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RB is a cell cycle-regulated repressor of cohesin-dependent loop formation

ABSTRACT: Chromatin organization and cell cycle are tightly intertwined in eukaryotes, but the underlying mechanisms remain unclear. We investigated how the retinoblastoma protein (RB), a key cell cycle regulator localized to chromatin insulators, influences chromatin architecture. Utilizing chromosome conformation capture, we revealed that RB loss increased the number and size of cohesin-dependent loops and strengthened topologically associating domains (TADs). Mechanistically, RB regulated the chromatin distribution of cohesin, which extensively colocalized with RB in the human genome. Active RB evicted cohesin from RB-bound TAD boundaries and repressed insulator activity, thereby enhancing the expression of non-E2F target genes central to cell adhesion. We identify RB's centrality in the interplay between cell cycle and chromatin organization and propose a novel RB function in safeguarding chromatin architecture and thereby transcription.

ORGANISM(S): Homo sapiens (human) Homo sapiens

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PROVIDER: S-BSST1221 | biostudies-other |

REPOSITORIES: biostudies-other

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RB is a cell cycle-regulated repressor of cohesin-dependent loop formation

Project description:Chromatin organization and cell cycle are tightly intertwined in eukaryotes, but the underlying mechanisms remain unclear. We investigated how the retinoblastoma protein (RB), a key cell cycle regulator, influences chromatin architecture. Chromosome conformation capture assays reveal that RB loss increases the number and size of cohesin-dependent loops and strengthens topologically associating domains (TADs). RB shows extensive colocalization with cohesin in the human genome, and it impacts cohesin’s distribution on chromatin. Active RB evicted cohesin from RB-bound TAD boundaries, repressed insulator activity, and enhanced the expression of non-E2F target genes that are important for cell adhesion. We conclude that RB has a central role in the interplay between cell cycle and chromatin organization. This role safeguards chromatin architecture and controls transcription.

2024-11-01 | E-MTAB-13566 | biostudies-arrayexpress

RB is a cell cycle-regulated repressor of cohesin-dependent loop formation

Project description:RB is a cell cycle-regulated repressor of cohesin-dependent loop formation

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The SMC1-SMC3 cohesin heterodimer structures DNA through supercoiling-dependent loop formation.

Project description:Cohesin plays a critical role in sister chromatid cohesion, double-stranded DNA break repair and regulation of gene expression. However, the mechanism of how cohesin directly interacts with DNA remains unclear. We report single-molecule experiments analyzing the interaction of the budding yeast cohesin Structural Maintenance of Chromosome (SMC)1-SMC3 heterodimer with naked double-helix DNA. The cohesin heterodimer is able to compact DNA molecules against applied forces of 0.45 pN, via a series of extension steps of a well-defined size ≈130 nm. This reaction does not require ATP, but is dependent on DNA supercoiling: DNA with positive torsional stress is compacted more quickly than negatively supercoiled or nicked DNAs. Un-nicked torsionally relaxed DNA is a poor substrate for the compaction reaction. Experiments with mutant proteins indicate that the dimerization hinge region is crucial to the folding reaction. We conclude that the SMC1-SMC3 heterodimer is able to restructure the DNA double helix into a series of loops, with a preference for positive writhe.

| S-EPMC3695518 | biostudies-literature

DNA sequence-dependent mechanics and protein-assisted bending in repressor-mediated loop formation.

