The eukaryotic genome is organized within cells as chromatin. For proper information output, higher-order chromatin structures can be regulated dynamically. How such structures form and behave in ...various cellular processes remains unclear. Here, by combining super-resolution imaging (photoactivated localization microscopy PALM) and single-nucleosome tracking, we developed a nuclear imaging system to visualize the higher-order structures along with their dynamics in live mammalian cells. We demonstrated that nucleosomes form compact domains with a peak diameter of ∼160 nm and move coherently in live cells. The heterochromatin-rich regions showed more domains and less movement. With cell differentiation, the domains became more apparent, with reduced dynamics. Furthermore, various perturbation experiments indicated that they are organized by a combination of factors, including cohesin and nucleosome-nucleosome interactions. Notably, we observed the domains during mitosis, suggesting that they act as building blocks of chromosomes and may serve as information units throughout the cell cycle.
Display omitted
•We visualized chromatin structures and their dynamics in live mammalian cells•Nucleosomes form compact chromatin domains in live cells and move coherently•The domains are organized by nucleosome-nucleosome interactions and cohesin•The domains exist during mitosis and act as building blocks of chromosomes
How a genome is organized and behaves in live cells remains unclear. Nozaki et al. visualized little bunches of chromatin, “chromatin domains,” and their dynamic behavior in live mammalian cells. The domains can work as “Lego blocks” of chromosomes to maintain genetic information throughout the cell cycle.
Recent studies of genome-wide chromatin interactions have revealed that the human genome is partitioned into many self-associating topological domains. The boundary sequences between domains are ...enriched for binding sites of CTCC-binding factor (CTCF) and the cohesin complex, implicating these two factors in the establishment or maintenance of topological domains. To determine the role of cohesin and CTCF in higher-order chromatin architecture in human cells, we depleted the cohesin complex or CTCF and examined the consequences of loss of these factors on higher-order chromatin organization, as well as the transcriptome. We observed a general loss of local chromatin interactions upon disruption of cohesin, but the topological domains remain intact. However, we found that depletion of CTCF not only reduced intradomain interactions but also increased interdomain interactions. Furthermore, distinct groups of genes become misregulated upon depletion of cohesin and CTCF. Taken together, these observations suggest that CTCF and cohesin contribute differentially to chromatin organization and gene regulation.
The investigation of cohesin binding sites throughout different mammalian genomes by ChIP-sequencing has been fundamental to discover how cohesin and CTCF collaborate to form chromatin loops and to ...gain insight in the intricate regulation of cohesin. Here we describe a detailed ChIP protocol that has been successfully used for different cohesin subunits and cohesin regulators in various cell lines.
The kollerin complex, consisting of Scc2/Scc4 in yeast and Nipbl/Mau2 in vertebrates, is crucial for the chromatin-association of the cohesin complex and therefore for the critical functions of ...cohesin in cell division, transcriptional regulation and chromatin organisation. Despite the recent efforts to determine the genomic localization of the kollerin complex in different cell lines, major questions still remain unresolved, for instance where cohesin is actually loaded onto chromatin. Further, Nipbl seems to have also additional roles, for instance as transcription factor.This chapter summarizes our current knowledge on kollerin function and the recent studies on the genomic localization of Scc2, highlighting and critically discussing controversial data.
Consistent with the diverse roles of the cohesin complex in chromosome biology, mutations in genes encoding cohesin and its regulators are found in different types of cancer and in developmental ...disorders such as Cornelia de Lange Syndrome. It is so far considered that the defects caused by these mutations result from altered function of cohesin in regulating gene expression during development. Chromatin conformation analyses have established the importance of cohesin for the architecture of developmental gene clusters and in vivo studies in mouse and zebrafish demonstrated how cohesin defects lead to gene misregulation and to malformations similar to the related human syndromes. Here we present our current knowledge on cohesin's involvement in gene expression, highlighting molecular and mechanistic consequences of pathogenic mutations in the Cornelia de Lange syndrome.
