legislator and former prosecutor with the Middlesex District Attorney's Office, Koutoujian has led the Middlesex Sheriff's Office since 2011.
"During his tenure as sheriff, he has overseen the implementation of several statewide and nationally recognized initiatives including a specialized treatment unit for incarcerated military veterans, a medication assisted treatment program and the launch of a groundbreaking new housing unit for incarcerated young adults (ages 18-24)," the release states.
Sheriff Koutoujian is also currently in his second year of a two-year term as President of the Massachusetts Sheriffs' Association (MSA).
Photo Caption: Middlesex Sheriff Peter J. Koutoujian (left) was sworn in as vice president of the Major County Sheriffs of America (MCSA) by President Grady Judd on Thursday, February 14, 2019 during the MCSA winter conference meeting in Washington, D.C. (Courtesy Photo)


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From Masstown, the highway runs east and northeast for 87 kilometres (54 mi) past the towns of Truro, Westville, Stellarton and New Glasgow to Sutherlands River.
Highway 104 passes to the north of Truro. This stretch was section was originally built in the 1960s, and upgraded to a 4-lane divided freeway that opened in the early 1990s. This section is a 4-lane divided freeway and has a posted speed limit of 110 km/h.
Notice the hillside stands of maple, from which farmers harvest maple syrup in the springtime, during the short period when sap rises from the tree's roots to its branches and leaves. The Indians first harvested the sweet sap by cutting a diagonal channel in the bark, gathering the liquid sap into birch bark containers, and then boiling it by placing not rocks in a wooden trough. From 25 litres of sap, and hours of boiling, you end up with one litre of maple syrup.
Along the north coast of Nova Scotia, along the Northumberland Strait, is the Poctou Lowlands ecosystem. This area was favoured by settlers, who liked its fertile soft rock geography, and by shipbuilders who like the area's fine harbours, with gentle rivers leading up to virgin pine stands. When coal was discovered around New Glasgow, it triggered the growth of five area towns.
Early settlers were attracted by rich fertile farmlands along the West River, leading the the coast at New Glasgow. Settlers that arrived later had to clear the poorer uplands, which were later abandoned, and are now being reclaimed by forest species. Before the advent of commercial fertilizers in the 1950s, farmers would come to nearby Limerock (just north of the highway) and burn limestone to "sweeten" their fields (lower its acidity). The vase shaped trees in the river are white elms, which took root in the silt after seeds were deposited during spring floods.
The grey stone structure west of the bridge (on the south side) is the Museum of Industry, which marks and remembers an abandoned New Glasgow coal mine. As early as 1839, miners descended 240 metres (800 feet) down, to work the 12 metres (39 ft) thick Foord seam of coal, said to be the world's thickest coal seam. The area's coal mines closed down after competition from oil fuels, and from underground explosions over the 1950s.
Trans-Canada Highway Itinerary Map
Use mouse to drag/move map. Click on "+" or "–" to zoom in or out. "Satellite" combines map & photo.


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SN 4 | EP 4 | The Finale
Cathy is visited by her estranged father; Sean develops mixed feelings about his kidney surgery; Andrea gets an opportunity of a lifetime.
Available: Amazon.com, Google Play, iTunes Store, VUDU, YouTube
Acclaimed actress Laura Linney stars in this comedy-drama as Minneapolis schoolteacher Cathy Jamison, whose reserved, structured way of life is turned upside down after she is diagnosed with terminal cancer. Gone are the days of pleasing everyone else; now, for the first time in her life, she is going to choose the path that most suits her needs. And that means, in part, telling her immature husband, spoiled son and outspoken brother to grow up. Cathy, meanwhile, wants to act up ... and along the way she forms new bonds with some unexpected people.
Comedy drama
Laura Linney,
Cathy Jamison
Oliver Platt,
Paul Jamison
John Benjamin Hickey,


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Pbx/exd proteins modulate the DNA binding affinities and specificities of Hox proteins and contribute to the execution of Hox-dependent developmental programs in arthropods and vertebrates. Pbx proteins also stably heterodimerize and bind DNA with Meis and Pknox1-Prep1, additional members of the TALE (three-amino-acid loop extension) superclass of homeodomain proteins that function on common genetic pathways with a subset of Hox proteins. In this study, we demonstrated that Pbx and Meis bind DNA as heterotrimeric complexes with Hoxb1 on a genetically defined Hoxb2 enhancer, r4, that mediates the cross-regulatory transcriptional effects of Hoxb1 in vivo. The DNA binding specificity of the heterotrimeric complex for r4 is mediated by a Pbx-Hox site in conjunction with a distal Meis site, which we showed to be required for ternary complex formation and Meis-enhanced transcription. Formation of heterotrimeric complexes in which all three homeodomains bind their cognate DNA sites is topologically facilitated by the ability of Pbx and Meis to interact through their amino termini and bind DNA without stringent half-site orientation and spacing requirements. Furthermore, Meis site mutation in the Hoxb2 enhancer phenocopies Pbx-Hox site mutation to abrogate enhancer-directed expression of a reporter transgene in the murine embryonic hindbrain, demonstrating that DNA binding by all three proteins is required for trimer function in vivo. Our data provide in vitro and in vivo evidence for the combinatorial regulation of Hox and TALE protein functions that are mediated, in part, by their interdependent DNA binding activities as ternary complexes. As a consequence, Hoxb1 employs Pbx and Meis-related proteins, as a pair of essential cofactors in a higher-order molecular complex, to mediate its transcriptional effects on an endogenous Hox response element.
