60 research outputs found
Spatial enhancer clustering and regulation of enhancer-proximal genes by cohesin
In addition to mediating sister chromatid cohesion during the cell cycle, the cohesin complex associates with CTCF and with active gene regulatory elements to form long-range interactions between its binding sites. Genome-wide chromosome conformation capture had shown that cohesin's main role in interphase genome organization is in mediating interactions within architectural chromosome compartments, rather than specifying compartments per se. However, it remains unclear how cohesin-mediated interactions contribute to the regulation of gene expression. We have found that the binding of CTCF and cohesin is highly enriched at enhancers and in particular at enhancer arrays or “super-enhancers” in mouse thymocytes. Using local and global chromosome conformation capture, we demonstrate that enhancer elements associate not just in linear sequence, but also in 3D, and that spatial enhancer clustering is facilitated by cohesin. The conditional deletion of cohesin from noncycling thymocytes preserved enhancer position, H3K27ac, H4K4me1, and enhancer transcription, but weakened interactions between enhancers. Interestingly, ∼50% of deregulated genes reside in the vicinity of enhancer elements, suggesting that cohesin regulates gene expression through spatial clustering of enhancer elements. We propose a model for cohesin-dependent gene regulation in which spatial clustering of enhancer elements acts as a unified mechanism for both enhancer-promoter “connections” and “insulation.
Nuclear entropy, angular second moment, variance and texture correlation of thymus cortical and medullar lymphocytes: Grey level co-occurrence matrix analysis
A Zebrafish Model of Roberts Syndrome Reveals That Esco2 Depletion Interferes with Development by Disrupting the Cell Cycle
The human developmental diseases Cornelia de Lange Syndrome (CdLS) and Roberts Syndrome (RBS) are both caused by mutations in proteins responsible for sister chromatid cohesion. Cohesion is mediated by a multi-subunit complex called cohesin, which is loaded onto chromosomes by NIPBL. Once on chromosomes, cohesin binding is stabilized in S phase upon acetylation by ESCO2. CdLS is caused by heterozygous mutations in NIPBL or cohesin subunits SMC1A and SMC3, and RBS is caused by homozygous mutations in ESCO2. The genetic cause of both CdLS and RBS reside within the chromosome cohesion apparatus, and therefore they are collectively known as “cohesinopathies”. However, the two syndromes have distinct phenotypes, with differences not explained by their shared ontology. In this study, we have used the zebrafish model to distinguish between developmental pathways downstream of cohesin itself, or its acetylase ESCO2. Esco2 depleted zebrafish embryos exhibit features that resemble RBS, including mitotic defects, craniofacial abnormalities and limb truncations. A microarray analysis of Esco2-depleted embryos revealed that different subsets of genes are regulated downstream of Esco2 when compared with cohesin subunit Rad21. Genes downstream of Rad21 showed significant enrichment for transcriptional regulators, while Esco2-regulated genes were more likely to be involved the cell cycle or apoptosis. RNA in situ hybridization showed that runx1, which is spatiotemporally regulated by cohesin, is expressed normally in Esco2-depleted embryos. Furthermore, myca, which is downregulated in rad21 mutants, is upregulated in Esco2-depleted embryos. High levels of cell death contributed to the morphology of Esco2-depleted embryos without affecting specific developmental pathways. We propose that cell proliferation defects and apoptosis could be the primary cause of the features of RBS. Our results show that mutations in different elements of the cohesion apparatus have distinct developmental outcomes, and provide insight into why CdLS and RBS are distinct diseases
Dosage Effects of Cohesin Regulatory Factor PDS5 on Mammalian Development: Implications for Cohesinopathies
Cornelia de Lange syndrome (CdLS), a disorder caused by mutations in cohesion proteins, is characterized by multisystem developmental abnormalities. PDS5, a cohesion protein, is important for proper chromosome segregation in lower organisms and has two homologues in vertebrates (PDS5A and PDS5B). Pds5B mutant mice have developmental abnormalities resembling CdLS; however the role of Pds5A in mammals and the association of PDS5 proteins with CdLS are unknown. To delineate genetic interactions between Pds5A and Pds5B and explore mechanisms underlying phenotypic variability, we generated Pds5A-deficient mice. Curiously, these mice exhibit multiple abnormalities that were previously observed in Pds5B-deficient mice, including cleft palate, skeletal patterning defects, growth retardation, congenital heart defects and delayed migration of enteric neuron precursors. They also frequently display renal agenesis, an abnormality not observed in Pds5B−/− mice. While Pds5A−/− and Pds5B−/− mice die at birth, embryos harboring 3 mutant Pds5 alleles die between E11.5 and E12.5 most likely of heart failure, indicating that total Pds5 gene dosage is critical for normal development. In addition, characterization of these compound homozygous-heterozygous mice revealed a severe abnormality in lens formation that does not occur in either Pds5A−/− or Pds5B−/− mice. We further identified a functional missense mutation (R1292Q) in the PDS5B DNA-binding domain in a familial case of CdLS, in which affected individuals also develop megacolon. This study shows that PDS5A and PDS5B functions other than those involving chromosomal dynamics are important for normal development, highlights the sensitivity of key developmental processes on PDS5 signaling, and provides mechanistic insights into how PDS5 mutations may lead to CdLS
