Written by Dr Adele Murrell,University of Bath, UK

A long time ago, when we still used restriction enzymes to identify polymorphisms and the term “epigenetic” was used to dismiss quirky data that did not obey the central dogma of ‘DNA-makes-RNA-makes-protein’, we used to think of genes as linear sequences of bases in a sea of junk DNA. At bit more than a decade ago, just as the human genome sequencing project was nearing completion, the term “epigenetic” was confined to instances of stable mitotic inheritance of gene expression changes that could not be attributed to changes in nucleotide sequence. DNA methylation and histone modifications associate with gene expression changes and most epigenetic studies involved analysis of DNA methylation and possibly some histone acetylation. Nowadays, in order to be a card-carrying epigeneticist, one has to bear in mind that the linear nucleotide gene sequences are actually wrapped around nucleosomes to form chromatin that adopts open and closed conformations to regulate gene expression. Chromatin itself is further packaged into spatially segregated megabase-sized domains and sub-megabase-sized topological domains which may well form the architectural scaffold of chromosomes. Gene regulatory elements such as promoters and enhancers separated by long genomic distances can be brought closer by chromatin folding (looping) and the base of these loops are conceptualised as topological associated domains (TADs).

Initial studies of chromatin looping conformation focused on single loci. These studies identified a pivotal role for the 11 zinc finger CCCTC- binding factor, CTCF, in shaping chromatin loops and demonstrated that cohesin co-localised with CTCF to stabilise loops^1-5. Cohesin was also shown to interact with large promoter-enhancer complexes such as Mediator⁶. In the last two years our understanding of chromatin organisation has been further increased by studies that have taken a genome-wide approach. It has been noted that architectural proteins CTCF and/or cohesin binding sites are enriched at boundaries between TADS^7;8. However, CTCF and cohesin sites are also present within TADS and may therefore not be the only determinants of TAD structure.

In the June issue of Cell this year, Phillips-Cremins et al. identified that distinct combinations of CTCF, CTCF, cohesin, and Mediator work in a combinatorial manner to functionally organize chromatin in a cell-type-specific manner at the submegabase-length scale⁹. This was done using a 5C-seq strategy⁷，以在小鼠胚胎干细胞（ESC）和神经祖细胞（NPC）（NPC）（NPC）中（NPC）中的七个基因组区域（NPC）产生染色质相互作用的高分辨率图。这项研究值得注意的是，该分辨率在4KB片段水平上，该水平大大高于以前的HI-C技术（平均40 KB分辨率），因此揭示了亚洲较低相关的域。结果表明，某些TAD在不同的细胞类型之间是不变的，但是在亚群水平的TAD中揭示了染色体质组织的许多细胞特异性差异。⁹. Interactions over short genomic distances (<100kb) were predominantly between promoters and enhancers and were bridged by cohesin and Mediator. At intermediate distances (<300) CTCF, cohesin and Mediator were often the bridging proteins, while at 600 – 1000kb Mediator complexes seem to hold loops together. CTCF alone or CTCF together with cohesin bridged interactions that were larger than 1MB. It will be interesting to see whether the paradigms for hierarchical differences in chromatin topology as observed between ESC and NCPs will be applicable in all cell types. My prediction is that it will. In fact, earlier this year Seitan et al found that depletion of cohesin in non-cycling thymocytes has no effect on the formation of Mb-compartmentalisation of the genome, but does affect specific promoter-enhancer interactions,¹⁰which would fit in with the above observation of interactions at shorter genomic distances. Interestingly, reduction of interactions at the sub-megabase level increased or reinforced interactions within the larger domains suggesting that conformational changes in one region influence the conformation at a wider region¹⁰.

The frequency of occurrence of aberrant chromatin topography in disease is still unknown. Since chromatin organisation is a ubiquitous feature of all cells, mutations in genes that affect chromatin organisation are expected to be rare. Cohesin subunits and associated proteins are involved in a spectrum of developmental disorders known collectively as cohesinopathies. The most well-known cohesinopathy is Cornelia de Lange syndrome (CdLS). Although cohesin is best known for its role in holding sister chromatids together in mitosis, most CdLS patients show alterations in gene regulation rather than mitotic defects¹¹. The Bickmore lab has found that NIPBL, a protein responsible for loading the cohesin complex onto chromatin during S-phase, may have a role in regulating higher order chromatin structure, independent of CTCF and cohesin¹². CdLS patients with mutations in theNIPBLgene show chromatin decompaction, which is visible by fluorescentin situhybridisation¹². No mutations had been reported in CTCF until recently. Gregor et al have now discovered mutations in theCTCFgene in individuals with intellectual impairment and growth defects¹³. These mutations result in functional haplo-insuffiency ofCTCF, and transcriptome data from affected patients identified several down-regulated genes. Consultation of ChIA-PET interaction data showed that the down-regulated genes in these patients could feasibly be due to changes in chromatin topology¹³.