Hox genes encode homeodomain-containing transcription factors that play critical roles in specifying positional information along several embryonic axes. A growing body of evidence supports a role for several members of the TALE (three-amino-acid loop extension) class of homeoproteins as essential contributors to Hox developmental programs in arthropods and vertebrates. Hox interactions with TALE proteins of the Pbx/exd subtype have been most extensively studied (22); they result in enhanced DNA binding affinities or specificities in vitro (7-9, 16, 19, 28-30, 37, 40). Genetic analyses of both mice andDrosophila melanogaster provide strong in vivo support that Pbx and Hox function in concert during development (33) through response elements containing their cognate DNA binding sites (7, 20, 22, 32). In addition to cooperative DNA binding activity, the genetic interactions of Hox and Pbx/exd proteins appear to involve, in part, regulation of their subcellular distributions (1, 21).
Aside from Pbx-Hox interactions, a cooperative function among TALE family members has also been shown to be critical for transcriptional processes. Several recent reports have shown that Pbx proteins dimerize and bind DNA with Meis-hth (4, 10, 17) and Pknox1-Prep1 (2, 3, 17), members of an ancient subclass of TALE proteins evolutionarily related to but distinct from Pbx/exd (5, 11, 24). Indeed, heterodimeric TALE homeoprotein complexes are directly implicated by biochemical analyses in the regulated expression of several genes (2-4, 39). Further support for their convergent function comes from studies of Drosophila, where the homolog of Meis (homothorax) displays genetic epistatis with exd and regulates its nuclear entry (6, 27, 34).
An additional layer of complexity arises from genetic and biochemical evidence demonstrating Hox interactions with Meis-like proteins. InDrosophila, homothorax affects nuclear localization and functions in common genetic pathways with a subset of Hox proteins (6, 27, 34). In murine myeloid leukemias, bothMEIS and HOX genes are activated by retroviral insertions, providing genetic support for the cooperative interactions of their respective gene products in mammalian neoplasias (25). Recent retroviral gene transfer experiments provide more direct genetic evidence that Meis1 and Hoxa9 collaborate in myeloid oncogenesis (18), and Hoxa9 and Meis1 have been shown in vitro to physically interact and bind DNA as heterodimers (38).
Thus far, investigations have primarily focused on the functional significance of dimeric Pbx-Hox, Pbx-Meis, and Meis-Hox interactions. These studies suggest two possible, but not necessarily mutually exclusive, models for integrating Hox and TALE protein functions. Representatives of the Pbx, Meis, and Hox homeoprotein families may compete with each other to establish a hierarchy of heterodimers or, alternatively, they may cooperatively enter into higher-order DNA binding complexes. In this report, we demonstrate that Hoxb1 binds native enhancers in vitro in a higher-order trimeric complex with Pbx and Meis. Unlike previous reports of Hox and TALE trimeric interactions, our results showed that the Meis component contributes to the DNA binding specificity of the heterotrimeric complex by binding a Meis cognate DNA site and is not simply tethered by Pbx to the complex. Furthermore, DNA binding by the Meis component appears to be essential for transcriptional activity of the trimeric complex in vivo, since the Meis site mutation phenocopies the Pbx-Hox site mutation to abrogate enhancer-directed expression of a reporter transgene in the developing hindbrain. Therefore, Hoxb1 simultaneously binds DNA with Pbx and Meis-related proteins and employs these paired TALE homeoproteins as essential cofactors to mediate its transcriptional effects on hindbrain enhancers in vivo.
Plasmid constructions and mutagenesis.Constructs for the expression of wild-type and mutant Pbx and Hox proteins under the control of SP6 or cytomegalovirus (CMV) promoters have been described in previous studies (8, 9). The full-length murine MEIS1 cDNA (25) was cloned into pCMV1 for use in transient-transcription assays. Deletion constructs of Pbx1 (Pbx1CT and Pbx1NT) and Meis1 (MeisCT and MeisNT) were constructed by standard cloning or PCR. A 900-bp (BamHI to StuI) upstream region of the Hoxb2 r4 enhancer element was amplified by PCR and subcloned into the BGZ40 lacZ reporter vector (42) containing a minimal human β-globin promoter. The 900-bp element spanned the three Krox20 and consensus Pbx-Hox sites, as reported previously (20, 36). Mutations of the Meis and Pbx-Hox sites in the Hoxb2 r4 element were performed by overlap extension PCR.
