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Integrative analysis of gene amplification in Drosophila follicle cells: parameters of origin activation and repression

In metazoans, how replication origins are specified and subsequently activated is not well understood. Drosophila amplicons in follicle cells (DAFCs) are genomic regions that undergo rereplication to increase DNA copy number. We identified all DAFCs by comparative genomic hybridization, uncovering two new amplicons in addition to four known previously. The complete identification of all DAFCs enabled us to investigate these in vivo replicons with respect to parameters of transcription, localization of the origin recognition complex (ORC), and histone acetylation, yielding important insights into gene amplification as a metazoan replication model. Significantly, ORC is bound across domains spanning 10 or more kilobases at the DAFC rather than at a specific site. Additionally, ORC is bound at many regions that do not undergo amplification, and, in contrast to cell culture, these regions do not correlate with high gene expression. As a developmental strategy, gene amplification is not the predominant means of achieving high expression levels, even in cells capable of amplification. Intriguingly, we found that, in some strains, a new amplicon, DAFC-22B , does not amplify, a consequence of distant repression of ORC binding and origin activation. This repression is alleviated when a fragment containing the origin is placed in different genomic contexts.


Integrative analysis of gene amplification in Drosophila follicle cells: parameters of origin activation and repression
The initiation of DNA replication occurs from discrete genomic regions called replication origins. Although the protein complexes that bind DNA and license origins for replication are known and evolutionarily conserved, the properties that enable a DNA sequence to function as a replication origin in metazoans are poorly delineated (Gilbert 2004(Gilbert , 2010Cvetic and Walter 2005;Mechali 2010). One reason is that analysis of metazoan replication origins has, until recently, proceeded from molecular characterization of a small number of identified origins (Aladjem 2007). For example, the DHFR locus in Chinese hamster ovary cells has been studied extensively using a variety of methods; replication at this locus initiates in a broad initiation zone containing multiple inefficient initiation sites (Hamlin et al. 2010). Recent genome-wide origin mapping studies in Drosophila, mouse, and human cell culture have greatly increased the number of identified metazoan origins (Cadoret et al. 2008;Sequeira-Mendes et al. 2009;Karnani et al. 2010;MacAlpine et al. 2010;Mesner et al. 2011), revealing that most origins coincide with active transcription units-specifically, the transcription start site. These data confirm conclusions from analyses of individual replication origins that there is no sequence-specific motif for origin recognition complex (ORC) binding or origin specification in metazoans. However, how local transcription affects replication initiation remains unknown, apart from a few specific examples (Danis et al. 2004;Mesner and Hamlin 2005;Xie and Orr-Weaver 2008).
Developmentally regulated gene amplification is an important origin discovery tool and model for investigating the regulation of metazoan replication origins in vivo (Claycomb and Orr-Weaver 2005). Specific genomic regions are amplified through replication-based mechanisms to increase gene copy number via either chromosomal excision followed by extrachromosomal amplification or repeated bidirectional replication initiation from an endogenous chromosomal locus. Gene amplification increases the DNA template to allow for sufficient levels of gene products required at high levels, such as rRNA in frog oocytes and cocoon proteins in Sciarid fly salivary glands. In Drosophila, two chorion gene clusters on the third and X chromosomes are amplified in ovarian follicle cells, somatic epithelial cells that surround the oocyte and secrete the components of the eggshell (Spradling 1981). Amplification enables the eggshell proteins to be produced in a short developmental period. When gene amplification is abolished-as in female-sterile mutants of replication factors such as ORC2, MCM6, and DBF4-these flies lay inviable eggs with thin eggshells, attributed to the inadequate template for transcription of eggshell genes (Landis et al. 1997;Landis and Tower 1999;Schwed et al. 2002).
Drosophila follicle cell gene amplification occurs by a DNA rereplication-based mechanism, making it a powerful model for investigating metazoan origin function (Park et al. 2007). In the follicle cell amplicons, repeated rounds of initiation of DNA replication from specific amplification origins produce gradients of increased DNA copy number. DNA replication during amplification uses the same known initiation factors that act in the typical S phase, such as ORC, Cdt1, and the MCM complex. In addition, gene amplification is amenable to diverse experimental approaches to study replication initiation. First, the process occurs within the context of developing egg chambers that are morphologically distinct and can be isolated for experimental analysis, allowing replication events to be studied in the context of development. Gene amplification begins during follicle cell differentiation after genomic replication has ended. Therefore, methods to assay DNA replication, including quantitative PCR (qPCR) or immunofluorescence of the nucleotide analog bromodeoxyuridine (BrdU) to visualize newly replicated DNA, can be used to assess the precise timing of replication events and to separate initiation and elongation phases at individual amplicons. Finally, Drosophila genetic tools for introducing DNA at ectopic sites allow one to delineate cis requirements for gene amplification.
In the context of gene amplification, the relationship between replication and transcription has two facets: how replication and increased DNA copy number affect transcription, as well as how local transcription affects origin selection and activation. Although gene amplification is considered a strategy to augment gene expression to high levels, how frequently amplification is used to achieve this output and whether amplification always leads to high expression levels are unknown. This latter question is especially important for evaluating the consequences of gene amplification in cancer cells. Sequencing cancer genomes has revealed a high frequency of chromosomally integrated gene amplification (Meyerson et al. 2010). Investigating the relationship between increased DNA copy number and gene expression in the developmentally programmed context of follicle cell gene amplification may shed light on this relationship in cancer cells.
To investigate gene amplification as a developmental strategy and metazoan replication model, we isolated pure populations of amplification stage follicle cells to identify both the complete catalog of follicle cell amplicons with array-based comparative genomic hybridization (aCGH) and analyze transcription on a genome-wide scale using next-generation sequencing. This provides the first highquality transcriptional profile of an amplifying differenti-ated tissue. Because synchronous gene amplification in follicle cells begins at a specific developmental stage, these amplification origins can be analyzed precisely with respect to transcription, ORC localization, and histone modifications. Finally, we investigate the determinants of origin activation at one amplification origin by exploiting its property of strain-specific amplification.

