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Lasting impressions? Imprinted gene clusters in the mouse

Genomic imprinting in mammals was recognized around 25 years ago from two very different experimen‐ tal approaches in the mouse. From nuclear transplantation experiments, it was shown that both a paternal genome and a maternal genome were required in order for normal development of an embryo to birth1,2. At the same time, sophisticated genetic techniques were being used to generate mice that were chromo‐ somally balanced, but had inherited both copies of a single chromosome or part of a chromosome from one parent only. Uniparental inheritance of some chromosome regions (called imprinting regions), but not others, resulted in the birth of mice with abnormal phenotypes3. The finding that phenotypic effects could be localized to particular chromosome regions implied that specific genes were involved, and this led to the startling conclusion that, contrary to Mendel’s principles, some mammalian genes functioned differ‐ ently depending on whether they had been inherited from the mother or the father. From further genetic experiments, 13 imprinting regions on eight autosomes were identified. The developmental phenotypes associated with uniparental inheritance of these regions ranged from pre‐ and post‐natal growth defects, reduced viability as well as postnatal behavioural deficits (Figure 1). Furthermore, the genetic experiments in the mouse indicated that imprinting may affect human disease and a number of human disorders with developmental defects involving growth and/or neurological anomalies resulting from mutations in im‐ printed genes or affecting epigenetic imprinting marks have been identified.


Imprinting control regions
Imprinted genes are thought to be associated in clusters so that the imprinted expression of all the genes in the cluster can be controlled by a master regulator. A great deal of work has been undertaken over the years to identify these regulators, called imprinting control regions (ICRs), and there is much ongoing work to understand how they function. Within each well-studied cluster, an ICR that regulates the imprinted expression of all the genes within the cluster has been found ( Figure 2). ICRs act in cis, usually bidirectionally, to silence genes in the cluster. ICRs are up to several kilobases in size, are abundant in the dinucleotide CpG, and are differentially methylated. ICRs are active when unmethylated and inactive when methylated. Methylation at ICRs is acquired in the germline; for some clusters, methylation is acquired in the maternal germline and, in others, methylation is acquired in the paternal germline. This DNA methylation constitutes the imprint, and the allele that acquires methylation is described as imprinted.
Before methylation marks can be set in the male and female germlines, pre-existing methylation marks have to be erased. This process occurs in primordial germ cells and is very poorly understood. However, there has been a very recent breakthrough reporting that activation-induced cytidine deaminase (AID) has a critical function in this process, although the mechanism of action remains to be established 6 .

Imprinted genes
The first three imprinted genes were found in 1991, and, so far, just over 130 imprinted loci have been identified in the mouse 4 , representing a small subset of around 0.5% of the total gene complement. The vast majority of imprinted genes not only map to the imprinting regions previously defined by phenotype studies, but also tend to occur in clusters. Clusters of imprinted genes have been identified in 11 of the 13 imprinting regions. Most imprinting regions contain a single cluster of imprinted genes, except for the region on distal chromosome 7 where two distinct clusters are known. The clusters can vary in size from 100 kb or so to nearly 2 Mb and four clusters contain more than 10 imprinted genes 5 ( Figure 1). All clusters described so far comprise paternally and maternally expressed genes and both protein-coding and non-protein-coding genes.
Favoured sites for expression of imprinted genes are the placenta and the brain. Misexpression of individual imprinted genes is associated with prenatal and/or early postnatal loss of viability and growth abnormalities, and behavioural anomalies, reminiscent of phenotypes associated with uniparental inheritance of imprinting regions. Effects on prenatal growth may be due to placental and/or fetal defects. Behavioural effects attributable to individual imprinted genes range from abnormalities in suckling and overall activity in newborns, to defects in maternal behaviour in caring for young, to a late onset anomaly in exploratory behaviour.