DNA binding and transcriptional assays.Proteins for DNA binding were produced in vitro from SP6 expression plasmids by using a coupled reticulocyte lysate system, as described previously (8). DNA binding reactions were performed at 4°C for 30 min in a 15-μl reaction mixture under conditions reported previously (8) and subjected to an electrophoretic mobility shift assay (EMSA) with 6% polyacrylamide gels in 0.25× Tris-borate-EDTA buffer. DNA probes consisted of gel-purified, end-labeled, double-stranded oligonucleotides encoding the Hoxb2 r4 enhancer (20) or portions of the Hoxb1 autoregulatory element (ARE) (32). Synthetic probes for the evaluation of Pbx-Meis DNA binding requirements contained consensus Pbx and Meis sites (underlined) in various configurations (e.g., 5′-CCCTGCCTTGATTGACAGTTGCGCCTG-3′ for nongapped sites and 5′-CTGCCTTGATGCCTGGTGACAGTTGCGC-3′ for N6-gapped sites). Actual sequences of DNA probes are available upon request.
Transient-transcription assays were performed essentially as described previously (12). COS-7 cells were seeded at 105cells per 35-mm-diameter dish in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 2 mM l-glutamine, penicillin (100 U/ml), and streptomycin (100 μg/ml) 16 to 24 h prior to transfection. Cells were transfected by a calcium phosphate coprecipitation procedure with different combinations of expression plasmids encoding Pbx1a (2 μg), Hoxb1 (500 ng), or Meis1 (2 μg) along with reporter (1 μg) and internal control (pCMV-βgal) plasmids. The reporter plasmid consisted of the firefly luciferase gene, driven by a simian virus 40 early promoter, and one copy of theHoxb2 r4 enhancer or Hoxb1 ARE. Total DNA concentration per dish was kept constant with nonspecific DNA. Luciferase activity was measured in light units with a Monolight 2010 luminometer; β-galactosidase activity was used to normalize luciferase activity to account for differences in transfection efficiency.
Immunoprecipitations and Western blots.In vitro-translated proteins (2 to 10 μl of reticulocyte lysates) were added to 200 μl of immunoprecipitation buffer (50 mM HEPES at pH 7.9, 250 mM NaCl, 5 mM dithiothreitol, 1% bovine serum albumin, 0.1% Triton X-100) and incubated at 4°C for 3 h with antibody (1 μg/100 μl) and an additional 2 h with 20 μl of protein G-Sepharose beads. Beads were precipitated and washed 10 times with immunoprecipitation buffer. The precipitated proteins were boiled in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer and analyzed by SDS-PAGE. Western blotting was performed as described previously (10) with an anti-Prep1 antiserum (Santa Cruz Biotechnology) on whole-cell lysates of hindbrain tissues microdissected from 9.5-embryonic-day embryos.
Transgenic mice.DNA constructs, as linearized inserts lacking vector sequences, were microinjected into fertilized mouse eggs generated from crosses of F1 hybrids (C57BL/6J × CBA). Microinjected eggs were implanted into pseudopregnant females, and embryos were harvested at 9.5 days postconception (dpc). Embryos were fixed in 4% paraformaldehyde (in phosphate-buffered saline) for 30 minutes, washed in buffer W (phosphate-buffered saline, 0.01% deoxycholate, 0.02% Nonidet P-40), and stained for 1 to 4 h at 37°C in X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) (1 mg/ml) with 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, and 2 mM MgCl2 in buffer W.
The amino termini of Pbx and Meis are necessary and sufficient for their dimerization in solution.To distinguish among alternative models for the function of TALE homeodomain proteins Pbx and Meis, we evaluated several features of their dimeric interactions. Since Pbx and Meis-related proteins form stable complexes in solution in the absence of DNA (2, 10), dimerization was assessed by coprecipitation analyses. We first tested whether Hox proteins were capable of displacing Meis from Pbx in solution, a potential requirement for a competitive heterodimerization model. Increasing concentrations of Hoxb1 did not reduce the amount of Meis1 that coprecipitated with Pbx1 (Fig. 1B), indicating that Hoxb1 was unable to compete with Meis1a for Pbx1a interaction in solution. Furthermore, under these conditions, we were unable to demonstrate coprecipitation of Hoxb1 with the TALE heterodimer, consistent with previous observations that Pbx and Hox interactions are weak in solution but markedly stable in the presence of cognate DNA (8).
Since Pbx-Hox interactions require sequences exclusively within the extended Pbx homeodomain (31), we next evaluated whether Pbx-Meis dimerization may also require one or both homeodomains. Deletion of either the Pbx1a or the Meis1a carboxy terminus containing the respective homeodomains did not abrogate their coprecipitation. In fact, coprecipitation of the amino termini was observed without either homeodomain. In contrast, removal of the amino-terminal regions of Pbx1a or Meis1a prevented coprecipitation of the Pbx-Meis complex (Fig.1). Attempts to further refine Pbx1a dimerization requirements by the deletion of additional C-terminal sequences from Pbx1NT abrogated coprecipitation (data not shown). Therefore, the amino-terminal portions of both Pbx and Meis were necessary and sufficient for their dimerization in solution, as assessed by this coprecipitation assay (Fig. 1). The required amino-terminal regions encompassed amino acid segments with predicted helical features and constituted the only domains conserved between Pbx and Meis-Prep1 proteins outside of their homeodomains. This portion of Pbx1 is required for cooperative DNA binding with Meis and Prep1 (2, 10, 17) but is separate and distinct from that required for optimal interactions with Hox partners (8, 10).