Cohesin-dependent globules and heterochromatin shape 3D genome architecture in S. pombe
Eukaryotic genomes are folded into three-dimensional structures, such as self-associating topological domains, the borders of which are enriched in cohesin and CCCTC-binding factor (CTCF) required for long-range interactions1-7. How local chromatin interactions govern higher-order folding of chromatin fibers and the function of cohesin in this process remain poorly understood. Here we perform genome-wide chromatin conformation capture (Hi-C) analysis8 to explore the high-resolution organization of the Schizosaccharomyces pombe genome, which despite its small size exhibits fundamental features found in other eukaryotes9. Our analyses of wild type and mutant strains reveal key elements of chromosome architecture and genome organization. On chromosome arms, small regions of chromatin locally interact to form “globules”. This feature requires a function of cohesin distinct from its role in sister chromatid cohesion. Cohesin is enriched at globule boundaries and its loss causes disruption of local globule structures and global chromosome territories. By contrast, heterochromatin, which loads cohesin at specific sites including pericentromeric and subtelomeric domains9-11, is dispensable for globule formation but nevertheless affects genome organization. We show that heterochromatin mediates chromatin fiber compaction at centromeres and promotes prominent interarm interactions within centromere-proximal regions, providing structural constraints crucial for proper genome organization. Loss of heterochromatin relaxes constraints on chromosomes, causing an increase in intra- and inter-chromosomal interactions. Together, our analyses uncover fundamental genome folding principles that drive higher-order chromosome organization crucial for coordinating nuclear functions
Unravelling higher order chromatin organisation through statistical analysis
Recent technological advances underpinned by high throughput sequencing have
given new insights into the three-dimensional structure of mammalian genomes.
Chromatin conformation assays have been the critical development in this area,
particularly the Hi-C method which ascertains genome-wide patterns of intra and
inter-chromosomal contacts. However many open questions remain concerning the
functional relevance of such higher order structure, the extent to which it varies, and
how it relates to other features of the genomic and epigenomic landscape.
Current knowledge of nuclear architecture describes a hierarchical organisation
ranging from small loops between individual loci, to megabase-sized self-interacting
topological domains (TADs), encompassed within large multimegabase chromosome
compartments. In parallel with the discovery of these strata, the ENCODE project has
generated vast amounts of data through ChIP-seq, RNA-seq and other assays applied
to a wide variety of cell types, forming a comprehensive bioinformatics resource.
In this work we combine Hi-C datasets describing physical genomic contacts with
a large and diverse array of chromatin features derived at a much finer scale in the
same mammalian cell types. These features include levels of bound transcription
factors, histone modifications and expression data. These data are then integrated
in a statistically rigorous way, through a predictive modelling framework from the
machine learning field. These studies were extended, within a collaborative project, to
encompass a dataset of matched Hi-C and expression data collected over a murine
neural differentiation timecourse.
We compare higher order chromatin organisation across a variety of human cell
types and find pervasive conservation of chromatin organisation at multiple scales.
We also identify structurally variable regions between cell types, that are rich in active
enhancers and contain loci of known cell-type specific function. We show that broad
aspects of higher order chromatin organisation, such as nuclear compartment domains,
can be accurately predicted in a variety of human cell types, using models based upon
underlying chromatin features. We dissect these quantitative models and find them
to be generalisable to novel cell types, presumably reflecting fundamental biological
rules linking compartments with key activating and repressive signals. These models
describe the strong interconnectedness between locus-level patterns of local histone
modifications and bound factors, on the order of hundreds or thousands of basepairs,
with much broader compartmentalisation of large, multi-megabase chromosomal
regions.
Finally, boundary regions are investigated in terms of chromatin features and
co-localisation with other known nuclear structures, such as association with the
nuclear lamina. We find boundary complexity to vary between cell types and link
TAD aggregations to previously described lamina-associated domains, as well as
exploring the concept of meta-boundaries that span multiple levels of organisation.
Together these analyses lend quantitative evidence to a model of higher order genome
organisation that is largely stable between cell types, but can selectively vary locally,
based on the activation or repression of key loci
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