Cohesin Components Research Articles

Assessing cell lines with inducible depletion of cohesin and condensins components through analysis of metaphase chromosome morphology.

One of the most productive strategies for finding the functions of proteins is to study the consequences of loss of protein function. For this purpose, cells or organisms with a knockout of the gene encoding the protein of interest are obtained. However, many proteins perform important functions and cells or organisms could suddenly lose fitness when the function of a protein is lost. For such proteins, the most productive strategy is to use inducible protein degradation systems. A system of auxin-dependent protein degradation is often implemented. To use this system, it is sufficient to introduce a transgene encoding a plant-derived auxin-dependent ubiquitin ligase into mammalian cells and insert a sequence encoding a degron domain into the gene of interest. A crucial aspect of development of cell lines engineered for inducible protein depletion is the selection of cell clones with efficient auxin-dependent degradation of the protein of interest. To select clones induced by depletion of the architectural chromatin proteins RAD21 (a component of the cohesin complex) and SMC2 (a component of the condensin complex), we propose to use the morphology of metaphase chromosomes as a convenient functional test. In this work, we obtained a series of clones of human HAP1 cells carrying the necessary genetic constructs for inducible depletion of RAD21 and SMC2. The degradation efficiency of the protein of interest was assessed by flow cytometry, Western blotting and metaphase chromosome morphology test. Based on our tests, we showed that the clones we established with the SMC2 degron effectively and completely lose protein function when induced by auxin. However, none of the HAP1 clones we created with the RAD21 degron showed complete loss of RAD21 function upon induction of degradation by auxin. In addition, some clones showed evidence of loss of RAD21 function even in the absence of induction. The chromosome morphology test turned out to be a convenient and informative method for clone selection. The results of this test are in good agreement with flow cytometry analysis and Western blotting data.

Chromatin assembly factor-1 preserves genome stability in ctf4Δ cells by promoting sister chromatid cohesion.

Chromatin assembly and the establishment of sister chromatid cohesion are intimately connected to the progression of DNA replication forks. Here we examined the genetic interaction between the heterotrimeric chromatin assembly factor-1 (CAF-1), a central component of chromatin assembly during replication, and the core replisome component Ctf4. We find that CAF-1 deficient cells as well as cells affected in newly-synthesized H3-H4 histones deposition during DNA replication exhibit a severe negative growth with ctf4Δ mutant. We dissected the role of CAF-1 in the maintenance of genome stability in ctf4Δ yeast cells. In the absence of CTF4, CAF-1 is essential for viability in cells experiencing replication problems, in cells lacking functional S-phase checkpoint or functional spindle checkpoint, and in cells lacking DNA repair pathways involving homologous recombination. We present evidence that CAF-1 affects cohesin association to chromatin in a DNA-damage-dependent manner and is essential to maintain cohesion in the absence of CTF4. We also show that Eco1-catalyzed Smc3 acetylation is reduced in absence of CAF-1. Furthermore, we describe genetic interactions between CAF-1 and essential genes involved in cohesin loading, cohesin stabilization, and cohesin component indicating that CAF-1 is crucial for viability when sister chromatid cohesion is affected. Finally, our data indicate that the CAF-1-dependent pathway required for cohesion is functionally distinct from the Rtt101-Mms1-Mms22 pathway which functions in replicated chromatin assembly. Collectively, our results suggest that the deposition by CAF-1 of newly-synthesized H3-H4 histones during DNA replication creates a chromatin environment that favors sister chromatid cohesion and maintains genome integrity.

Abstract A054: Accurate prediction of cohesin-mediated 3D genome organization using 2D chromatin features