Pbx and Meis bind DNA without stringent half-site orientation and spacing requirements.The observations that Pbx and Meis dimerize through their amino termini suggested the possibility that their DNA binding activity may not be constrained by stringent half-site spacing or orientation requirements. Thus, Pbx-Meis may be similar to MATα2, a yeast TALE homeodomain protein that dimerizes through its flexible amino terminus (15). Towards this end, Pbx-Meis heterodimeric binding activity was examined on synthetic oligonucleotides containing Pbx and Meis half sites with varied spacing and orientation. Pbx-Meis heterodimers tolerated separation of their half sites by 3 or 6 nucleotides (Fig.2A, lanes 2 and 3). Steady-state binding was more robust on sites separated by 6 nucleotides and was dependent on intact half sites (Fig. 2A, lanes 3 to 5). Significant, but less robust, binding also occurred on DNA probes with half sites in various inverted orientations (Fig. 2A, lanes 6 to 8). Although an extensive analysis of site configurations was not conducted, at a minimum these studies demonstrated that Pbx-Meis heterodimers are more tolerant of alterations in half-site spacing and orientation (Fig. 2B) than are Pbx-Hox heterodimers (8). This flexibility is likely facilitated by the stable interactions of Pbx and Meis through their unique amino-terminal dimerization motifs.
Two hindbrain enhancers contain conserved Meis sites in proximity to Pbx-Hox sites.The flexible character of Pbx-Meis interactions raised the possibility that Pbx may simultaneously interact with Meis as well as Hox proteins in a higher-order molecular complex in which each component contacts DNA. Furthermore, the rigid half-site requirements of Pbx-Hox dimers, in contrast to the less stringent requirements of Pbx-Meis dimers, suggested that potential trimeric binding sites may consist of a core Pbx-Hox site separated from a Meis site by variable distances. This possibility was investigated by evaluating genetically characterized Hox enhancers for the presence of Meis sites flanking Pbx-Hox sites. Two enhancers were discovered to meet these criteria (Fig. 3). Both have been shown in genetic studies to mediate the in vivo transcriptional effects of Hoxb1 in conjunction with Pbx cofactors during hindbrain development. The Hoxb1 ARE is necessary for the autoregulation of Hoxb1 expression in rhombomere 4 of the hindbrain (32). The Hoxb2 r4 element functions to direct Hoxb1 cross-regulation of the Hoxb2 gene in the same anatomic site (20). Both enhancers contain Pbx-Hox sites as previously reported (20, 32). However, they also both contain Meis sites which have strikingly similar configurations and which are upstream of, and in reverse orientation with respect to, their Pbx-Hox sites (Fig. 3).
Trimeric Meis-Pbx-Hox complexes assemble on the Hoxb2r4 enhancer and demonstrate a requirement for an intact Meis site.We performed EMSA with a DNA probe spanning the Pbx-Hox and Meis sites in the Hoxb2 r4 enhancer to determine whether this element could support the formation of trimeric Meis-Pbx-Hoxb1 DNA binding complexes. On the Hoxb2 r4 enhancer, Pbx1a-Meis1a heterodimers displayed measurable cooperative binding (Fig.4A, lane 1). More robust dimeric binding was observed in reactions containing Pbx1a and Hoxb1 (Fig. 4A, lane 2). However, when all three proteins were present in the binding reaction, the predominant complex displayed a lower mobility than did heterodimers (Fig. 4A, lane 3). This was accompanied by a substantial reduction in the amount of dimeric complex detected, strongly suggesting that in the presence of all three proteins, simple heterodimeric binding of Hoxb1 with Pbx1a was not favored. Rather, the appearance of a slower-migrating complex suggested that Hoxb1 preferentially entered into a higher-order DNA binding complex containing both Pbx1a and Meis1a. Consistent with this possibility, the abundance of the slower-migrating complex (Fig. 4A, lane 3) was comparable to that of the Pbx-Meis complex (lane 1).
The requirement for DNA binding by each component of the heterotrimeric complex was evaluated by using probes containing mutations of the Meis or Pbx-Hox binding sites. Mutation of the Pbx-Hox site completely abrogated DNA binding by all heterodimeric and heterotrimeric complexes (data not shown). Mutation of the Meis site in r4 abrogated formation of the trimer but not the Pbx1a-Hoxb1 heterodimer (Fig. 4A, lane 5 versus 6), indicating that DNA binding by the Meis component was necessary to form a ternary complex on DNA. Interestingly, the amount of dimer observed on the mutant r4 element was markedly reduced in the presence of Meis1a (Fig. 4A, lane 6), but this was not associated with the concomitant appearance of the slower-migrating complex seen with the wild-type probe (lane 3). This observation suggested that, in solution, the three proteins formed a trimeric complex whose DNA binding requirements were more stringent than those of Pbx-Hox or Pbx-Meis heterodimers.