Project description:As the chief informational molecule of life, DNA is subject to extensive physical manipulations. The energy required to deform double-helical DNA depends on sequence, and this mechanical code of DNA influences gene regulation, such as through nucleosome positioning. Here we examine the sequence-dependent flexibility of DNA in bacterial transcription factor-mediated looping, a context for which the role of sequence remains poorly understood. Using a suite of synthetic constructs repressed by the Lac repressor and two well-known sequences that show large flexibility differences in vitro, we make precise statistical mechanical predictions as to how DNA sequence influences loop formation and test these predictions using in vivo transcription and in vitro single-molecule assays. Surprisingly, sequence-dependent flexibility does not affect in vivo gene regulation. By theoretically and experimentally quantifying the relative contributions of sequence and the DNA-bending protein HU to DNA mechanical properties, we reveal that bending by HU dominates DNA mechanics and masks intrinsic sequence-dependent flexibility. Such a quantitative understanding of how mechanical regulatory information is encoded in the genome will be a key step towards a predictive understanding of gene regulation at single-base pair resolution.

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2D gel electrophoresis reveals dynamics of t-loop formation during the cell cycle and t-loop in maintenance regulated by heterochromatin state.

Project description:Linear chromosome ends are capped by telomeres that have been previously reported to adopt a t-loop structure. The lack of simple methods for detecting t-loops has hindered progress in understanding the dynamics of t-loop formation and its function in protecting chromosome ends. Here, we employed a classical two-dimensional agarose gel method (2D gel method) to innovatively apply to t-loop detection. Briefly, restriction fragments of genomic DNA were separated in a 2D gel, and the telomere sequence was detected by in-gel hybridization with telomeric probe. Using this method, we found that t-loops are present throughout the cell cycle, and t-loop formation tightly couples to telomere replication. We also observed that t-loop abundance positively correlates with chromatin condensation, i.e. cells with less compact telomeric chromatin (ALT cells and trichostatin A (TSA)-treated HeLa cells) exhibited fewer t-loops. Moreover, we observed that telomere dysfunction-induced foci, ALT-associated promyelocytic leukemia bodies, and telomere sister chromatid exchanges are activated upon TSA-induced loss of t-loops. These findings confirm the importance of the t-loop in protecting linear chromosomes from damage or illegitimate recombination.

| S-EPMC6484139 | biostudies-literature

Cohesin and condensin extrude DNA loops in a cell cycle-dependent manner.

Project description:Loop extrusion by structural maintenance of chromosomes (SMC) complexes has been proposed as a mechanism to organize chromatin in interphase and metaphase. However, the requirements for chromatin organization in these cell cycle phases are different, and it is unknown whether loop extrusion dynamics and the complexes that extrude DNA also differ. Here, we used Xenopus egg extracts to reconstitute and image loop extrusion of single DNA molecules during the cell cycle. We show that loops form in both metaphase and interphase, but with distinct dynamic properties. Condensin extrudes DNA loops non-symmetrically in metaphase, whereas cohesin extrudes loops symmetrically in interphase. Our data show that loop extrusion is a general mechanism underlying DNA organization, with dynamic and structural properties that are biochemically regulated during the cell cycle.

| S-EPMC7316503 | biostudies-literature

Cell cycle-dependent nucleosome occupancy at cohesin binding sites in yeast chromosomes.

Project description:In the budding yeast, cohesin is loaded onto the chromosome during the late G1 phase, establishes sister chromatid cohesion concomitant with DNA replication, and dissociates by the telophase. Here, using oligonucleotide tiling arrays, we show that, at the anaphase, nearly all of the cohesin binding sites contain nucleosome-free regions. The majority of these sites remain nucleosome-free throughout the cell cycle, consistent with the suggestion of a DNA-binding anchoring protein present at these sites, although such a region could also serve as part of a marker for the binding of cohesin in the next cell cycle. However, a third of these sites are remodeled in the G1 phase, being reoccupied by nucleosomes by the G1/S boundary, though their subsequent removal in the S phase appears to be independent of DNA replication. Whether this difference is a result of other functions of cohesin or of the chromatin remains to be elucidated.

