Wolf Reik - Head of Laboratory
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Epigenetic gene regulation: control by the centre

Imprinted genes are only expressed from either the maternal or the paternal allele. Most imprinted genes occur in clusters, and their expression and silencing is often regulated by imprinting centres located within the cluster. Our recent work shows that there are at least two different types of imprinting centre. The first controls higher order chromatin structure, and partitions genes into expressed or repressed domains. The second controls repressive chromatin modifications through a non-coding RNA.

There are about 80 imprinted genes in the mouse genome, and most of them are conserved in humans. They have important roles in mammalian development, including the regulation of fetal growth (see Constancia), postnatal adaptations and metabolism (see Kelsey), and adult behaviour (see Wilkinson). Most imprinted genes are clustered in the genome, and within a cluster share regulatory elements such as enhancers or chromatin boundary elements (also called insulators). The clusters can be up to 1Mb or so in size and contain several imprinted genes. An important discovery was that of 'imprinting centres' (IC), that is regulatory elements in the cluster that control expression and imprinting of the majority or all genes in a cluster. How imprinting centres work is not known.

We are interested in a cluster of imprinted genes approximately 1 Mb in size which is located on distal chromosome 7 in the mouse, and on 11p15.5 in the human where genetic or epigenetic defects in the cluster are associated with the fetal overgrowth and cancer condition - Beckwith-Wiedemann syndrome. The cluster contains approximately 14 imprinted genes, and can be further subdivided into two domains (Figure 1).

Figure 1 (Click to enlarge)

Representation of the distal chromosome 7 imprinting cluster with the IC1 and IC2 subdomains. Filled boxes: imprinted genes; open boxes: non imprinted genes; DMRs (differentially methylated regions) are shown as methylated (filled) or undermethylated (open).

The centromeric domain is regulated by IC1 and contains the paternally expressed fetal growth enhancer Igf2, and the Ins2 gene which is paternally expressed in yolk sac. The telomeric domain is regulated by IC2 and contains several maternally expressed genes that are involved in growth suppression, such as for example the cell cycle suppressor Cdkn1c. The two ICs are the only regions in the cluster which have differential DNA methylation inherited from the parental germlines; IC1 is methylated in sperm but not in the oocyte and this is maintained throughout development, while IC2 is methylated in oocyte not sperm. IC1 has been previously defined as a chromatin insulator, which blocks the access of enhancers to gene promoters. Since the enhancers for the Igf2 gene are located about 100kb downstream of the gene, and the IC1 insulator is located between enhancers and the gene, it should block transcription of Igf2. This is indeed the case and transcriptional blocking of Igf2 is mediated by the insulator protein CTCF which binds to IC1. While this is the case on the maternal allele, on which Igf2 is silent, on the paternal allele IC1 is methylated and CTCF cannot bind to the DNA, allowing the distal enhancers to access the Igf2 promoters for transcription to occur.

These insights raise the important question of how the distal enhancers communicate with the Igf2 promoters, and how the insulator disrupts this communication. Adele Murrell in the lab reasoned that higher order chromatin structure or 'looping' could be involved (Figure 2).

Figure 2 (Click to enlarge)

The non-coding RNA model for imprinting in the IC2 region. The promoter region of the non-coding transcript Kcnq1ot1 (wavy arrow) has a DNA methylation germline imprint originating in the oocyte. Early postnatal expression of the Kcnq1ot1 transcript results in regional repressive histone modifications on the paternal chromosome, which are maintained in the extraembryonic tissues, leading to paternal silencing of genes near IC2.

She tested this model by tagging an engineered protein onto the IC1. On the maternal chromosome, this protein was then detected not only on IC1, but also associated with a region just upstream of Igf2, called DMR1. On the paternal chromosome, the protein was detected associated with IC1 and a region at the end of the Igf2 gene, called DMR2. This shows that IC1 is in close physical contact with the Igf2 gene. On the maternal allele, this results in Igf2 residing in a repressed chromatin loop away from the enhancers, while on the paternal allele, Igf2 is outside this loop and comes to be located close to the enhancers. These observations result in a model for the epigenetic control of insulators by organising higher order chromatin interactions. It will now be interesting to see what protein factors might be involved in mediating these interactions.

IC2 is a promoter region for a long non-coding antisense transcript, Kcnq1ot1, which is paternally expressed. This transcript is needed for the silencing of the neighbouring imprinted genes on the paternal chromosome. Annabelle Lewis and Kohzoh Mitsuya in the lab found that imprinting of these genes is maintained in the absence of DNA methylation in the placenta (but not the embryo). They reasoned that an epigenetic marking system other than DNA methylation might be involved in maintenance of imprints. It was found that the paternal chromosome domain is marked by repressive histone methylation, whereas the maternal domain is marked by activating modifications. Importantly, deletion of the promoter of Kcnq1ot1 abolished the repressive histone marks on the paternal chromosome. These observations lead to a model in which expression of the non-coding RNA in early embryos results in targeting of repressive histone marks to the region, which serve to stably silence adjacent genes (Figure 3).