Abstract The three-dimensional (3D) genome organization influences diverse nuclear processes. Chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) and High-throughput chromatin conformation capture (Hi-C) are powerful methods used to study the 3D genome organization. However, these two experiments are costly, time-consuming, require tens to hundreds of millions of cells, and challenging to optimize and analyze. Predicting ChIA-PET/Hi-C data using cheaper ChIP-Seq data and other easily obtainable features is a useful alternative. Also, it is well-established that the cohesin protein complex is a key determinant of 3D genome organization. Considering this, we present Chromatin Interaction Predictor (ChIPr), a suite of regression models based on deep neural networks (DNN), random forest, and gradient boosting. ChIPr predicts cohesin-mediated chromatin interaction strength between any two given loci in the genome. It was trained mainly using two top-tiered ENCODE cell lines GM12878 and K562 and tested on other cell lines (H1 and HepG2). Comprehensive tests showed that ChIPr predictions correlated well with the original ChIA-PET data at the peak-level resolution and at bin sizes 25kb and 5kb. In addition, ChIPr accurately captured most of the cell-type-dependent loops identified by both, ChIA-PET and Hi-C data, respectively. Extensive feature testing highlighted genomic distance and RAD21 (a cohesin component) ChIP-Seq signals as the most important inputs contributing to ChIPr, in determining the chromatin interaction strength. A standard ChIPr model requires three experimental inputs: ChIP-Seq signals for RAD21, H3K27ac (enhancer/active chromatin mark), and H3K27me3 (inactive chromatin mark). While a reduced model requires a single experiment input: ChIP-Seq signals for RAD21 and performs equally well. Additionally, the use of ChIPr was further extended to predict the contact maps for several prostate cancer (PCa) cell lines whose ChIA-PET data were not available on the ENCODE portal, for instance, RWPE1 and VCaP. We generated the necessary ChIP-seq data for these cell lines to use as input to the model to further examine the changes in the 3D interactions of PCa driver genes and also their isolated neighbourhoods and decipher useful biological insights about the gene regulatory networks involved. Integrative analysis revealed novel insights into the role of CTCF motif, its orientation, and CTCF binding on the prevalence and strength of cohesin-mediated chromatin interactions (~50% - 80% of the RAD21 interactions were enriched with CTCF, < 15% of the interactions had no CTCF ChIP-seq binding in both of the two peaks). Lastly, we also noticed a subset of RAD21 interactions with CTCF binding in only one or none of the two anchor peaks, which were significantly enriched with enhancers. Taken together, this study outlines the general features of genome folding and opens new avenues to analyze spatial genome organization in specimens with limited cell numbers. Citation Format: Khyati Chandratre. Accurate prediction of cohesin-mediated 3D genome organization using 2D chromatin features [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A054.

Investigating the Role of HDAC8 in CD4 +T Cells

Abstract T cell exclusion restricts immune checkpoint blockade (ICB) efficacy for some hepatocellular carcinoma (HCC) patients. Epigenetic therapy provides new opportunities to rewire transcriptional programs. We have recently elucidated that pharmacological inhibition of histone deacetylase 8 (HDAC8) resulted in increased CD8 +T cell infiltration and enhanced ICB therapy efficacy in HCC. Moreover, this combined immunotherapy induced durable survival benefits even after tumor rechallenge accompanied by elevated memory T cells (Sci Transl Med, 2021). In this study, we found that HDAC8 inhibition increased memory T cells in HCC tumor-bearing mice. Interestingly, HDAC8 knockdown in tumor cells did not influence the effect of pharmacological HDAC8 inhibition in T cell activation and cytotoxic function, suggesting an immune cell-intrinsic role of HDAC8. HDAC8 was highly expressed in T cells instead of other immune cells. Notably, HDAC8 inhibition upregulated memory-related gene expression in CD4 +T cells, but not in CD8 +T cells. Besides, HDAC8 inhibition increased acetylation of structural maintenance of chromosome 3 (SMC3ac), a ring-shaped cohesin component in topologically entrapping enhancers and promoters which may modulate gene expression. Moreover, inhibiting HDAC8 increased SMC3ac binding onto the targeting gene promoter region and induced mRNA expression in CD4-expressing Jurkat cells. These data suggest that HDAC8 inhibition exerts memory T cell formation through SMC3ac-mediated epigenome reprogramming in CD4 +T cells, thus providing insights into developing effective cancer immunotherapy. Acknowledgment: This work is supported by the RGC Collaborative Research Fund (C4045-18W), Li Ka Shing Foundation, TRS T11-706/18-N and EpiHK.

Essential role of MESP1-RING1A complex in cardiac differentiation.