The presence of all three homeodomain proteins in the trimeric complex that formed on the wild-type r4 probe was verified by the inclusion of specific antibodies in the binding reactions. Supershifted complexes were observed with monoclonal antibodies directed against each of the input proteins, demonstrating that all three proteins were present in the slower-migrating complex (Fig. 4B, lanes 2 to 4). Small amounts of residual dimeric complexes in lane 4 represent Pbx-Meis heterodimers that do not contain Hoxb1 and thus were not supershifted by the anti-Flag antibody. Furthermore, the formation of a ternary complex was dependent on specific dimerization motifs in each protein (Fig. 4C). Heterodimeric Pbx-Hox complexes, but not trimeric complexes, were detected when EMSA was performed with mutants Pbx1CT (Fig. 4C, lane 2) or Meis1CT (lane 3), each of which is defective in Pbx-Meis dimerization (Fig. 1B). Similarly, a Hoxb1 hexapeptide mutant was incapable of entering into a trimeric complex with Pbx-Meis heterodimers (Fig. 4C, lane 4).
Heterotrimeric complexes also preferentially assemble on a subportion of the Hoxb1 ARE that contains Meis and Pbx-Hox sites.As noted above, we discovered a strikingly similar configuration of Meis and Pbx-Hox consensus sites upon examination of a second genetically defined Hox enhancer, the Hoxb1 ARE (Fig.3). This enhancer contains three conserved Pbx-Hox consensus sites (r1 to r3), one of which, r3, has been shown to support the assembly of a trimeric Prep1-Pbx1-Hoxb1 complex in vitro (3). Consistent with these previous observations, we observed that the intact ARE supported the binding of a Meis1a-Pbx1a-Hoxb1 complex under our EMSA conditions (Fig. 5). A Pbx1a-Hoxb1 heterodimer formed on the ARE in the absence of Meis1a (Fig. 5, lane 2), similar to results obtained with the Hoxb2 enhancer. With the addition of Meis1a, heterodimer binding was markedly reduced, and a trimeric complex was observed (Fig. 5, lane 3). To evaluate the specific sequences within the Hoxb1 ARE that are required for the formation of a trimeric Hoxb1-Pbx1a-Meis1a complex, we performed EMSA with ARE subfragments as probes. Trimer formation was found to be most robust on a probe containing the conserved block 1 (b1) and the adjacent Pbx-Hox consensus repeat element r3 (Fig. 5). In contrast to previous studies (3), the r3 element alone did not support trimer binding, although Pbx1a-Hoxb1 heterodimers bound robustly to this element (Fig. 5, lanes 11 and 12). The amount of Pbx1a-Hoxb1 dimer detected on the r3 element was markedly reduced in the presence of Meis1a (Fig. 5, lane 12), but this was not associated with the concomitant appearance of a slower-migrating complex, as seen with the b1-r3 subfragment (lane 9). These findings again suggested that in the presence of all three proteins, Hox-Pbx heterodimers converted in solution to trimers with increased DNA binding requirements. Such trimers, however, did not appear to be sufficiently stable to withstand immunoprecipitation under our conditions (Fig. 1B). Since no trimeric complex formed on the r3 element alone, the sequences in b1 were deduced to be critical for trimer binding and, in fact, to encode the consensus Meis site described above (Fig. 3). Taken together, these data suggested that heterotrimer formation on DNA was dependent on the recognition of cognate DNA sites by each respective homeodomain in the complex. Therefore, two genetically defined response elements for Hoxb1 support the in vitro binding of higher-order molecular complexes containing Hoxb1 and the TALE class homeodomain proteins Pbx1a and Meis1a. Our analyses indicated that all three proteins are capable of binding DNA as a trimeric complex on these enhancer elements and that the Meis component contributes substantially to the DNA binding specificity of the ternary complex.
Trimeric complexes display Meis-dependent transcriptional activity.The potential functional consequences of trimeric Hox-Pbx-Meis interactions on the Hoxb2 r4 enhancer were first tested in transient-transcription assays. For these studies, we employed a reporter gene containing a single copy of theHoxb2 r4 enhancer upstream from a simian virus 40 early promoter. When coexpressed, Pbx1a and Meis1a displayed no activation above background levels, comparable to the results obtained with Hoxb1 alone or with Hoxb1 coexpressed with Pbx1a (Fig.6). In contrast, cotransfection of all three homeodomain proteins resulted in a severalfold increase in transcription above the baseline (Fig. 6). When similar analyses were performed with a reporter gene containing a mutant Meis site in theHoxb2 enhancer, no activation above the background was observed when all three homeodomain proteins were cotransfected. This provides in vivo evidence that trimer-enhanced transcriptional function requires the recognition of an appropriate binding site by the Meis component, consistent with our observations that the assembly of a trimeric complex on this enhancer in EMSA is dependent upon an intact Meis site. Previous studies employing reporter genes containing multiple copies of the r3 element of the ARE provided evidence that the Meis-related protein Prep1 enhanced transcriptional activation of a Pbx1-Hoxb1 complex (2). We obtained similar evidence for an accessory role of Meis1a with a reporter gene containing a single ARE. This reporter showed 10-fold-higher transcriptional activity upon cotransfection of all three proteins than Pbx1a and Meis1a alone (Fig.6). On this element, coexpressed Pbx1a and Hoxb1 (in the absence of exogenous Meis1a) were also capable of activating transcription, consistent with previous observations (12). However, this activity was reproducibly two- to threefold lower and probably reflects the presence of endogenous Meis-Prep1 proteins in COS-7 cells (Fig.7C) that are limiting for the nuclear entry of Pbx, which requires Meis-related proteins, as demonstrated with orthologous proteins in Drosophila (27, 34).