CGH identifies two new follicle cell amplicons
To identify all of the amplified regions in follicle cells, we employed an aCGH strategy, an expansion of a previous study that used cDNA microarrays containing <50% of all Drosophila genes and no intergenic regions (Claycomb et al. 2004). Before follicle cells begin gene amplification, they undergo three rounds of endoreduplication, or chromosomal replication without intervening mitoses, to reach 16C DNA copy levels. We used flow cytometry to isolate 16C nuclei, which encompass a mixed population enriched for amplification stages spanning exit from the endocycle, the start of amplification (stage 10), and the stage immediately prior to egg deposition. DNA isolated from 16C nuclei was competitively hybridized with diploid embryonic DNA on genome-wide tiling microarrays containing one 60-mer probe approximately every 600 base pairs (bp), representing all single-copy chromosomal sequences. Because of this high-density coverage, we are confident that we identified all of the amplicons present in euchromatin in these cells. Using this approach, we confirmed the presence of the four previously known follicle cell amplicons and identified two new follicle cell amplicons: DAFC-22B and DAFC-34B (Drosophila amplicon in follicle cell, followed by cytological position) (Fig. 1A).
Like the four previously identified amplicons, DAFC-22B and DAFC-34B show a gradient of replicated DNA that spans ;100 kb ( Fig. 1B,C). The maximum aCGH enrichment ratios for DAFC-22B and DAFC-34B are less than those of the chorion amplicons DAFC-7F and DAFC-66D but are comparable with DAFC-30B and DAFC-62D (Table  1). As expected, the genomic regions in DAFC-22B and DAFC-34B contain genes. The 23 genes within the amplified region of DAFC-34B are generally <5 kb in length, and one gene, Vm34Ca, located in the peak of amplification, encodes a structural component of the vitelline membrane, the innermost layer underlying the Drosophila eggshell (Mindrinos et al. 1985). Notably, there is just one 60-kb gene of unknown function-CG7337-in the most amplified region of DAFC-22B, thus differing from the genomic organization of the other follicle cell amplicons, which contain small genes that are ;1 kb in length encoding eggshell proteins and enzymes.
Amplified genes are not necessarily highly expressed, and highly expressed gene regions are not typically amplified We performed high-throughput RNA sequencing (RNAseq) to assess expression of genes in the precisely defined amplified regions. The isolation of RNA from follicle cells undergoing gene amplification presents a unique technical challenge from isolating DNA from sorted nuclei, because these cells must be separated from the nurse cell and oocyte as well as mitotic and endocycling follicle cell populations, while preserving cell integrity and RNA quality. We isolated RNA from 16C follicle cells recovered by FACS and performed Illumina sequencing to uncover a global view of transcript levels in amplification stage follicle cells. Transcript profiles for the chorion amplicons DAFC-66D and DAFC-7F show high expression levels of chorion genes in these regions (Fig. 2), although some of the chorion genes, such as cp16, are expressed at lower levels than others (Supplemental Table 1). These results correspond precisely with gene expression studies using developmental Northern blots and in situ hybridization, validating the accuracy of our RNA-seq quantification (Griffin-Shea et al. 1982). Other genes within the 100-kb amplified gradients of DAFC-66D or DAFC-7F were expressed at low levels. Thus, being located within an amplicon does not, by default, lead to abundant expression, nor does it, for expressed genes, enhance expression to uniform levels. Although gene amplification can promote high expression of some genes, additional regulatory mechanisms fine-tune the levels, developmental timing, and spatial control of gene expression.  The four nonchorion amplicons show distinct transcript profiles. Like the chorion amplicons, DAFC-34B shows high expression levels for at least two genes in the amplified region (Fig. 2C). The vitelline membrane gene Vm34Ca had the highest expression value in our RNA-seq data set, although, unlike the chorion amplicons, there is not a cluster of highly expressed genes in the 10-kb most amplified region of DAFC-34B. In contrast, DAFC-30B and DAFC-62D contain genes that are only moderately expressed by RNA-seq (Fig. 2B,D), which may reflect the very narrow developmental window in which they are expressed. Claycomb et al. (2004) showed that genes within DAFC-30B and DAFC-62D-such as CG13113 and yellow-g2, respectively-display high expression levels across all follicle cells in only one or two stages of egg chamber development, unlike the chorion genes, which show more sustained high expression levels throughout several stages (Parks et al. 1986;Parks and Spradling 1987). Additionally, DAFC-30B contains genes at the edge of the amplified gradient that are more highly expressed than genes in the central region, a unique feature among the six amplicons. For DAFC-22B, we found that CG7337 is expressed at low levels ( Fig. 2A). It is possible that gene amplification is necessary for even these low expression levels in follicle cells. However, CG7337 is expressed at comparable and even higher levels in other tissues where the 22B region is not amplified (Chintapalli et al. 2007). Another possibility is that amplification of this region does not augment gene expression. Instead, amplification may be the indirect consequence of transcription, which results in chromatin modifications or displacement of nucleosomes, permitting the region to serve as a replication origin (Eaton et al. 2010).
If high levels of transcription can create a chromatin domain that promotes gene amplification specifically in follicle cells, we might expect other genomic regions with highly expressed genes to become amplified. However, this does not appear to be the case, as there are hundreds of highly expressed genes and gene clusters, yet only six amplified regions (Fig. 3A). 16C follicle cells contain cells from stage 9 egg chambers, which have exited the endocycle but have not yet begun gene amplification. To address whether highly expressed genes measured by RNA-seq reflect robust stability of mRNAs transcribed at or prior to stage 9 rather than active transcription during gene amplification stages, we determined the localization of RNA polymerase II (RNAPII) by chromatin immunoprecipitation (ChIP) followed by hybridization to a microarray (ChIP-chip). We examined RNAPII localization in stage 10 egg chambers with the nurse cells manually removed, rather than in a mixed population of follicle cells, to assess whether RNAPII was active or poised for transcription at the precise onset of gene amplification. Although many genomic regions, including all six amplicons, show RNAPII localization at stage 10 and expression by RNA-seq (Fig. 2), only a limited number become amplified, demonstrating that active transcription is not sufficient for origin activation and gene amplification in follicle cells. Furthermore, these data reveal that gene amplification is not the exclusive means by which genes become highly expressed in follicle cells.