甚至没有提及破坏全球表观遗传基因调控的癌症或复杂的疾病，很明显，我们正处于理解异常染色质构象对疾病形成的贡献的边缘。下一步是开发易于用户友好的测定法，以鉴定各种患者组织样品上的异常染色质构象。

参考：

1. Hadjur, S., Williams, L.M., Ryan, N.K., Cobb, B.S., Sexton, T., Fraser, P., Fisher, A.G., and Merkenschlager, M. (2009). Cohesins form chromosomal cis-interactions at the developmentally regulated IFNG locus. Nature 460, 410-413.

2. Mishiro, T., Ishihara, K., Hino, S., Tsutsumi, S., Aburatani, H., Shirahige, K., Kinoshita, Y., and Nakao, M. (2009). Architectural roles of multiple chromatin insulators at the human apolipoprotein gene cluster. Embo J 28, 1234-1245.

3. Degner，S.C.，Verma-Gaur，J.，Wong，T.P.，Bossen，C.，Iverson，G.M.，Torkamani，A.，Vettermann，C.，Lin，Y.C.等。（2011）。CCCTC结合因子（CTCF）和粘着蛋白会影响Pro-B细胞中IGH基因座和反义转录的基因组结构。Proc Natl Acad Sci U S 108，9566-9571。

4. Chien，R.，Zeng，W.，Ball，A.R。和Yokomori，K。（2011）。粘着蛋白：哺乳动物基因调节中的临界染色质组织者。生物化学细胞Biol 89，445-458。

5. Nativio，R.，Wendt，K.S.，Ito，Y.，Huddleston，J.E.，Uribe-Lewis，S.，Woodfine，K.，Krueger，C.，Reik，W.。（2009）。在印迹的IGF2-H19基因座上，需要高阶染色质构象所需的粘蛋白。PLOS Genet 5，E1000739。

6. Kagey, M.H., Newman, J.J., Bilodeau, S., Zhan, Y., Orlando, D.A., van Berkum, N.L., Ebmeier, C.C., Goossens, J., Rahl, P.B., Levine, S.S., et al. (2010). Mediator and cohesin connect gene expression and chromatin architecture. Nature 467, 430-435.

7. Dixon，J.R.，Selvaraj，S.，Yue，F.，Kim，A.，Li，Y.，Shen，Y.，Hu，M.，Liu，J.S。和Ren，B.（2012）。通过分析染色质相互作用鉴定的哺乳动物基因组中的拓扑结构域。自然485，376-380。

8. Nora, E.P., Lajoie, B.R., Schulz, E.G., Giorgetti, L., Okamoto, I., Servant, N., Piolot, T., van Berkum, N.L., Meisig, J., Sedat, J., et al. (2012). Spatial partitioning of the regulatory landscape of the X-inactivation centre. Nature 485, 381-385.

9. Phillips-Cremins, J.E., Sauria, M.E., Sanyal, A., Gerasimova, T.I., Lajoie, B.R., Bell, J.S., Ong, C.T., Hookway, T.A., Guo, C., Sun, Y., et al. (2013). Architectural protein subclasses shape 3D organization of genomes during lineage commitment. Cell 153, 1281-1295.

10.面筋、V。福尔,一个詹,Y。,悉尼,R。Lajoie, B., Ing-Simmons, E., Lenhard, B., Giorgetti, L., Heard, E., Fisher, A., et al. (2013). Cohesin-based chromatin interactions enable regulated gene expression within pre-existing architectural compartments. Genome Res.

11. Castronovo, P., Gervasini, C., Cereda, A., Masciadri, M., Milani, D., Russo, S., Selicorni, A., and Larizza, L. (2009). Premature chromatid separation is not a useful diagnostic marker for Cornelia de Lange syndrome. Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology 17, 763-771.

12. Nolen, L.D., Boyle, S., Ansari, M., Pritchard, E., and Bickmore, W.A. (2013). Regional chromatin decompaction in Cornelia de Lange syndrome associated with NIPBL disruption can be uncoupled from cohesin and CTCF. Hum Mol Genet.

13. Gregor, A., Oti, M., Kouwenhoven, E.N., Hoyer, J., Sticht, H., Ekici, A.B., Kjaergaard, S., Rauch, A., Stunnenberg, H.G., Uebe, S., et al. (2013). De novo mutations in the genome organizer CTCF cause intellectual disability. American journal of human genetics 93, 124-131.