Heart development is controlled by a complex transcriptional network composed of transcription factors and epigenetic regulators. Mutations in key developmental transcription factor MESP1 and chromatin factors, such as PRC1 and cohesin components, have been found in human congenital heart diseases (CHDs), although their functional mechanism during heart development remains elusive. Here, we find that MESP1 interacts with RING1A/RING1, the core component of PRC1. RING1A depletion impairs human cardiomyocyte differentiation, and cardiac abnormalities similar to those in patients with MESP1 mutations were observed in Ring1A knockout mice. Mechanistically, MESP1 associates with RING1A to activate cardiogenic genes through promoter-enhancer interactions regulated by cohesin and CTCF and histone acetylation mediated by p300. Importantly, CHD mutations of MESP1 significantly affect such mechanisms and impair target gene activation. Together, our results demonstrate the importance of MESP1-RING1A complex in heart development and provide insights into the pathogenic mechanisms of CHDs caused by mutations in MESP1, PRC1, and cohesin components.

Turning coldspots into hotspots: targeted recruitment of axis protein Hop1 stimulates meiotic recombination in Saccharomyces cerevisiae.

The DNA double-strand breaks that initiate meiotic recombination are formed in the context of the meiotic chromosome axis, which in Saccharomyces cerevisiae contains a meiosis-specific cohesin isoform and the meiosis-specific proteins Hop1 and Red1. Hop1 and Red1 are important for double-strand break formation; double-strand break levels are reduced in their absence and their levels, which vary along the lengths of chromosomes, are positively correlated with double-strand break levels. How axis protein levels influence double-strand break formation and recombination remains unclear. To address this question, we developed a novel approach that uses a bacterial ParB-parS partition system to recruit axis proteins at high levels to inserts at recombination coldspots where Hop1 and Red1 levels are normally low. Recruiting Hop1 markedly increased double-strand breaks and homologous recombination at target loci, to levels equivalent to those observed at endogenous recombination hotspots. This local increase in double-strand breaks did not require Red1 or the meiosis-specific cohesin component Rec8, indicating that, of the axis proteins, Hop1 is sufficient to promote double-strand break formation. However, while most crossovers at endogenous recombination hotspots are formed by the meiosis-specific MutLγ resolvase, crossovers that formed at an insert locus were only modestly reduced in the absence of MutLγ, regardless of whether or not Hop1 was recruited to that locus. Thus, while local Hop1 levels determine local double-strand break levels, the recombination pathways that repair these breaks can be determined by other factors, raising the intriguing possibility that different recombination pathways operate in different parts of the genome.

Meiosis-specific cohesin complexes display essential and distinct roles in mitotic embryonic stem cell chromosomes

BackgroundCohesin is a chromosome-associated SMC–kleisin complex that mediates sister chromatid cohesion, recombination, and most chromosomal processes during mitosis and meiosis. However, it remains unclear whether meiosis-specific cohesin complexes are functionally active in mitotic chromosomes.ResultsThrough high-resolution 3D-structured illumination microscopy (3D-SIM) and functional analyses, we report multiple biological processes associated with the meiosis-specific cohesin components, α-kleisin REC8 and STAG3, and the distinct loss of function of meiotic cohesin during the cell cycle of embryonic stem cells (ESCs). First, we show that STAG3 is required for the efficient localization of REC8 to the nucleus by interacting with REC8. REC8-STAG3-containing cohesin regulates topological properties of chromosomes and maintains sister chromatid cohesion. Second, REC8-cohesin has additional sister chromatid cohesion roles in concert with mitotic RAD21-cohesin on ESC chromosomes. SIM imaging of REC8 and RAD21 co-staining revealed that the two types of α-kleisin subunits exhibited distinct loading patterns along ESC chromosomes. Third, knockdown of REC8 or RAD21-cohesin not only leads to higher rates of premature sister chromatid separation and delayed replication fork progression, which can cause proliferation and developmental defects, but also enhances chromosome compaction by hyperloading of retinoblastoma protein–condensin complexes from the prophase onward.ConclusionsOur findings indicate that the delicate balance between mitotic and meiotic cohesins may regulate ESC-specific chromosomal organization and the mitotic program.