Cohesin complexes mediate sister-chromatid cohesion in dividing cells but may also contribute to gene regulation in postmitotic cells. How cohesin regulates gene expression is not known. Here we ...describe cohesin-binding sites in the human genome and show that most of these are associated with the CCCTC-binding factor (CTCF), a zinc-finger protein required for transcriptional insulation. CTCF is dispensable for cohesin loading onto DNA, but is needed to enrich cohesin at specific binding sites. Cohesin enables CTCF to insulate promoters from distant enhancers and controls transcription at the H19/IGF2 (insulin-like growth factor 2) locus. This role of cohesin seems to be independent of its role in cohesion. We propose that cohesin functions as a transcriptional insulator, and speculate that subtle deficiencies in this function contribute to 'cohesinopathies' such as Cornelia de Lange syndrome.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The cohesin complex is crucial for chromosome segregation during mitosis and has recently also been implicated in transcriptional regulation and chromatin architecture. The NIPBL protein is required ...for the loading of cohesin onto chromatin, but how and where cohesin is loaded in vertebrate cells is unclear. Heterozygous mutations of NIPBL were found in 50% of the cases of Cornelia de Lange Syndrome (CdLS), a human developmental syndrome with a complex phenotype. However, no defects in the mitotic function of cohesin have been observed so far and the links between NIPBL mutations and the observed developmental defects are unclear. We show that NIPBL binds to chromatin in somatic cells with a different timing than cohesin. Further, we observe that high-affinity NIPBL binding sites localize to different regions than cohesin and almost exclusively to the promoters of active genes. NIPBL or cohesin knockdown reduce transcription of these genes differently, suggesting a cohesin-independent role of NIPBL for transcription. Motif analysis and comparison to published data show that NIPBL co-localizes with a specific set of other transcription factors. In cells derived from CdLS patients NIPBL binding levels are reduced and several of the NIPBL-bound genes have previously been observed to be mis-expressed in CdLS. In summary, our observations indicate that NIPBL mutations might cause developmental defects in different ways. First, defects of NIPBL might lead to cohesin-loading defects and thereby alter gene expression and second, NIPBL deficiency might affect genes directly via its role at the respective promoters.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Our understanding of the organization of the chromatin fiber within the cell nucleus has made great progress in the last few years. High-resolution techniques based on next-generation sequencing as ...well as optical imaging that can investigate chromatin conformations down to the single cell level have revealed that chromatin structure is highly heterogeneous at the level of the individual allele. While TAD boundaries and enhancer-promoter pairs emerge as hotspots of 3D proximity, the spatiotemporal dynamics of these different types of chromatin contacts remain largely unexplored. Investigation of chromatin contacts in live single cells is necessary to close this knowledge gap and further enhance the current models of 3D genome organization and enhancer-promoter communication. In this review, we first discuss the potential of single locus labeling to study architectural and enhancer-promoter contacts and provide an overview of the available single locus labeling techniques such as FROS, TALE, CRISPR-dCas9 and ANCHOR, and discuss the latest developments and applications of these systems.
Marked by incomplete division of the embryonic forebrain, holoprosencephaly is one of the most common human developmental disorders. Despite decades of phenotype-driven research, 80-90% of ...aneuploidy-negative holoprosencephaly individuals with a probable genetic aetiology do not have a genetic diagnosis. Here we report holoprosencephaly associated with variants in the two X-linked cohesin complex genes, STAG2 and SMC1A, with loss-of-function variants in 10 individuals and a missense variant in one. Additionally, we report four individuals with variants in the cohesin complex genes that are not X-linked, SMC3 and RAD21. Using whole mount in situ hybridization, we show that STAG2 and SMC1A are expressed in the prosencephalic neural folds during primary neurulation in the mouse, consistent with forebrain morphogenesis and holoprosencephaly pathogenesis. Finally, we found that shRNA knockdown of STAG2 and SMC1A causes aberrant expression of HPE-associated genes ZIC2, GLI2, SMAD3 and FGFR1 in human neural stem cells. These findings show the cohesin complex as an important regulator of median forebrain development and X-linked inheritance patterns in holoprosencephaly.
Cohesin is a ring-shaped multiprotein complex that is crucial for 3D genome organization and transcriptional regulation during differentiation and development. It also confers sister chromatid ...cohesion and facilitates DNA damage repair. Besides its core subunits SMC3, SMC1A, and RAD21, cohesin in somatic cells contains one of two orthologous STAG subunits, STAG1 or STAG2. How these variable subunits affect the function of the cohesin complex is still unclear. STAG1- and STAG2-cohesin were initially proposed to organize cohesion at telomeres and centromeres, respectively. Here, we uncover redundant and specific roles of STAG1 and STAG2 in gene regulation and chromatin looping using HCT116 cells with an auxin-inducible degron (AID) tag fused to either STAG1 or STAG2. Following rapid depletion of either subunit, we perform high-resolution Hi-C, gene expression, and sequential ChIP studies to show that STAG1 and STAG2 do not co-occupy individual binding sites and have distinct ways by which they affect looping and gene expression. These findings are further supported by single-molecule localizations via
stochastic optical reconstruction microscopy (
STORM) super-resolution imaging. Since somatic and congenital mutations of the STAG subunits are associated with cancer (STAG2) and intellectual disability syndromes with congenital abnormalities (STAG1 and STAG2), we verified STAG1-/STAG2-dependencies using human neural stem cells, hence highlighting their importance in particular disease contexts.
PDF
