Rad21 is the core subunit of the cohesin complex involved in directing genome organization

The ring-shaped cohesin complex is an important factor regulating genome structure. It is thought to mediate the formation of chromatin loops and topologically associating domains (TADs) by loop extrusion. However, the regulation of association between cohesin and chromatin is poorly understood. In this study, we directly visualized cohesin loading after up-regulation of cohesin subunit Rad21 by identifying the formation of vermicelli-like structures via live cell super-resolution imaging. We also reveal that cohesin loading can be promoted by Rad21-loader interactions and accumulated contacts were shown at TAD corners while inter-TAD interactions increased after vermicelli formation, indicating that Rad21 is an important determinant of chromatin structure. Moreover, we find that cohesin saddle on topologically associating domains by FISH assay, which is consistent with the CTCF/cohesin-anchored chromatin loop model. Importantly, expression of Rad21 is strictly controlled, and aberrant expression of Rad21 leads to the formation of Rad21 “beads” in the nucleus. In summary, our observations provided important new biological insights into the mechanism of cohesin loading and its functions.

Eco1-dependent cohesin acetylation anchors chromatin loops and cohesion to define functional meiotic chromosome domains

Cohesin organizes the genome by forming intra-chromosomal loops and inter-sister chromatid linkages. During gamete formation by meiosis, chromosomes are reshaped to support crossover recombination and two consecutive rounds of chromosome segregation. Here we show that Eco1 acetyltransferase positions both chromatin loops and sister chromatid cohesion to organize meiotic chromosomes into functional domains in budding yeast. Eco1 acetylates the Smc3 cohesin subunit in meiotic S phase to establish chromatin boundaries, independently of DNA replication. Boundary formation by Eco1 is critical for prophase exit and for the maintenance of cohesion until meiosis II, but is independent of the ability of Eco1 to antagonize the cohesin-release factor, Wpl1. Conversely, prevention of cohesin release by Wpl1 is essential for centromeric cohesion, kinetochore monoorientation and co-segregation of sister chromatids in meiosis I. Our findings establish Eco1 as a key determinant of chromatin boundaries and cohesion positioning, revealing how local chromosome structuring directs genome transmission into gametes.

Rad21l1 cohesin subunit is dispensable for spermatogenesis but not oogenesis in zebrafish

During meiosis I, ring-shaped cohesin complexes play important roles in aiding the proper segregation of homologous chromosomes. RAD21L is a meiosis-specific vertebrate cohesin that is required for spermatogenesis in mice but is dispensable for oogenesis in young animals. The role of this cohesin in other vertebrate models has not been explored. Here, we tested if the zebrafish homolog Rad21l1 is required for meiotic chromosome dynamics during spermatogenesis and oogenesis. We found that Rad21l1 localizes to unsynapsed chromosome axes. It is also found between the axes of the mature tripartite synaptonemal complex (SC) in both sexes. We knocked out rad21l1 and found that nearly all rad21l1-/- mutants develop as fertile males, suggesting that the mutation causes a defect in juvenile oogenesis, since insufficient oocyte production triggers female to male sex reversal in zebrafish. Sex reversal was partially suppressed by mutation of the checkpoint gene tp53, suggesting that the rad21l1 mutation activates Tp53-mediated apoptosis or arrest in females. This response, however, is not linked to a defect in repairing Spo11-induced double-strand breaks since deletion of spo11 does not suppress the sex reversal phenotype. Compared to tp53 single mutant controls, rad21l1-/- tp53-/- double mutant females produce poor quality eggs that often die or develop into malformed embryos. Overall, these results indicate that the absence of rad21l1-/- females is due to a checkpoint-mediated response and highlight a role for a meiotic-specific cohesin subunit in oogenesis but not spermatogenesis.

Visualizing looping of two endogenous genomic loci using synthetic zinc-finger proteins with anti-FLAG and anti-HA frankenbodies in living cells

In eukaryotic nuclei, chromatin loops mediated through cohesin are critical structures that regulate gene expression and DNA replication. Here we demonstrate a new method to visualize endogenous genomic loci using synthetic zinc-finger proteins harboring repeat epitope tags (ZF probes) for signal amplification via binding of tag-specific intracellular antibodies, or frankenbodies, fused with fluorescent proteins. We achieve this in two steps. First, we develop an anti-FLAG frankenbody that can bind FLAG-tagged proteins in diverse live-cell environments. The anti-FLAG frankenbody complements the anti-HA frankenbody, enabling two-color signal amplification from FLAG and HA-tagged proteins. Second, we develop a pair of cell-permeable ZF probes that specifically bind two endogenous chromatin loci predicted to be involved in chromatin looping. By coupling our anti-FLAG and anti-HA frankenbodies with FLAG- and HA-tagged ZF probes, we simultaneously visualize the dynamics of the two loci in single living cells. This reveals close association between the two loci in the majority of cells, but the loci markedly separate upon the triggered degradation of the cohesin subunit RAD21. Our ability to image two endogenous genomic loci simultaneously in single living cells provides a proof-of-principle that ZF probes coupled with frankenbodies are useful new tools for exploring genome dynamics in multiple colors.