Inner Nuclear Protein Matrin-3 Coordinates Hematopoietic Cell Transcription and Differentiation By Stabilizing Chromatin Architecture

The nucleus is spatially organized by chromosome and interchromatin functional components. Global reorganization of chromatin interactions and compartmentalization occurring during differentiation requires proper chromosome positioning, but the involvement of nuclear components in this process remains largely underexplored. In particular, blood cell development exemplifies a coordinated process accompanied by dramatic chromatin reorganization, thereby providing a model in which to interrogate chromatin dynamics during differentiation. Here, we show that an abundant inner nuclear protein Matrin-3 (Matr3) plays a critical role in the maintenance of chromatin structure and has a broad effect on erythroid cell differentiation by coordinating gene expression.First, we deleted the entire gene body by CRISPR/Cas9 in mouse erythroleukemia (MEL) cells. The Matr3 knockout (KO) cells proliferate normally and exhibit morphological changes on differentiation suggestive of accelerated maturation. Consistently, erythroid-specific genes were expressed at a higher level in MEL Matr3 KO cells than in parental cells. The consequences of Matr3 deletion were also determined in G1ER cells, in which differentiation is conditional on activation of GATA-1. To assess the global impact of Matr3 loss on erythroid cell maturation, we measured global RNA expression changes. Erythroid-specific genes were expressed at a much higher level upon differentiation of Matr3 KO cells. Differentiation is typically accompanied by specific changes in nuclear architecture. Using super-resolution microscopy, we observed that heterochromatin protein 1α (HP1α) was more dispersed and irregular in appearance in Matr3 KO cells, suggesting that Matr3 loss alters morphological boundaries of heterochromatin.Analysis of the interactions between different regions of chromatin identifies topologically associating domains and classifies the genome into two compartments (A and B). The A and B compartments correspond to the structures and characteristics of known euchromatin and heterochromatin, respectively. We next explored global chromatin structure using a high-throughput chromosome conformation capture (Hi-C) assay. In Matr3 KO cells, insulation at the domain boundaries was reduced, and the compartment strengths between the B compartments became stronger, while those between A-type domains were reduced. Remarkably, we found that these changes in cells lacking Matr3 were similar to changes in chromatin contact during differentiation. To access the genomic features at a higher resolution, we performed the assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq). Notably, the newly opened regions in Matr3 KO, as compared to parental, cells were enriched for GATA motifs, which are generally more accessible in differentiated erythroid cells.Architectural proteins function cooperatively to organize chromatin. Using affinity purification followed by mass spectrometry and immunoblotting, we found that Matr3 interacts with proteins involved in chromatin remodeling, such as CTCF and cohesin. To identify whether Matr3 loss alters chromatin occupancy of its interacting partners, we performed ChIP-seq for CTCF and the core cohesin component Rad21. In the absence of Matr3, occupancy of CTCF and Rad21 was perturbed in a subset of genomic regions. Moreover, destabilization of CTCF and cohesin binding correlated with altered transcription and accelerated erythroid differentiation. Most sites with disrupted CTCF and Rad21 binding during differentiation were also sensitive to the absence of the scaffold protein Matr3.Our data demonstrate that the nucleoplasmic protein Matr3 stabilizes the binding of the architectural proteins (CTCF and cohesin) to chromatin and serves to maintain chromatin structure. We speculate that Matr3 negatively regulates cell fate transitions by maintaining cellular state through fine-tuning the binding of CTCF/cohesin to chromatin and associated 3D interactions. Our work reveals a previously unrecognized role of Matr3 in chromatin organization and responses to developmental cues. DisclosuresNo relevant conflicts of interest to declare.

Cohesin promotes HSV-1 lytic transcription by facilitating the binding of RNA Pol II on viral genes