ORC binding in amplicons localizes in a domain to the most amplified region
Although we found that abundant and active transcription is not sufficient for gene amplification, it was nevertheless possible that transcription influences the localization of ORC. For example, recent studies in Drosophila cell culture have shown that ORC associates predominantly with the promoters of active genes (MacAlpine et al. 2004. To investigate a possible correlation between transcription and ORC localization in follicle cells, as well as determine the localization of ORC with respect to the 100-kb amplified gradients of the six amplicons, we performed ChIP-chip experiments using an antibody specific to ORC2. We used hand-sorted stage 10 egg chambers to obtain a stage-specific profile of ORC localization at the start of gene amplification (stage 10) and examined ORC binding in regions showing high expression and RNAPII localization. The dec-1 locus and vitelline membrane gene cluster at 26A are highly expressed in follicle cells (Fig. 3B,C). These regions do not exhibit significant levels of ORC binding or an increase in DNA copy number, emphasizing that high expression levels can be, and are most frequently, achieved in follicle cells without gene amplification.
We computationally identified the top scoring regions of ORC enrichment using the MA2C program (Song et al. 2007) and found that, although the amplicons were among the top scoring of the 99 regions, ORC localized to other discrete genomic regions that were not amplified (Fig. 4). ORC localization alone is not sufficient for gene amplification in follicle cells. Other regulatory mechanisms are likely necessary to establish full helicase loading and activate replication initiation. Alternatively, ORC localization at these nonamplifying sites may play a role independent of replication initiation, such as cohesin loading or maintenance of heterochromatic silencing (Pak et al. 1997;Huang et al. 1998;Takahashi et al. 2004). Additionally, although roughly two-thirds of the ORC-bound sites overlapped with transcription units (65 of 99), only onetenth corresponded to regions that were expressed at a reads per kilobase per million (RPKM) of >3 (10 of 99), marking a notable difference from Drosophila cell culture, where ORC-binding sites are associated predominantly with active promoters .
The six follicle cell amplicons show differences in the number of rounds of replication initiation. To ask whether differences in ORC localization might be responsible for differences in amplification levels, we examined the ORCbinding profile at the six amplicons. We found that ORC localizes to all follicle cell amplicons in a region centered at the peak of amplification, corresponding to a large zone ;10-30 kb in length, depending on the amplicon ( Fig. 2; Table 1). Previous studies by ChIP-qPCR suggested that ORC was bound to specific sites in the DAFC-62D and DAFC-66D amplicons (Austin et al. 1999;Xie and Orr-Weaver 2008); thus, it was striking to find ORC localized to broad domains. As the status of ORC binding for DAFC-7F and DAFC-30B was previously unknown, these studies contribute four new ORC-bound amplification origins to the catalog of metazoan replication origins. The chorion amplicons, which reach the highest copy number levels, also show the largest domain of ORC localization. However, the relationship between the width of ORC binding and magnitude of gene amplification is not precisely correlated for the other four amplicons.
In addition to ORC localization at the onset of gene amplification (stage 10), we also examined ORC-binding profiles by ChIP-chip at the six amplicons in stages immediately prior (stage 9 and early stage 10) to examine potential dynamics of ORC localization that could not be identified using 16C DNA alone. The ORC-binding profiles of the four nonchorion amplicons were remarkably similar between the two samples (Supplemental Fig. 1). In contrast, ORC enrichment dramatically increased from preamplification to amplification stages at the two chorion amplicons, suggesting that an active mechanism promotes ORC Figure 3. Gene amplification is not required for high follicle cell gene expression. (A) Gene expression by RNA-seq was analyzed in 100-kb windows, and the 90th percentile of highly expressed 100-kb regions was plotted and is designated by black boxes. Chromosome 3L is depicted in A. Many genomic regions show high expression but are not amplified. (B,C) Two-hundred-kilobase regions of two nonamplified genomic loci with highly expressed genes. Sequence reads from RNA-seq of purified follicle cells as well as localization of RNAPII, ORC, and tetra-acetylated H4 by ChIP-chip from stage 10 egg chambers are shown as in Figure 2. 7C contains the defective chorion 1 gene (dec-1), and 26A contains a cluster of genes encoding components of the vitelline membrane. Bars below the tetraacetylated H4 values represent significant regions of enrichment, as calculated by the MA2C program, using a P-value cutoff of 0.00001 and a bandwidth of 500 bp.