Hoxb2 enhancer function in the hindbrain requires Meis in addition to Pbx-Hox consensus binding sites.We next evaluated whether Meis-related cofactors contribute to function of the Hoxb2 r4 enhancer in directing rhombomere-restricted expression in the mouse hindbrain. lacZ reporter transgenes were constructed with 5′ flanking regions of the Hoxb2 gene (Fig. 7A) containingcis-acting elements required for its up-regulated expression in rhombomeres 3 to 5 (called r3 to r5), following 8.5 dpc of development (20, 36). In addition to the 180-bp r4 enhancer which directs r4-restricted expression, we included three Krox20 sites that direct Hoxb2 expression in r3 and r5 to serve as internal controls for comparing transgene expression in r4. Transgenic embryos were evaluated at 9.5 to 10 dpc, when r3 expression is beginning to wane but r4 expression is maximal (36, 41). Embryos microinjected with constructs containing wild-type sequences displayed intense reporter staining in r4 and its associated neural crest, which migrates into the second branchial arch (Fig. 7B). Less staining was seen in r3 and r5, as was expected at this stage of development (36, 41). Embryos that were transgenic for constructs containing mutations in the Pbx-Hox consensus site of the r4 enhancer showed persistent lacZ expression in r3 and r5 but no expression in r4 (Fig. 7B). This is consistent with previous studies (20) reporting the contributions of a direct cross-regulatory interaction of Hoxb1 and Pbx cofactors toHoxb2 expression in r4. A similar loss of r4 and branchial-arch staining, but not r3 or r5 staining, was seen in transgenic embryos microinjected with constructs containing Meis site mutations (Fig. 7A and B) that abrogate in vitro DNA binding by Meis-Pbx-Hoxb1 trimers as well as by Meis-enhanced transcription. Therefore, mutations of the Meis site in the Hoxb2 r4 enhancer phenocopy Pbx-Hox site mutations, indicating that appropriate transgene expression requires DNA binding by Meis-like proteins as well as Pbx and Hox proteins.
These studies provide in vitro and in vivo evidence that Hoxb1 binds DNA simultaneously with Pbx and Meis-related TALE homeodomain proteins as cofactors to mediate its transcriptional effects on theHoxb2 r4 hindbrain enhancer. Although trimeric associations of Hox and TALE proteins have recently been reported on two other enhancer elements (3, 39), our studies are the first to demonstrate that DNA binding by the Meis component contributes to the specificity of ternary Hox-TALE homeoprotein complexes and is obligatory for their in vivo functions (Fig.8). We had previously hypothesized that Pbx may simultaneously interact with and bind DNA with Hox and Meis partners, based on observations that Pbx employs distinctly different domains for interactions with Hox versus Meis-related proteins (10). However, the preferences of both Hox and Meis partners to assume a position on DNA immediately 3′ to Pbx in heterodimeric complexes appeared to be inconsistent with the formation of higher-order complexes in which all three homeodomains contact their cognate DNA sites. In the current study, we provided a solution to this topological predicament by demonstrating that Pbx-Meis heterodimers are capable of binding DNA without stringent half-site orientation and spacing requirements. The amino-terminal portions of Pbx and Meis are necessary and sufficient for their stable heterodimerization, presumably leaving their respective homeodomains free to bind DNA half sites in various configurations. This property allows Pbx-Meis-Hox heterotrimeric complexes to assemble on DNA sites consisting of a Pbx-Hox consensus core sequence flanked by a distant Meis site. This configuration satisfies the stringent half-site DNA binding requirements of the Pbx-Hox component, while simultaneously permitting DNA binding by the more flexible Meis component.