Cohesin subunit Rad21 binds to the HSV-1 genome near CTCF insulator sites during latency in vivo

Herpes Simplex Virus 1 (HSV-1) is a human pathogen that has the ability to establish a lifelong infection in the host. During latency, HSV-1 genomes are chromatinized and are abundantly associated with histones in sensory neurons, yet the mechanisms that govern the latent-lytic transition remain unclear. We hypothesize that the latent-lytic switch is controlled by CTCF insulators, positioned within the HSV-1 latent genome. CTCF insulators, together with the cohesin complex, have the ability to establish and maintain chromtin loops that allow distance separated gene regions to be spatially oriented for transcriptional control. In this current study, we demonstrated that the cohesin subunit Rad21 was recruited to latent HSV-1 genomes near four of the CTCF insulators during latency. We showed that the CTCF insulator known as CTRS1/2, positioned downstream from the essential transactivating IE region of ICP4 was only enriched in Rad21 prior to but not during latency, suggesting that the CTRS1/2 insulator is not required for the maintenance of latency. Further, deletion of the CTRL2 insulator, positioned downstream from the LAT enhancer, resulted in a loss of Rad21 enrichment at insulators flanking the ICP4 region at early times post-infection in mice ganglia, suggesting that these insulators are interdependent. Finally, deletion of the CTRL2 insulator resulted in a loss of Rad21 enrichment at the CTRL2 insulator in a cell-type specific manner, and this loss of Rad21 enrichment was correlated to decreased LAT expression, suggesting that Rad21 recruitment to viral genomes is important for efficient gene expression. Importance CTCF insulators are important for transcriptional control and increasing evidence suggests that that CTCF insulators, together with the cohesin complex, regulate viral transcription in DNA viruses. The CTCF-cohesin interaction is important for the formation of chromatin loops, structures that orient distance separated elements in close spatial proximity for transcriptional control. Herpes Simplex Virus 1 (HSV-1) has seven putative CTCF insulators that flank the LAT and the IE, indicating that CTCF insulators play a role in the transition from latency to reactivation. Contributions from the work presented here include the finding that CTCF insulators in HSV-1 genomes are differentially enriched in the cohesin subunit Rad21, suggesting that CTCF-cohesin interactions could be establishing and anchoring chromatin loop structures to control viral transcription.

The Myc-associated zinc finger protein (MAZ) works together with CTCF to control cohesin positioning and genome organization

The Myc-associated zinc finger protein (MAZ) is often found at genomic binding sites adjacent to CTCF, a protein which affects large-scale genome organization through its interaction with cohesin. We show here that, like CTCF, MAZ physically interacts with a cohesin subunit and can arrest cohesin sliding independently of CTCF. It also shares with CTCF the ability to independently pause the elongating form of RNA polymerase II, and consequently affects RNA alternative splicing. CTCF/MAZ double sites are more effective at sequestering cohesin than sites occupied only by CTCF. Furthermore, depletion of CTCF results in preferential loss of CTCF from sites not occupied by MAZ. In an assay for insulation activity like that used for CTCF, binding of MAZ to sites between an enhancer and promoter results in down-regulation of reporter gene expression, supporting a role for MAZ as an insulator protein. Hi-C analysis of the effect of MAZ depletion on genome organization shows that local interactions within topologically associated domains (TADs) are disrupted, as well as contacts that establish the boundaries of individual TADs. We conclude that MAZ augments the action of CTCF in organizing the genome, but also shares properties with CTCF that allow it to act independently.

Effects of forced cohesin eviction and retention on X-inactivation and autosomes

SUMMARYDepletion of architectural factors globally alters chromatin structure, but only modestly affects gene expression. We revisit the structure-function relationship using the inactive X chromosome (Xi) as a model. We investigate cohesin imbalances by forcing its depletion or retention using degron-tagged RAD21 (cohesin subunit) or WAPL (cohesin release factor). Interestingly, cohesin loss disrupts Xi superstructure, unveiling superloops between escapee genes, with minimal effect on gene repression. By contrast, forced cohesin retention markedly affects Xi superstructure and compromises spreading of Xist RNA-Polycomb complexes, attenuating Xi silencing. Effects are greatest at distal chromosomal ends, where looping contacts with the Xist locus are weakened. Surprisingly, cohesin loss created an “Xi superloop” and cohesin retention created “Xi megadomains” on the active X. Across the genome, a proper cohesin balance protects against aberrant inter-chromosomal interactions and tempers Polycomb-mediated repression. We conclude that a balance of cohesin eviction and retention regulates X-inactivation and inter-chromosomal interactions across the genome.