Origin activity in Drosophila
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H4 acetylation corresponds to the magnitude of gene amplification
To investigate further the molecular determinants of differential ORC localization and origin activation among the six amplicons, we examined histone modifications on a genome-wide scale. Two independent studies have found enrichment of hyperacetylated H3 and H4 at follicle cell amplicons (Aggarwal and Calvi 2004;Hartl et al. 2007). Loss-of-function mutant clones of the histone deacetylase Rpd3 resulted in increased acetylation levels and showed inappropriate genomic replication in amplification stage egg chambers. Furthermore, follicle cell amplification using a reporter construct of DAFC-66D could be inhibited by tethering Rpd3 to the region (Aggarwal and Calvi 2004). We performed ChIP-chip on stage 10 egg chambers using an antibody against tetra-acetylated H4, which recognizes acetylated Lys 5, Lys 8, Lys 12, and Lys 16. Enrichment of tetra-acetylated H4 was found at significant levels only at the two chorion amplicons (Fig. 2). Notably, tetra-acetylated H4 was enriched across many sites in the genome but was not sufficient for ORC localization or gene amplification. Because this antibody recognizes all four acetylated lysines, we used antibodies specific for single residues to test their specificity at the amplicons. Whereas acetylated H4K5, H4K12, and H4K16 antibodies showed only modest enrichment at the amplicons by ChIP-qPCR, acetylated H4K8 was most significantly enriched at the chorion amplicons (Supplemental Fig. 2; data not shown).
Given that there are significant peaks of H4 acetylation only at the chorion amplicons, we wanted to test for functionality of this modification. To analyze whether H4 acetylation levels could influence amplification levels, we used the amplification reporter system developed by the Calvi laboratory (Aggarwal and Calvi 2004). The reporter designated TT1 contains the 3.8-kb minimal origin with ACE3 and Orib from DAFC-66D next to UAS, which can be bound by GAL4 and GAL4 DNA-binding domain (DBD) fusion proteins. We expressed the Rpd3 fusion protein via a heat-shock promoter to determine quantitatively the effect of tethering the histone deacetylase to TT1 on DNA copy number and enrichment of H4K8. We used qPCR with transgene-specific primers to get a more accurate measure of DNA copy number changes than previous analyses, which used quantification of immunofluorescence images and Southern blots. We specifically examined H4K8 acetylation because this mark showed the highest levels of enrichment at the amplicons by ChIP-qPCR, and we reasoned that we could more confidently observe differences in acetylation levels due to experimental treatment.
Following 1-h heat-shock induction of a GAL4 DBD TRpd3 fusion, amplification was completely abolished in stage 10 as well as pooled egg chambers of stages 11 and 12, without affecting the endogenous amplicons (Fig. 5A). We examined stage 10 egg chambers specifically for acetylated H4K8 after induction of the GAL4 DBD TRpd3 fusion and found that this histone mark was significantly reduced at TT1 but was essentially unchanged at the endogenous amplicons (Fig. 5B). Thus, these results indicate that acetylation of H4K8 is necessary for amplification of the TT1 transgene, an effect that may be specific to TT1 and the chorion amplicons because the other four amplicons amplify without showing significant enrichment of H4 acetylation.