One role we observed for Meis is to increase the DNA binding requirements of the heterotrimeric complex. This was most evident by the suppression of Pbx-Hox binding to dimeric sites upon the inclusion of Meis in DNA binding reaction mixtures, presumably due to an increased requirement for DNA recognition by all three proteins. Thus, ternary complexes were not observed on DNA probes consisting of Pbx-Hox sites alone. On two natural enhancers, site-directed mutation or deletion of the flanking Meis site abrogated the in vitro DNA binding of trimeric Meis-Pbx-Hox complexes. These data demonstrate that Meis makes essential contributions to the binding specificity of the ternary complex, thereby preventing its recognition of simple dimeric Pbx-Hox sites. The specific mechanisms by which Meis affects the DNA binding requirements of the ternary complex were not established by our studies. Preliminary studies suggest that Meis does not measurably affect the DNA binding selectivity of Hoxb1, which appears to be comparable whether Hoxb1 is acting as a trimer with Pbx-Meis or as a heterodimer with Pbx (14). Furthermore, deletion of Meis's homeodomain does not completely impair its ability to suppress Pbx-Hox binding to dimeric sites (14), indicating that the increased binding specificity of trimeric complexes is not due solely to their requirements for a cognate Meis site. One possibility is that Meis modulates the DNA binding properties of Pbx, particularly since portions of Pbx that are necessary for dimeric interactions with Meis in solution are directly upstream of the Pbx homeodomain. In the absence of a cognate Meis site, the conformation of the Pbx homeodomain N-terminal arm may be sufficiently altered by a tethered but non-DNA-bound Meis partner to abrogate ternary complex formation on Pbx-Hox sites. The DNA binding site requirements of Meis in a ternary complex with Pbx1a and Hoxb1 were not addressed in detail. Both enhancers contained similarly configured Meis sites, which showed inverted orientations with respect to nearby consensus Pbx-Hox sites. Interestingly, the distance separating Meis and Pbx-Hox sites was precisely one (r4) or two (ARE) DNA helical turns. It is not yet clear how rigid this spacing requirement may be in accommodating the possible topological features of the ternary homeodomain protein complex.
Our observation that Meis contributes to the DNA binding requirements of ternary complexes differs from previous observations and raises the possibility that its contributions may vary with different enhancers or under different cellular conditions. Berthelsen et al. (3) showed by EMSA that a trimeric complex consisting of Pbx1, Hoxb1, and the Meis-related protein Prep1 formed on the r3 element of theHoxb1 ARE. Since the DNA binding selectivity of the ternary complex was identical to that of Pbx-Hoxb1, the findings of Berthelsen et al. were most consistent with tethering of Prep1 to a DNA-bound Pbx-Hoxb1 heterodimer without a requirement for Prep1 homeodomain binding to DNA (Fig. 8). However, it is notable that DNA binding by the Prep1-containing ternary complex was enhanced upon deletion of the Prep1 homeodomain, suggesting that the homeodomain partially suppressed Pbx-Hoxb1 binding and thus contributed at some level to the selectivity of the ternary complex. Using Meis1 instead of Prep1, we were unable to demonstrate the formation of a comparable complex on r3 alone. We observed formation of a trimeric complex on the Hoxb1 ARE, but the sequences within block 1 as well as the consensus Pbx-Hox site in r3 were required. The discrepant results likely reflect variations in EMSA conditions and/or protein preparations but not inherent differences between Prep1 and Meis1, since we obtained comparable results with either protein under our DNA binding conditions (14). Swift et al. (39) have also observed tethering of Meis to a DNA-bound heterodimer with the B element of the elastase enhancer, which mediates the effects of homeodomain protein PDX1 in pancreatic acinar cells (Fig. 8). Notably, ternary Pbx-Meis-PDX1 complexes were detected in the nuclear extracts of acinar cell lines by EMSA with the B element as a probe. Unfortunately, comparable studies have not detected endogenous Hoxb1-containing complexes in the nuclear extracts of embryos with probes representative of either Pbx-Hoxb1 sites (10) or the Hoxb2 r4 enhancer (14). These studies consistently detect heterodimeric Pbx-Meis or Pbx-Prep1 complexes; the lack of ternary complexes presumably reflects the low abundance of Hoxb1 in embryonic extracts. Nevertheless, in vitro studies leave open the possibility that tethering of the Meis-Prep1 component of a heterotrimer can occur under some conditions, but the potential functional implications of this effect in vivo remain to be determined.
The most compelling evidence that DNA binding by a Meis-related protein is required for the in vivo function of ternary complexes is provided by our analysis of the requirements for function of theHoxb2 r4 enhancer in rhombomere 4 of the developing hindbrain. Elegant genetic studies have demonstrated that this enhancer directs the appropriate expression of the Hoxb2 gene in response to Hoxb1 cross-regulation in rhombomere 4 at approximately 8.5 to 10 days of hindbrain development (20). Extensive mapping showed that a consensus Pbx-Hox site was essential for r4 enhancer-mediated expression in rhombomere 4, but not in rhombomeres 3 and 5, which was consistent with our own observations. However, these earlier studies also indicated that the Pbx-Hox site was sufficient for r4-directed expression, a conclusion that conflicts with our current findings that mutation of the flanking Meis site phenocopies Pbx-Hox site mutation. This disparity may be accounted for by the fact that the previous studies employed synthetic elements that were not in a natural configuration and that contained iterated copies of Pbx-Hox sites (20). Since Meis crossbinds to Pbx-Hox consensus sites (10), synthetic elements containing them in tandem resemble the natural tripartite Meis-Pbx-Hox elements identified here and, in fact, weakly support DNA binding by ternary Hoxb1 complexes in vitro (Fig. 5, lane 15). This may also account for the Meis-mediated enhancement of the expression of reporter genes containing similar multimerized configurations of the ARE r3 site (2). Our studies demonstrate a consistent requirement for the Meis site in vitro and in vivo, but it is not yet clear which of the various Meis-Prep1 family members may be directly responsible for r4 enhancer function in the developing hindbrain. Both Meis and Prep1 proteins are expressed in the hindbrain at embryonic day 9.5 by Western blot analyses (Fig. 7C). Since Meis genes display dynamic expression profiles during embryonic development (26), a more precise determination of the in vivo roles of individual Meis-related proteins in r4 functions will require studies with mice that are nullizygous for one or more of the Meis genes.