BackgroundHerpes Simplex Virus type I (HSV-1) is a large double-stranded DNA virus that enters productive infection in epithelial cells and reorganizes the host nucleus. Cohesin, a major constituent of interphase and mitotic chromosomes comprised of SMC1, SMC3, and SCC1 (Mcd1/Rad21), SCC3 (SA1/SA2), have diverse functions, including sister chromatid cohesion, DNA double-stranded breaks repair, and transcriptional control. Little is known about the role of cohesin in HSV-1 lytic infection.MethodsWe measured the effect on HSV-1 transcription, genome copy number, and viral titer by depleting cohesin components SMC1 or Rad21 using RNAi, followed by immunofluorescence, qPCR, and ChIP experiments to gain insight into cohesin's function in HSV-1 transcription and replication.ResultsHere, we report that cohesion subunits SMC1 and Rad21 are recruited to the lytic HSV-1 replication compartment. The knockdown results in decreased viral transcription, protein expression, and maturation of viral replication compartments. SMC1 and Rad21 knockdown leads to the reduced overall RNA pol II occupancy level but increased RNA pol II ser5 phosphorylation binding on viral genes. Consistent with this, the knockdown increased H3K27me3 modification on these genes.ConclusionsThese results suggest that cohesin facilitates HSV-1 lytic transcription by promoting RNA Pol II transcription activity and preventing chromatin's silencing on the viral genome.

USP13 interacts with cohesin and regulates its ubiquitination in human cells

Cohesin is a multiprotein ring complex that regulates 3D genome organization, sister chromatid cohesion, gene expression, and DNA repair. Cohesin is known to be ubiquitinated, although the mechanism, regulation, and effects of cohesin ubiquitination remain poorly defined. We previously used gene editing to introduce a dual epitope tag into the endogenous allele of each of 11 known components of cohesin in human HCT116 cells. Here we report that mass spectrometry analysis of dual-affinity purifications identified the USP13 deubiquitinase as a novel cohesin-interacting protein. Subsequent immunoprecipitation/Western blots confirmed the endogenous interaction in HCT116, 293T, HeLa, and RPE-hTERT cells; demonstrated that the interaction occurs specifically in the soluble nuclear fraction (not in the chromatin); requires the ubiquitin-binding domains (UBA1/2) of USP13; and occurs preferentially during DNA replication. Reciprocal dual-affinity purification of endogenous USP13 followed by mass spectrometry demonstrated that cohesin is its primary interactor in the nucleus. Ectopic expression and CRISPR knockout of USP13 showed that USP13 is paradoxically required for both deubiquitination and ubiquitination of cohesin subunits in human cells. USP13 was dispensable for sister chromatid cohesion in HCT116 and HeLa cells, whereas it was required for the dissociation of cohesin from chromatin as cells transit through mitosis. Together these results identify USP13 as a new cohesin-interacting protein that regulates the ubiquitination of cohesin and its cell cycle regulated interaction with chromatin.

Cohesin Components Stag1 and Stag2 Differentially Influence Haematopoietic Mesoderm Development in Zebrafish Embryos

Cohesin is a multiprotein complex made up of core subunits Smc1, Smc3, and Rad21, and either Stag1 or Stag2. Normal haematopoietic development relies on crucial functions of cohesin in cell division and regulation of gene expression via three-dimensional chromatin organization. Cohesin subunit STAG2 is frequently mutated in myeloid malignancies, but the individual contributions of Stag variants to haematopoiesis or malignancy are not fully understood. Zebrafish have four Stag paralogues (Stag1a, Stag1b, Stag2a, and Stag2b), allowing detailed genetic dissection of the contribution of Stag1-cohesin and Stag2-cohesin to development. Here we characterize for the first time the expression patterns and functions of zebrafish stag genes during embryogenesis. Using loss-of-function CRISPR-Cas9 zebrafish mutants, we show that stag1a and stag2b contribute to primitive embryonic haematopoiesis. Both stag1a and stag2b mutants present with erythropenia by 24 h post-fertilization. Homozygous loss of either paralogue alters the number of haematopoietic/vascular progenitors in the lateral plate mesoderm. The lateral plate mesoderm zone of scl-positive cells is expanded in stag1a mutants with concomitant loss of kidney progenitors, and the number of spi1-positive cells are increased, consistent with skewing toward primitive myelopoiesis. In contrast, stag2b mutants have reduced haematopoietic/vascular mesoderm and downregulation of primitive erythropoiesis. Our results suggest that Stag1 and Stag2 proteins cooperate to balance the production of primitive haematopoietic/vascular progenitors from mesoderm.

Whole Exome Sequencing Identifies Cohesin Component STAG1 Mutation in de novo Acute Myeloid Leukemia (FAB M2): A Pilot Study with Cytogenetics, Clinical and Prognostic Implications.