DAFC-22B exhibits strain-specific amplification
In the process of characterizing the amplification properties of DAFC-22B, we examined gene amplification at this locus in a number of genetic backgrounds. Surprisingly, we found that DAFC-22B displays strain-specific amplification. By aCGH, we found that, even in two closely related strains where all five other amplicons are common, DAFC-22B amplifies in OrR TOW (an Oregon R strain maintained in our laboratory) and is not ampli-fied in OrR MOD (an Oregon R strain adopted for the modENCODE project) (Fig. 6A,B). This finding enabled us to test the link between amplification, transcription, and histone acetylation, as well as define the requirements for ORC localization at this locus.
To characterize the amplification properties of DAFC-22B more closely, we examined the timing of origin activation as well as expression of the gene in the amplified locus. We determined the stage-specific replication profile of DAFC-22B by hand-sorting OrR TOW egg chambers to isolate genomic DNA and performing qPCR quantification of DNA copy levels. We found that replication initiation increased from stages 10B through 13, resulting in approximately fourfold amplification by stage 13 (Supplemental Fig. 3). Because DAFC-22B coincides with a gene of which the 59 end of one isoform (D-isoform) is in the peak of amplification, we examined stage-specific gene expression of CG7337 in both strains for possible differences that might explain the presence or absence of amplification at the region. Intriguingly, despite the presence of four times the amount of DNA template in OrR TOW compared with OrR MOD , we found that there was no difference in overall CG7337 expression levels between the amplifying and Figure 5. Tethering Rpd3 to the amplification reporter TT1 eliminates its amplification and reduces H4K8 acetylation. (A) qPCR with transgene-specific primers was used to determine copy number of the TT1 transgene in different genetic backgrounds and experimental conditions. TT1 amplification is repressed upon Rpd3 expression (heat shock) and tethering to TT1 at stage 10 and pooled stages 11 and 12 egg chamber samples. (B) Levels of H4K8 acetylation at TT1 and representative primers from endogenous amplicons in stage 10 by ChIP-qPCR. The arrow points to reduction in acetylated H4K8 levels upon Rpd3 expression and tethering to TT1.
To elucidate the mechanism of differential gene amplification at DAFC-22B, we performed ChIP-chip with the ORC2 antibody on the strain that does not amplify DAFC-22B in stage 10 egg chambers and found that ORC is absent at DAFC-22B despite the same ORC localization for the other five amplicons (Fig. 6A,B). Because the determinants of ORC binding are poorly understood in metazoans, we sought to uncover the difference between the two strains responsible for the difference in amplification to gain a better understanding of how ORC binds to specific DNA regions to promote replication initiation. We performed ChIP-chip on stage 10 egg chambers to examine H4 tetraacetylation at DAFC-22B and observed similar patterns between the two strains (Fig. 6A,B). Thus, although levels of H4 acetylation influence the magnitude of gene amplification at the chorion amplicons, acetylated H4 alone is not sufficient for replication initiation and does not appear to be responsible for differential DAFC-22B amplification.

Repression of origin activation is responsible for strain-specific differences in DAFC-22B amplification
In Drosophila, cis requirements for amplification can be determined using P-element-mediated transformation to examine amplification of a test sequence at an ectopic site. Because of this capability, we generated transposons containing the 12-kb ORC-binding zone from OrR TOW as well as the equivalent region from OrR MOD by PCR (fragment A in Fig. 7A) to test amplification. In addition, DAFC-22B Figure 6. DAFC-22B exhibits strain-specific amplification that correlates with the ability of the region to bind ORC. (A,B) Follicle cell DNA copy levels as well as ORC and tetra-acetylated H4 by ChIP-chip from stage 10 egg chambers are shown as in Figure 2 for two different strains: OrR TOW and OrR MOD . DAFC-22B is not amplified in the OrR MOD strain, and this region does not show ORC localization. Tetra-acetylated H4 is observed in similar patterns in both amplifying and nonamplifying strains. (C) cDNA was generated from different egg chamber stages and analyzed by qPCR. Fold enrichment is relative to Rps17 and normalized to the stage 1-9 sample. There is no difference in expression levels for the gene in DAFC-22B (CG7337) for any developmental stage.
is the only amplified region that, in cell culture, also shows ORC binding, although the peaks of ORC localization in follicle cells and cell culture do not overlap. Thus, we also amplified the 10-kb sequence containing the cell culture ORC-binding sites from OrR TOW to test for amplification (fragment B in Fig. 7A). We flanked the 22B sequences with Figure 7. Genetic analysis of cis control elements for differential DAFC-22B amplification reveals distant repression of origin activation. (A) The DAFC-22B regions for P-element-mediated transformation and testing sufficiency for amplification are shown in black bars. Fragment A was PCR-amplified from OrR TOW (amplifying) and OrR MOD (nonamplifying) flies. Fragment B was amplified from OrR TOW flies. These sequences integrated into random sites in the genome of a strain that does not amplify the endogenous 22B locus. ORC2 ChIP-chip data are shown for OrR TOW . ORC2 ChIP-seq data from KC cells are also shown (scale is sequence tag density). The circle represents the position of qPCR primers that detect amplification of fragment A. The star represents the position of qPCR primers that detect amplification of fragment A or B. (B) The TOW A fragment shows amplification using both sets of primers. The stage 1-8 egg chambers serve as a control, because amplification does not begin until stage 10. (C) The TOW B fragment shows amplification using only the star set of primers because the endogenous 22B sequence recognized by the circle primers does not amplify. (D) The MOD A fragment shows amplification using both sets of primers. (E) ORC2 ChIP-qPCR was performed on stage 10 egg chambers from transgenic flies carrying the MOD A fragment. The primers designated 22B endogenous do not amplify the MOD A transgene.