| S-EPMC2268989 | biostudies-literature

An extrinsic motor directs chromatin loop formation by cohesin

Project description:The ring-shaped cohesin complex topologically entraps two DNA molecules to establish sister chromatid cohesion. Cohesin also shapes the interphase chromatin landscape with wide-ranging implications for gene regulation, and cohesin is thought to achieve this by actively extruding DNA loops without topologically entrapping DNA. The 'loop extrusion' hypothesis finds motivation from in vitro observations - whether this process underlies in vivo chromatin loop formation remains untested. Here, using the budding yeast S. cerevisiae, we generate cohesin variants that have lost their ability to extrude DNA loops but retain their ability to topologically entrap DNA. Analysis of these variants suggests that in vivo chromatin loops form independently of loop extrusion. Instead, we find that transcription promotes loop formation, and acts as an extrinsic motor that expands these loops and defines their ultimate positions. Our results necessitate a re-evaluation of the loop extrusion hypothesis. We propose that cohesin, akin to sister chromatid cohesion establishment at replication forks, forms chromatin loops by DNA-DNA capture at places of transcription, thus unifying cohesin's two roles in chromosome segregation and interphase genome organization.

| S-SCDT-10_1038-S44318-024-00202-5 | biostudies-other

A cell cycle-dependent co-repressor mediates photoreceptor cell-specific nuclear receptor function.

Project description:Photoreceptor cell-specific nuclear receptor (PNR) (NR2E3) acts as a sequence-specific repressor that controls neuronal differentiation in the developing retina. We identified a novel PNR co-repressor, Ret-CoR, that is expressed in the developing retina and brain. Biochemical purification of Ret-CoR identified a multiprotein complex that included E2F/Myb-associated proteins, histone deacetylases (HDACs) and NCoR/HDAC complex-related components. Ret-CoR appeared to function as a platform protein for the complex, and interacted with PNR via two CoRNR motifs. Purified Ret-CoR complex exhibited HDAC activity, co-repressed PNR transrepression function in vitro, and co-repressed PNR function in PNR target gene promoters, presumably in the retinal progenitor cells. Notably, the appearance of Ret-CoR protein was cell-cycle-stage-dependent (from G1 to S). Therefore, Ret-CoR appears to act as a component of an HDAC co-repressor complex that supports PNR repression function in the developing retina, and may represent a co-regulator class that supports transcriptional regulator function via cell-cycle-dependent expression.

| S-EPMC1794400 | biostudies-literature

miR-449a causes Rb-dependent cell cycle arrest and senescence in prostate cancer cells.

Project description:MicroRNAs (miRNAs) are a class of small non-coding RNAs (ncRNAs) that regulate gene expression by repressing translation or triggering the degradation of complementary mRNA sequences. Certain miRNAs have been shown to function as integral components of the p53 and/or retinoblastoma (Rb) regulatory networks. As such, miRNA dysregulation can have a profound effect on cancer development. Previous studies have shown that miR-449a is down-regulated in human prostate cancer tissue and possesses potential tumor suppressor function. In the present study, we identify miR-449a-mediated growth arrest in prostate cancer cells is dependent on the Rb protein. We show that mutant Rb prostate cancer cells (DU- 145) are resistant to cell cycle arrest and cellular senescence induced by miR- 449a, while overexpression of wild-type Rb in DU-145 sublines (DU-1.1 and B5) restores miR-449a function. In silico analysis of 3'UTR regions reveal a putative miR-449a target site in the transcript of Cyclin D1 (CCND1); an oncogene involved in directly regulating Rb activity and cell cycle progression. Luciferase 3'UTR reporter constructs and inhibitory oligonucleotides confirm that Cyclin D1 is a direct downstream target of miR-449a. We also reveal that miR-449a suppresses Rb phosphorylation through the knockdown of Cyclin D1 and previously validated target HDAC1. By targeting genes involved in controlling Rb activity, miR- 449a regulates growth and senescence in an Rb-dependent manner. These data indicate that miR-449a is a miRNA component of the Rb pathway and its tumor suppressor-like effects, in part, depends on Rb status in prostate cancer cells.

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