The clinical implications of cohesin gene complex mutation in acute myeloid leukemia (AML) are not well characterized. In the present study, a cohort of 152 de novo unselected adult AML patients underwent conventional and molecular cytogenetic analysis for chromosomal aberrations. Further, we examined the frequency and clinical implications of mutations in cohesin gene complex STAG1, STAG2, RAD21, SMC1, and SMC3 using whole exome sequencing as a pilot study in 10 de novo patients with AML-FAB M2. Among the 10 cases, we identified a functionally heterozygous mutation in exon16 of STAG1 in one patient (10%), however no mutation was observed in STAG2, RAD21, SMC1, and SMC3. Sanger sequencing analysis for exon 16 of STAG1 in the remaining 142 AML cases did not reveal any further mutations, which underlined the observation that mutations took place throughout the cohesin gene complex without presence of a mutational hot spot region. The present study identified a positive correlation between serum bilirubin, LDH, and hematological parameters such as Hb, WBC, and platelet count with STAG1 mutation. Our data suggest that the cohesin complex may represent an attractive therapeutic target for future preclinical and clinical studies. However, more studies with a larger number of patients should be performed prospectively to determine the pathogenic involvement of STAG 1 mutation in AML patients.

The meiosis-specific cohesin component stromal antigen 3 promotes cell migration and chemotherapeutic resistance in colorectal cancer.

Chromosome instability is one of the hallmarks of cancer. Stromal antigen (STAG) 3 is a core component of the meiosis-specific cohesin complex, which regulates sister chromatid cohesion. Although aberrantly activated genes encoding the cohesin complex have been identified in cancers, little is known about the role of STAG3 in colorectal cancer (CRC). Here, we evaluated the prognostic impact and role of STAG3 in CRC. Analysis of 172 CRC surgical specimens revealed that high STAG3 expression was associated with poor prognosis. STAG3 knockdown inhibited cell migration and increased drug sensitivity to oxaliplatin, 5-fluorouracil, irinotecan hydrochloride hydrate, and BRAF inhibitor in CRC cell lines. The enhanced drug sensitivity was also confirmed in a human organoid established from a CRC specimen. Moreover, suppression of STAG3 increased γH2AX foci. Particularly, in BRAF-mutant CRC cells, STAG3 silencing suppressed the expression of snail family transcriptional repressor 1 and phosphorylation of extracellular signal-regulated kinase via upregulation of dual-specificity phosphatase 6. Our findings suggest that STAG3 is related to poor clinical outcomes and promotes metastasis and chemotherapeutic resistance in CRC. STAG3 may be a novel prognostic marker and potential therapeutic target for CRC.

Abstract IA16: Genetic mouse models and organoids: Synergy for functional genomics of bladder cancer genes