Origin activity in Drosophila
Suppressor of Hairy wing [Su(HW)]-binding sites to minimize position effect variability (Lu and Tower 1997). Sequencing the 22B transposon constructs revealed that no major rearrangements were present between the two strains, and no cytological rearrangements were observed in polytene squashes of chromosome 2 heterozygous for the two strain backgrounds that could account for repression in OrR MOD (data not shown). By restriction mapping, we confirmed that the transposon constructs from each strain contained unrearranged DNA relative to the genomic strain. The DAFC-22B transposons were injected into a genetic background, w 1118 , where the endogenous DAFC-22B locus does not amplify. Thus, in addition to using transposon-specific primers to detect amplification, the DAFC-22B sequence that is on the transposon can be used to test amplification, whereas the DAFC-22B sequence that is not on the transposon serves as a control for no endogenous DAFC-22B amplification.
When we tested for DAFC-22B amplification in transgenic lines where the test sequence had integrated randomly into the genome, we found that the fragments bearing the egg chamber and cell culture ORC-binding sequences derived from OrR TOW were both sufficient to support amplification at an ectopic site (Fig. 7B,C). As the two sequences are partially overlapping, these results indicate that, minimally, a 5-kb sequence is sufficient for amplification. Remarkably, we found that, in the two independent transformants recovered, the egg chamber ORC-binding sequence derived from the OrR MOD strain, which does not amplify DAFC-22B at the endogenous locus, was also capable of supporting amplification to levels comparable with the transgene derived from OrR TOW (Fig.  7D). To determine whether there is a peak of amplification within the 12-kb transgene derived from the OrR MOD strain, we assessed DNA copy levels by qPCR using primers spaced ;2 kb apart. We observed a modest increase at the center of the transgene compared with the distal ends (Supplemental Fig. 5). Importantly, we observed an increase in DNA copy levels from stage 10B to stage 13 egg chambers, recapitulating the developmental program of replication initiation at the endogenous 22B locus in OrR TOW . In addition, we examined ORC localization at the transgene derived from the OrR MOD strain and found levels of ORC enrichment consistent with that observed by ChIP-chip (Fig. 7E).
Thus, we uncovered an example of a sequence that is competent to function as a replication origin but is typically repressed for ORC localization and origin activation at its endogenous location. Because the transposon boundaries are ;5 kb away from the peak of ORC binding, repression of origin activation appears to be acting from a distance, and not directly at the central region of ORC binding. This repression can be reset when the DNA sequence is placed in new genomic contexts.

Discussion
We used Drosophila follicle cell gene amplification as a model system to study the molecular properties of metazoan replication origins. Although cell culture stud-ies have shown a strong relationship between actively transcribed promoters and replication origins, we found that high expression output and active transcription are not sufficient for ORC localization in the context of follicle cell amplification, an in vivo replication model system. Furthermore, ORC localization by itself is not sufficient for origin activation in amplifying follicle cells.
Our studies unexpectedly uncovered an example in which chromatin or DNA elements repress ORC binding and replication initiation at a distance. DAFC-22B provides a powerful opportunity to study the determinants of ORC binding in metazoans because the region displays strain-specific amplification that is correlated with ORC localization. By testing the amplification status of sequences generated from strains that do and do not amplify DAFC-22B at ectopic sites in transgenic flies, we found that sequences from both strains are capable of amplification. Thus, sequence differences within 5 kb of the ORC-binding zone are likely not responsible for differential ORC localization and origin activation. Instead, in strains that do not amplify DAFC-22B at the endogenous site, the replication origin-competent sequence is repressed, possibly by an epigenetic mechanism, from a distance of at least 5 kb away. The repression is not due to a trans-acting factor because DAFC-22B is not amplified at the endogenous locus in the genetic background in which the DAFC-22B transposons were introduced and found to amplify. We do not think that placing DNA in the context of Su(HW) insulator elements artificially makes any sequence replication-competent. Molecular analysis of DAFC-62D using the same transposon system demonstrated that various deletions in a 10-kb sequence, which was sufficient for ectopic amplification, eliminated origin activation (Xie and Orr-Weaver 2008).
A striking feature of the ORC-binding profiles for the six amplicons is that ORC localizes over broad domains of 10-30 kb. This contrasts with the localization pattern seen in diploid cell culture and with the other sites of ORC localization in the follicle cells . ORC binding in a zone in the amplicons also has been observed in the laboratories of Michael Botchan and Brian Calvi (pers. comm.). Notably, the chorion amplicons (DAFC-7F and DAFC-66D) show the largest domains of ORC binding, and they also undergo the most rounds of initiation. How might this molecular snapshot of ORC binding correspond to increased origin activation at the amplicons? The large domains of ORC binding may reflect ORC coating the DNA strands over tens of kilobases, and this could increase the probability of origin activation. Alternatively, because follicle cells are polyploid and have 16 copies of each DNA duplex when amplification initiates, ORC could be bound at slightly different sites within the amplicons on each of the 16 duplexes. Likewise, there may be cell-to-cell variability in the precise position of ORC localization, resulting in an apparent zone of binding. Differences in the levels of amplification could reflect the number of DNA helices that initiated DNA replication.
A distinct interpretation is that the increased ORC zone at the amplicons is a consequence of initiation rather than a cause. If more ORC molecules bind to the newly replicated DNA strands at the amplicons, this would explain the higher levels of ORC enrichment as well as the additional rounds of origin activation at the amplicons-in a sense, a feed-forward mechanism for replication. An active process of continuous ORC recruitment is an attractive model, particularly for the chorion amplicons, as there is a marked increase in ORC binding from preamplification to amplification stages at the chorion amplicons that is not observed at the other four loci (Supplemental Fig. 1).
We investigated the relationship between histone acetylation and gene amplification in Drosophila follicle cells and found that acetylation of H4-and, specifically, H4K8quantitatively correlated with levels of gene amplification, as demonstrated by amplification of a reporter upon Rpd3 tethering. At the endogenous amplicons, only the two chorion amplicons show overlap of the ORC-binding region with significant enrichment of H4 acetylation. Thus, H4 acetylation is not sufficient or necessary for ORC localization or origin firing. Instead, H4 acetylation levels may be required specifically for chorion gene amplification or may influence the number of rounds of origin firing in the context of regions undergoing replication initiation. Recent studies in Brian Calvi's laboratory also found that H4 acetylation correlates with the level of origin firing at the follicle cell amplicons. This work shows that multiple acetylated forms of H3 and H4 are enriched at amplicons, and these marks are regulated in a dynamic manner (B Calvi, pers. comm.).
One of the most consistent findings among recent genome-wide origin mapping studies is the significant number of origins that correspond to gene regions and, in particular, the transcription start sites of active genes (Cadoret et al. 2008;Sequeira-Mendes et al. 2009;MacAlpine et al. 2010). Additionally, a high-resolution study of replication timing in multiple cell lines using next-generation sequencing methods reported that genes expressed solely in one cell type were early-replicating exclusively in that cell type, a finding also supported by analysis of individual model replicons, suggesting a causal effect of transcription on early replication (Hansen et al. 2010). Although there are just six follicle cell amplicons to examine, compared with thousands of origins activated in the canonical S phase, all six ORC-binding amplification origins correspond to gene regions that show active transcription. However, there are hundreds of genes and gene clusters expressed at higher levels in follicle cells than even the moderately expressed genes of DAFC-30B and DAFC-62D, and these regions do not become amplified. Furthermore, highly expressed gene regions do not show a greater enrichment of ORC localization. Thus, active transcription is not sufficient for origin activation or ORC localization in amplifying follicle cells.
The identification of all of the amplified regions in Drosophila follicle cells, combined with genome-wide assessment of transcript levels, reveals that the simple model of gene amplification being a developmental strategy to promote high levels of gene expression, as seems to be the case for some genes within the two chorion amplicons, may not be an absolute one. When examined by RNA-seq of 16C follicle cells, the genes in DAFC-30B and DAFC-62D are expressed to moderate levels, and the genes in DAFC-22B are expressed to low levels. For the chorion genes and genes in DAFC-62D and DAFC-30B, the extra gene copies are necessary for full expression levels, as gene expression is reduced in amplification mutants (Landis et al. 1997;Schwed et al. 2002;Claycomb et al. 2004). As a gene regulatory strategy, gene amplification is absolutely essential, because these mutants lay inviable eggs with thin eggshells. However, amplification appears to have no effect on overall CG7337 expression levels between the amplifying and nonamplifying strains despite a fourfold difference in DNA template. Gene amplification and DNA copy number variations are significant features of cancer genomes. Although there is a strong correlation between these genetic alterations and cancer progression, our data highlight the importance of considering that increased DNA copy number may not always be coupled to enhanced gene expression.
Our studies have generated important insights regarding gene amplification as a developmental strategy and metazoan replication model. First, gene amplification is not always a driving force for abundant transcription. Our results of highly expressed genes that are not amplified demonstrate that promoter strength is the primary strategy for achieving high expression levels, even in a cell that is capable of gene amplification. Additionally, gene amplification does not uniformly lead to abundant expression levels. Gene amplification can augment gene expression, as is the case for the chorion genes, but it can also have no effect on expression, as for CG7337 at DAFC-22B. Second, we discovered an example of distant repression of ORC binding and origin activation. This finding validates the importance of replication origin discovery combined with close molecular analysis of individual origins. The strain specificity of DAFC-22B amplification likely reflects the widespread plasticity of origin specification and activation in metazoans, and will be a useful model replicon for future studies of poorly understood areas such as the regulation of dormant origins and cell type specificity of replication origin usage.