Rad21l1 cohesin subunit is dispensable for spermatogenesis but not oogenesis in zebrafish

Meiosis produces haploid gametes that will give rise to the next diploid generation. Chromosome segregation errors occurring at one or both meiotic divisions result in aneuploidy, which can lead to miscarriages or birth defects in humans. During meiosis I, ring-shaped cohesin complexes play important roles to aid in the proper segregation of homologous chromosomes. While REC8 is a specialized meiosis-specific cohesin that functions to hold sister chromatids together, the role of its vertebrate-specific paralog, RAD21L, is poorly understood. Here we tested if Rad21l1, the zebrafish homolog of human and mouse RAD21L, is required for meiotic chromosome dynamics during oogenesis and spermatogenesis. We found that Rad21l1 is an abundant component of meiotic chromosomes where it localizes to both the chromosome axes and the transverse filament of the synaptonemal complex (SC). Knocking out rad21l1 causes nearly the entire mutant population to develop as fertile males, suggesting the mutation triggers a sex reversal from female to male due to a failure in oocyte production. The rad21l1−/− mutant males display normal fertility at sexual maturity. Sex reversal was partially suppressed in the absence of tp53, suggesting that the rad21l1−/− mutation causes defects leading to a Tp53 dependent response, specifically in females. The rad21l1−/−;tp53−/− double mutant females produced elevated rates of decomposing eggs and deformed offspring compared to tp53−/− controls. This response, however, is not linked to a defect in repairing Spo11-induced double-strand breaks since deletion of Spo11 does not suppress the sex reversal phenotype. Overall, our data highlight an exceptional sexually dimorphic phenotype caused by knocking out a meiotic-specific cohesin subunit. We propose that Rad21l1 is required for maintaining the integrity of meiotic chromatin architecture during oogenesis.Author SummaryA prominent symptom of age-linked reproductive decline in women is the increased rate of miscarriage and birth defects due to aneuploidy. Aneuploidy can arise when chromosomes fail to segregate properly during meiosis, the process of creating haploid gametes from a diploid germ cell. Oocyte progression normally arrests prior to anaphase I, after homologous chromosomes have formed crossovers, but before ovulation, which triggers the first round of segregation. This prolonged arrest makes oocytes especially vulnerable to degradation of meiotic chromosome structure and homolog connections over time. Cohesin complexes play a major role in maintaining the meiotic chromosome architecture. Here we assess the role of the vertebrate-specific Rad21l1 cohesin subunit in zebrafish. We find that while males appear mostly unaffected by loss of Rad21l1, oocyte production is massively compromised, leading to sex reversion to males. Sex reversion can be partially prevented in the absence of Tp53, demonstrating that loss or Rad21l1 leads to a Tp53-dependent response in oocytes. Strikingly, double mutant rad21l1 tp53 females produce large numbers of poor quality eggs and malformed offspring. This demonstrates a cohesin-linked vulnerability in female meiosis not present in males and sheds light on a potential mechanism associated with the decline in female reproductive health.

Cohesin mutations are synthetic lethal with stimulation of WNT signaling

Mutations in genes encoding subunits of the cohesin complex are common in several cancers, but may also expose druggable vulnerabilities. We generated isogenic MCF10A cell lines with deletion mutations of genes encoding cohesin subunits SMC3, RAD21 and STAG2 and screened for synthetic lethality with 3,009 FDA-approved compounds. The screen identified several compounds that interfere with transcription, DNA damage repair and the cell cycle. Unexpectedly, one of the top ‘hits’ was a GSK3 inhibitor, an agonist of Wnt signaling. We show that sensitivity to GSK3 inhibition is likely due to stabilization of β-catenin in cohesin mutant cells, and that Wnt-responsive gene expression is highly sensitized in STAG2-mutant CMK leukemia cells. Moreover, Wnt activity is enhanced in zebrafish mutant for cohesin subunit rad21. Our results suggest that cohesin mutations could progress oncogenesis by enhancing Wnt signaling, and that targeting the Wnt pathway may represent a novel therapeutic strategy for cohesin mutant cancers.