Abstract The cancer genome sequencing projects have unveiled a myriad of novel genes mutated in human tumors, a fraction of which are recurrently altered. For many of these genes, a relevant challenge is understanding their function in normal cells and in tumors as well as revealing how mutations can be leveraged from a translational standpoint. To understand the involvement of novel bladder cancer genes in urothelial carcinogenesis, we have taken a three-way approach: 1) assessing the association of mutations with clinical-pathologic characteristics of the tumor, 2) generating conditional mouse models to recapitulate carcinogenesis in vivo, and 3) harnessing the potential of organoids as a model to understand their biology and function during malignant transformation. We have focused on two genes, STAG2 and RBM10. STAG2 codes for a component of cohesin, whose major role is chromosome segregation. However, the molecular pathology studies have indicated that mutations are mainly associated with bladder cancers of low-grade and stage lacking aneuploidy: mutations are present in 30-40% of low-grade papillary tumors and 12-15% of muscle-invasive tumors. We have generated a novel conditional Stag2-knockout mouse and have shown that STAG2 has unique functions during embryo development and that it is largely dispensable in the adult under the conditions tested. In the urothelium, Stag2 deletion is not sufficient to give rise to tumors; experiments are ongoing testing its cooperation with other bladder cancer oncogenes and tumor suppressor genes. RBM10 is involved in pre-mRNA splicing, and tumor-specific alterations involving NUMB mRNA exon inclusion have been demonstrated in lung cancer. RBM10 is mutated in 5-8% of bladder cancers, regardless of stage and grade, with a predominance among tumors with urothelial differentiation. We have generated a novel conditional Rbm10 knockout mouse, which has revealed that RBM10 is also required for normal embryo development and that it is largely dispensable in the adult. In the urothelium, Rbm10 deletion in the urothelium is not sufficient to give rise to tumors and experiments are ongoing testing its cooperation with other bladder cancer oncogenes and tumor suppressor genes. To facilitate the study of the role of these genes in bladder tumorigenesis, we have developed methods to consistently establish normal mouse urothelial organoids. These organoids can be maintained in culture uninterruptedly for more than one year, they are strictly dependent on EGF and Wnt signals, and they can be induced to differentiate upon growth factor depletion. PPAR gamma agonists and EGFR inhibitors further support the urothelial program. Differentiated organoids show downregulated expression of basal cell markers, upregulation of uroplakins and adhesion molecules, and acquisition of barrier function. RNA-Seq analyses (both bulk and single cell) have revealed novel pathways involved in normal urothelial differentiation. These organoids are powerful tools to accelerate the discovery of the functions of bladder cancer genes and facilitate drug screening. Overall, I will show how our group and our collaborators are able to exploit complementary features of genetic mouse models and organoids to accelerate research on the biology of these tumor suppressor genes and its translation. This work has been possible thanks to the collaboration of the following groups: N. Malats (CNIO, Madrid), A. Losada (CNIO, Madrid), J. Valcárcel (CRG, Barcelona), M. Hoglund (U. Lund, Lund), L. Dyrskjot (U. Aarhus, Aarhus), A. Hartmann (U. Erlangen, Germany), among others. Citation Format: Francisco X. Real. Genetic mouse models and organoids: Synergy for functional genomics of bladder cancer genes [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr IA16.

CTCF orchestrates long-range cohesin-driven V(D)J recombinational scanning.

RAG endonuclease initiates V(D)J recombination in progenitor (pro)-B cells1. Upon binding a recombination center (RC)-based JH, RAG scans upstream chromatin via loop extrusion, potentially mediated by cohesin, to locate Ds and assemble a DJH-based RC2. CTCF looping factor-bound elements (CBEs) within IGCR1 upstream of Ds impede RAG-scanning3–5; but their inactivation allows scanning to proximal VHs where additional CBEs activate rearrangement and impede scanning any further upstream5. Distal VH utilization is thought to involve diffusional RC access following large-scale Igh locus contraction6–8. Here, we test the potential of linear RAG-scanning to mediate distal VH usage in G1-arrested v-Abl-pro-B cell lines9, which undergo robust D-to-JH but little VH-to-DJH rearrangements, presumably due to lack of locus contraction2,5. Through an auxin-inducible approach10, we degrade the cohesin-component Rad2110–12 or CTCF12,13 in these G1-arrested lines. Rad21 degradation eliminated all V(D)J recombination and RAG-scanning-associated interactions, except RC-located DQ52-to-JH joining in which synapsis occurs by diffusion2. Remarkably, while CTCF degradation suppressed most CBE-based chromatin interactions, it promoted robust RC interactions with, and robust VH-to-DJH joining of, distal VHs, with patterns similar to those of “locus-contracted” primary pro-B cells. Thus, down-modulation of CTCF-bound scanning-impediment activity promotes cohesin-driven RAG-scanning across the 2.7Mb Igh locus.

Combined Cohesin-RUNX1 Deficiency Synergistically Perturbs Chromatin Looping and Causes Myelodysplastic Syndromes.