CGH
Two strains were used: OrR TOW and OrR MOD . 16C nuclei were isolated by FACS from OrR TOW and OrR MOD fattened females as described previously (Lilly and Spradling 1996). Genomic DNA was prepared using the DNeasy Blood and Tissue kit (Qiagen), digested with AluI and RsaI, and labeled using Invitrogen's BioPrime labeling kit. Slides were hybridized to custom Agilent tiling arrays with probes every 600 or 400 bp and washed as per Agilent's recommendations. Array intensities were mediannormalized across channels and smoothed by genomic windows of 10 kb using the Ringo package in R (Toedling et al. 2007).

Follicle cell isolation
16C follicle cells were isolated as described previously (Bryant et al. 1999;Cayirlioglu et al. 2003) with the following modifications. Ovaries from ;100 females were dissected in Grace's unsupplemented medium containing 10 mg/mL Hoechst 33342. After three washes in PBS to remove residual calcium, ovaries were trypsinized (0.5% final; Gibco) in PBS supplemented with 10 mg/mL Hoechst 33342 and filtered through 100-mm mesh into Grace's unsupplemented medium with 10 mg/mL Hoechst 33342. Four rounds of trypsinization were necessary to completely dissociate all follicle cells. FACS was used to isolate follicle cells based on ploidy using a MoFlo flow cytometer. Twohundred-fifty-thousand to 750,000 16C follicle cells were routinely recovered from 100 fattened females.