The r4 and ARE enhancers contain strikingly similar configurations of Pbx-Hox and flanking Meis sites that were found to be essential for in vitro DNA binding by ternary Meis-Pbx-Hoxb1 complexes. However, our in vivo studies focused on the r4 enhancer because the ARE is a complex regulatory element with multiple potential homeoprotein binding sites (32). Although we found that heterotrimer binding was most robust on the b1-r3 subportion of the ARE, weak binding was also detected on other portions as well. The in vivo requirements for each of the several conserved sequence motifs in the ARE for its function in the developing mouse hindbrain have been extensively evaluated (32). When individually mutated, none of the conserved elements of this enhancer was found to be essential for ARE activity in rhombomere 4. Mutation of repeat 3 had the most significant effect but only partially reduced enhancer function, to approximately 60% of wild-type activity, whereas b1 (containing a conserved Meis site) was dispensable for ARE function. These in vivo data appear to be consistent with our observations that Meis-Pbx-Hoxb1 ternary complexes bind in vitro, with differing affinities, to at least two portions of the ARE. This contrasts with the Hoxb2 r4 enhancer, in which mutation of either the Pbx-Hox or the Meis sites results in the complete loss of expression in rhombomere 4. Therefore, the ARE appears to be redundant in its composition, which complicates attempts to correlate the in vivo and in vitro contributions of individual elements.
The functional interrelationships of TALE proteins were initially suggested by studies of Drosophila showing that the Meis ortholog homothorax regulates the activation of the Pbx homolog exd through a posttranslational mechanism involving nuclear translocation (6, 27, 34). There is evidence for a similar mechanism in mammals (13, 35). These observations suggest that obligate heterodimerization with Meis-related proteins may be a generalized feature of Pbx/exd protein transcriptional function. In further support of this hypothesis, heterodimers consisting of Pbx paired with Meis or Pknox1-Prep1 are present in many adult and embryonic tissues (2, 10) and are implicated in the regulated expression of several genes (2-4, 10, 17, 39). Our studies demonstrate, however, that the heterodimerization of TALE proteins does not preclude their interaction with Hoxb1, thereby allowing DNA binding as a trimeric complex in which each component binds its cognate DNA site. These observations support a model in which Hoxb1 functionally interacts with preformed TALE protein heterodimers, perhaps serving as a specificity factor to direct ternary complexes to a subset of enhancers with appropriate binding sites (23).
In summary, our studies provide support at the molecular level for previous observations that each component of the TALE heterodimer interacts and functions on common genetic pathways with a subset of Hox proteins. Although its generality for Hox function remains to be determined, a trimeric model invoking a higher-order assembly of Hox and TALE proteins provides a molecular framework for integrating the functions of these developmentally important proteins.
These studies were supported by grants from the NIH (CA42971, AI-07290, and CA09151). C.A.S. was supported by postdoctoral fellowship funds from the Leukemia Society of America.
We acknowledge Yelena Marchuk and Yanru Chen for microinjections. We thank David Kingsley and Peter Rigby for providing the BGZ40lacZ reporter gene vector.
- Received 12 January 1999.
- Returned for modification 1 March 1999.
- Accepted 6 April 1999.
- Copyright © 1999 American Society for Microbiology


text:
Someone correct me if I'm wrong: it may be only 83 days left of summer. When June, a favorite month sped by in a haze of anguish, grief and exhaustion I decided to find the joy no matter what. I love summer and I love my life even when it's peppered with four-letter words. It would be easy peasy to drown in self-doubt and pity. To shake my fist at the gods of why is everything is upside down and a challenge? Of course it's not every single thing. It just feels that way from stress and not sleeping more than four hours in a row. It's what it's like to care for the sick and dying. In my case, it's my cats but it feels no different than a child, parent, spouse or friend. It's exhausting, heart-stopping, hand-wringing but with moments of peace and out-of-body bliss. The worst of times can be a blessing. Usually the insight comes after the event or transition is over and can be filtered in the rear-view mirror of memory. What if we didn't wait to grab the blessing? What if we loved every fucking word of our life right now? Wretched. Shitty. Tiresome. Depressing.
Confusing, overwhelming, nasty, scary.
Heart-opening, tiny joy. Awe-inspiring wondrous. Soul-nourishing. Quixotic. Awful to awesome.
Any word you identify with is yours. Those words make up of the story of you. Your life. Only your life. No one else. You are the writer. You get to choose the words. Change the words. Make up words. Now that's pretty fucking amazing.
Check out our Instagram page from our latest image/words. It's Cat Wisdom 101 and there are cats but also what inspires me in nature, art and life.