Features
October 2010 © 2010 The Biochemical Society Epigenetics methylation mark. This protein-coding transcript was expressed in oocytes at the time when methylation is established. Transcription through a number of other maternally methylated ICRs elsewhere in the genome was also found, suggesting that transcription from specific oocyte promoters may be a general requirement for maternal de novo methylation 9 .
Methylation occurs more frequently in the maternal germline than the paternal germline. In the mouse, 15 regions that acquire methylation during spermatogenesis or oogenesis, and thus are potential ICRs, have been identified and, of these, 12 acquired methylation during oogenesis and only three became methylated in spermatogenesis 10 . Six of these putative ICRs have been fully verified by functional testing and these are located in well-studied clusters of imprinted genes. Of these six ICRs, four (at the Gnas cluster on distal chromosome 2, at the PWS/AS cluster on central chromosome 7, at the Kcnq1 cluster on distal chromosome 7 and at the Igf2r/Airn cluster on proximal chromosome 17) acquire methylation in the female germline. The remaining two (the Igf2/H19 cluster on distal chromosome 7 and Following erasure, remethylation of the ICR must occur. Maternal methylation of ICRs occurs postnatally in the final stages of oocyte maturation and paternal methylation of ICRs takes place in male germ cells during fetal development. The mechanisms underlying the establishment of methylation at ICRs are not well understood, but the acquisition of methylation at ICRs in both male and female germlines is mediated by the DNA methyltransferase DNMT3a and its non-enzymatic cofactor DNMT3L 7,8 . Histone modifications appear to have a role for it has been shown that DNMT3L interacts with histone H3, but only when it is unmethylated at Lys-4. Thus, for methylation to occur, DNMT3L must interact with both DNMT3a and histone H3. Tandem repeat sequences at ICRs that are methylated in the maternal germline may play a part in attracting the methylation complex, and it has been shown recently that transcription also has a role in inducing methylation in the maternal germline. Thus transcription of a protein-coding transcript, called Nesp, across the ICR in the Gnas cluster ( Figure 2) was essential for induction of the maternal  Epigenetics which genes within a cluster are silenced by an active ICR have been described. One mechanism involves the active ICR acting as an insulator between genes on one side and their enhancers on the other together with higher-order chromatin interactions. Another mechanism involves long non-coding antisense RNAs that arise from the ICR. A third recently described mechanism involves a retrogene that arises from the ICR and either the retrogene or the ICR induces premature polyadenylation and loss of function of a protein-coding gene 10 .
An example of an intergenic ICR with an insulator function is the one at the Igf2/H19 cluster on distal mouse chromosome 7. This ICR is methylated and therefore inactive on the maternally derived chromosome, and unmethylated and active on the paternally derived chromosome ( Figure 2). The active ICR binds the zinc-finger transcription factor CTCF and forms a barrier or insulator between Igf2 and its downstream enhancers, silencing Igf2. In addition to CTCF, the protein complex cohesin is needed for the formation of chromatin loops so that Igf2 can be silenced 10 . When the ICR is methylated and inactive, Igf2 can interact with its enhancers and be expressed.
In recent years, it has become apparent that most of the mammalian genome is transcribed, and that most of the transcriptome is non-coding and of unknown function. Of the four fully verified maternally methylated ICRs, three (at the Gnas cluster on chromosome 2, at the Kcnq1 cluster on chromosome 7 and at the Igf2r/Airn cluster on chromosome 17) contain promoters for long non-coding RNAs that run antisense to a protein coding gene. The fourth maternally methylated ICR at the PWS/AS cluster lies within the transcription unit of a non-coding antisense transcript. Each of these non-coding antisense transcripts is paternally expressed from the active unmethylated ICR. These transcripts can be of extraordinary size; for example, the Airn transcript in the Igf2r/Airn cluster is 108 kb long and silences three genes dispersed over 300 kb of DNA and the Kcnq1ot1 transcript in the Kcnq1 cluster in distal mouse chromosome 7 is 90 kb long and silences 11 genes distributed over 800 kb 10 . Both of these very long RNAs that arise from their respective ICRs have been shown to be essential for silencing all of the protein-coding genes in cis in their respective clusters. It is these non-coding RNAs that are essential imprinting elements in the Igf2r/Airn and Kcnq1 clusters. A very active field of research is to uncover the mechanisms whereby expression of these long non-coding RNAs results in bidirectional gene silencing in cis. One major question concerns whether the long non-coding RNA itself is responsible for silencing or whether transcription of the non-coding RNA results in silencing. Recent work has suggested imprinted gene silencing in the placenta is brought about by the RNA itself 5 . However, in the embryo, the act of transcription of the non-coding RNA may bring about repression.   the Dlk1/Gtl2 cluster on distal chromosome 12) acquire methylation in the male germline ( Figure 2). These methylation marks set down at ICRs in the germline are extremely robust and resistant to the genome-wide wave of demethylation that occurs shortly after fertilization and are maintained in somatic cells throughout development. DNMT1 is essential for the maintenance of methylation. Specific histone modifications are also associated with methylated and unmethylated ICRs. Thus chromatin enriched for histone marks H3K9me3 and H4K20me3 has been found at DNA-methylated ICRs, whereas enrichment for H3K4me2 and H3K4me3 is found at unmethylated ICRs 8 . Several other proteins, including the zinc-finger protein ZFP57 and STELLA, also appear to play a role in the maintenance of methylation.

Modes of functioning of ICRs
Paternally methylated ICRs are located in intergenic regions, whereas maternally methylated ICRs lie in promoter regions, and this difference is probably relevant for the way that the ICRs function. Several mechanisms by