RNA isolation and RNA-seq
Total RNA was extracted from purified 16C follicle cells (from OrR TOW ) using TRIzol reagent (Invitrogen) according to the manufacturer's recommendations. One-hundred nanograms of RNA was used to generate an RNA-seq library using the mRNA-seq Sample Preparation kit from Illumina with the exception that no selection was used to isolate polyA RNA. Rather, RNA-seq libraries generated from total RNA were subjected to Duplex-Specific thermostable nuclease enzyme (DSN) normalization according to Illumina recommendation. Briefly, 100 ng of the RNA-seq library was treated with DSN after heat denaturation and subsequent renaturation to remove highly abundant rRNA and tRNA-derived transcripts. Two biological replicates with a correlation coefficient of 0.89 were used in the analysis. Expression is not normalized to DNA copy number.
RNA-seq reads were aligned to BDGP release 5/DM3 using ELAND and counts were normalized by the mRNA length to obtain the final RPKM values as in Nordman et al. (2011).
For the analysis of CG7337 expression, purified follicle cells were isolated using a protocol modified from Bryant et al. (1999). Two-hundred stage 13 egg chambers were dissected in ice-cold Schneider's medium supplemented with 10% FBS. Tissue was digested with 0.9 mL of 0.25% trypsin/EDTA and 0.1 mL of 50 mg/mL collagenase for 12 min at room temperature. The supernatant was strained through a 40-mm mesh and spun at 1000g for 7 min in the cold. TRIzol was added to the pellet for RNA isolation. RNA samples were reverse-transcribed with AMV reverse transcriptase (Promega). For cDNA analysis, Rps17 was used as the endogenous control.

ChIP
ChIP was performed with the following antibodies: RNAPII (Upstate Biotechnolgy, clone CTD4H8), which recognizes both phosphorylated and nonphosphorylated forms; ORC2 (Steve Bell); tetra-acetylated H4 (Active Motif, 39179); acetylated H4K5 (Upstate Biotechnology, 07-327); acetylated H4K8 (Upstate Biotechnology, 07-328); acetylated H4K12 (Upstate Biotechnology, 07-595); and acetylated H4K16 (Upstate Biotechnology, 07-329). The commercial histone modification antibodies used were ones validated by the modENCODE consortium for specificity. ChIP-qPCR was performed on 300 staged egg chambers per experiment as described (Xie and Orr-Weaver 2008). Likewise, for ChIP-chip, we isolated stage 10 egg chambers by hand when follicle cell gene amplification commences, and performed ChIP-chip. The starting number of egg chambers (;1200) produced enough material so that no amplification had to be performed on the samples. For ChIP-chip with the RNAPII antibody, stage 10 egg chambers were further dissected in half to remove the nurse cell population. All ChIP experiments were compared with input DNA. For hybridization to arrays, DNA was labeled using Invitrogen's BioPrime Total for Agilent aCGH labeling kit.
Peak calling for ORC ChIP-chip and TetraH4 ChIP-chip was done using the python MA2C package (Song et al. 2007). ORC peaks were identified using a P-value cutoff of 0.002 and a bandwidth of 5000 bp. TetraH4 peaks were identified using a P-value cutoff of 0.00001 and a bandwidth of 500 bp.

Reduction of histone acetylation on DAFC-66D transgenes
Transgenic lines carrying the TT1 and the hspTGAL4 DBD TRpd3 transposons were a gift from Brian Calvi (Aggarwal and Calvi 2004). Flies were crossed to introduce the two transposons into the same line; siblings that contained only TT1 (TT1/+) were kept as controls. One hour of heat shock at 37°C was used to overexpress the GAL4 fusion protein, followed by a 6-h recovery period. DNA copy levels were assessed for dissected stage 10 as well as pooled stages 11 and 12 egg chambers to ensure that expression of the fusion protein occurred prior to or during replication initiation of the reporter at stage 10.
qPCR Genomic DNA was isolated from staged egg chambers and quantified using absolute quantitative PCR as described (Claycomb et al. 2004) or relative quantification as described (Xie and Orr-Weaver 2008). Absolute quantification was used for the DAFC-22B replication profile, whereas relative quantification was used for all other experiments.

Transgenic fly construction
To test the cis requirements for amplification at DAFC-22B, we constructed transposons with the 12-kb ORC-binding region (stage 10) from OrR MOD (MOD4-9, MOD A in text), the 12-kb ORC-binding region (stage 10) from OrR TOW (TOW4-9, TOW A in text), and the 10-kb ORC-binding region (cell culture) from OrR TOW (TOW8-10, TOW B in text). The gap in probes in the DAFC-22B aCGH amplification profile represents a repeated DNA sequence not present in either OrR strain. In the transposons, the ORC-binding fragments were flanked by Su(HW)binding sites (SHWBS) to control for genomic position-specific integration effects. The sequences were PCR-amplified using exTaq DNA polymerase (Takara) and primers with AscI and AvrII sites on the forward and reverse sequences, respectively. These products were cloned into a modified PCRA vector with AscI and AvrII sequences engineered into the multiple cloning site (Lu et al. 2001). These plasmids are called PCRA_22B_MOD4-9, PCRA_ 22B_TOW4-9, and PCRA_22B_TOW8-10. These plasmids were digested with NotI and subjected to a partial XhoI digest to transfer the 12-kb or 10-kb inserts to the NotI and XhoI sites of Big Parent to generate BP_22B_MOD4-9, BP_22B_TOW4-9, and BP_22B_TOW8-10. These constructs were sent to BestGene, Inc., for injection. Two independent transformants were recovered for the MOD A line and one transformant each was recovered for the TOW A and TOW B lines.