Figure 2 (Click to enlarge)

The chromatin loop model for imprinting in the IC1 region. On the maternal chromosome, the H19 DMR and Igf2 DMR1 interact physically, placing Igf2 in repressive chromatin. On the paternal chromosome, the H19 DMR contacts Igf2 DMR2, placing the Igf2 gene outside the repressive chromatin loop, and near the enhancers (open circles) downstream of H19.

It will now be important to elucidate the molecular mechanism of this epigenetic marking process which involves non-coding RNA. This regulation of regional silencing by an imprinting centre bears striking similarities to the mechanism of X chromosome inactivation. This raises an interesting hypothesis by which the evolution of X inactivation and imprinting may be intricately linked. Comparative genomic and functional work is required to explore this hypothesis further.


Recent, selected publications

Smits G, Mungall AJ, Griffiths-Jones S, Smith P, Beury D, Matthews L, Rogers J, Pask AJ, Shaw G, VandeBerg JL, McCarrey JR, SAVOIR Consortium, Renfree MB, Reik W, Dunham I (2008) Conservation of the H19 noncoding RNA and H19-IGF2 imprinting mechanism in therians.
Nature Genetics 40 971-978
http://dx.doi.org/10.1038/ng.168

Lewis A, Green K, Dawson C, Redrup L, Huynh KD, Lee JT, Hemberger M, Reik W (2006) Epigenetic dynamics of the Kcnq1 imprinted domain in the early embryo.
Development 133 4203-4210
http://dx.doi.org/10.1242/dev.02612

Lewis AJ, Mitsuya K, Umlauf D, Smith P, Dean WL, Walter J, Higgins M, Feil R, Reik W (2004) Imprinting on distal chromosome 7 in the placenta involves repressive histone methylation independent of DNA methylation.
Nature Genetics 36 1291-1295
http://dx.doi.org/10.1038/ng1468

Murrell AM, Heeson S, Reik W (2004) Interaction between differentially methylated regions partitions the imprinted genes Igf2 and H19 into parent-specific chromatin loops.
Nature Genetics 36 889-893
http://dx.doi.org/10.1038/ng1402

Maher ER, Brueton LA, Bowdin SC, Luharia A, Cooper W, Cole TR, Macdonald F, Sampson JR, Barratt CL, Reik W, Hawkins MM (2003) Beckwith-Wiedemann syndrome and assisted reproduction technology (ART).
Journal of Medical Genetics 40 62-64
http://dx.doi.org/10.1136/jmg.40.1.62

Constancia M, Hemberger M, Hughes J, Dean WL, Ferguson-Smith AC, Fundele R, Stewart F, Kelsey GD, Fowden AL, Sibley C, Reik W (2002) Placental-specific IGF-II is a major modulator of placental and fetal growth.
Nature 417 945-948
http://dx.doi.org/10.1038/nature00819

Dean WL, Santos F, Stojkovic M, Zakhartchenko V, Walter J, Wolf E, Reik W (2001) Conservation of methylation reprogramming in mammalian development: aberrant reprogramming in cloned embryos.
Proceedings of the National Academy of Sciences of the United States of America 98 13734-13738
http://dx.doi.org/10.1073/pnas.241522698

Reik W, Dean WL, Walter J (2001) Epigenetic reprogramming in mammalian development.
Science 293 1089-1093
http://dx.doi.org/10.1126/science.1063443

Reik W, Walter J (2001) Evolution of imprinting mechanisms: the battle of the sexes begins in the zygote.
Nature Genetics 27 255-256
http://dx.doi.org/10.1038/85804

Constancia M, Dean WL, Lopes S, Moore TF, Kelsey GD, Reik W (2000) Deletion of a silencer element in Igf2 results in loss of imprinting independent of H19.
Nature Genetics 26 203-206
http://dx.doi.org/10.1038/79930

Oswald J, Engemann S, Lane N, Mayer W, Olek A, Fundele R, Dean WL, Reik W, Walter J (2000) Active demethylation of the paternal genome in the mouse zygote.
Current Biology 10 475-478
http://dx.doi.org/10.1016/S0960-9822(00)00448-6

Group Members

Wolf Reik - Head of Laboratory - Contact by email

Wendy Dean - Senior Research Associate - Contact by email

Heather Burgess - Research Assistant - Contact by email
Matthew Turley - Research Assistant - Contact by email

Miguel Branco - Post Doc - Contact by email
Cassandra Farthing - Postdoc - Contact by email
Gabriela Ficz - Postdoc - Contact by email
Christel Krueger - Postdoc - Contact by email
Joanna Marques - Postdoc - Contact by email
Masaaki Oda - Postdoc - Contact by email
Fatima Santos - Postdoc - Contact by email

Lizzie Perdeaux - PhD Student - Contact by email
Christian Popp - PhD Student - Contact by email
Andrew Keniry - PhD Student - Contact by email
Stefanie Seisenberger - PhD Student - Contact by email



Other Links

The Epigenome
Harwell Imprinting site
SAVOIR
CellCentric
University of Melbourne