STAG2 encodes a cohesin component and is frequently mutated in myeloid neoplasms, showing highly significant comutation patterns with other drivers, including RUNX1. However, the molecular basis of cohesin-mutated leukemogenesis remains poorly understood. Here we show a critical role of an interplay between STAG2 and RUNX1 in the regulation of enhancer-promoter looping and transcription in hematopoiesis. Combined loss of STAG2 and RUNX1, which colocalize at enhancer-rich, CTCF-deficient sites, synergistically attenuates enhancer-promoter loops, particularly at sites enriched for RNA polymerase II and Mediator, and deregulates gene expression, leading to myeloid-skewed expansion of hematopoietic stem/progenitor cells (HSPC) and myelodysplastic syndromes (MDS) in mice. Attenuated enhancer-promoter loops in STAG2/RUNX1-deficient cells are associated with downregulation of genes with high basal transcriptional pausing, which are important for regulation of HSPCs. Downregulation of high-pausing genes is also confirmed in STAG2-cohesin-mutated primary leukemia samples. Our results highlight a unique STAG2-RUNX1 interplay in gene regulation and provide insights into cohesin-mutated leukemogenesis. SIGNIFICANCE: We demonstrate a critical role of an interplay between STAG2 and a master transcription factor of hematopoiesis, RUNX1, in MDS development, and further reveal their contribution to regulation of high-order chromatin structures, particularly enhancer-promoter looping, and the link between transcriptional pausing and selective gene dysregulation caused by cohesin deficiency.This article is highlighted in the In This Issue feature, p. 747.

Polo-like kinase 4 and Stromal antigen 3 are not associated with recurrent pregnancy loss caused by embryonic aneuploidy

No genetic association with recurrent pregnancy loss (RPL) caused by embryonic aneuploidy has been found. Recent studies have indicated that the common genetic variant rs2305957, surrounding the PLK4 gene, contributes to mitotic-origin aneuploidy risk during human early embryo development. The decrease in meiosis-specific cohesin causes predivision of sister chromatids in the centromere and chromosome segregation errors. STAG3 is a component of cohesin and is a meiosis-specific gene. Our case-control study included 184 patients with RPL whose previous products of conception (POC) exhibited aneuploidy and 190 fertile control women without a history of miscarriage. We performed a genetic association study to examine the genotype distribution at PLK4 (rs2305957) and STAG3 in patients with RPL caused by aneuploidy compared with controls. Regarding STAG3, SNPs with a minor allele frequency (MAF) threshold > 0.05 that were predicted to be binding sites of transcription factors and that showed significant associations in expression quantitative trait locus (e-QTL) analysis were selected. No significant differences in the MAF or distribution in any model of PLK4 (rs2305957) and 5 selected tag SNPs in STAG3 were found between the patients and controls. A further genome-wide association study is needed since a combination of genetic risk alleles might be useful in predicting future age-dependent RPL caused by aneuploidy.

Foxl3, a sexual switch in germ cells, initiates two independent molecular pathways for commitment to oogenesis in medaka

Germ cells have the ability to differentiate into eggs and sperm and must determine their sexual fate. In vertebrates, the mechanism of commitment to oogenesis following the sexual fate decision in germ cells remains unknown. Forkhead-box protein L3 (foxl3) is a switch gene involved in the germline sexual fate decision in the teleost fish medaka (Oryzias latipes). Here, we show that foxl3 organizes two independent pathways of oogenesis regulated by REC8 meiotic recombination protein a (rec8a), a cohesin component, and F-box protein (FBP) 47 (fbxo47), a subunit of E3 ubiquitin ligase. In mutants of either gene, germ cells failed to undergo oogenesis but developed normally into sperm in testes. Disruption of rec8a resulted in arrest at a meiotic pachytenelike stage specifically in females, revealing a sexual difference in meiotic progression. Analyses of fbxo47 mutants showed that this gene regulates transcription factors that facilitate folliculogenesis: LIM homeobox 8 (lhx8b), factor in the germline α (figla), and newborn ovary homeobox (nobox). Interestingly, we found that the fbxo47 pathway ensures that germ cells do not deviate from an oogenic pathway until they reach diplotene stage. The mutant phenotypes together with the timing of their expression imply that germline feminization is established during early meiotic prophase I.

SNAI1-Driven Sequential EMT Changes Attributed by Selective Chromatin Enrichment of RAD21 and GRHL2.

Over two decades of research on cancer-associated epithelial-mesenchymal transition (EMT) led us to ascertain the occurrence of transitional intermediate states (collectively referred to as the EMT spectrum). Among the molecular factors that drive EMT, SNAI1 plays an indispensable role in regulating other core transcription factors, and this regulation is highly context-dependent. However, molecular investigation on this context-dependent regulation is still lacking. Using two ovarian cancer cell lines, we show that SNAI1 regulation on other core EMT-TFs switches from a repressive control in highly epithelial cells to an activation signaling in intermediate epithelial cells. Upon further scrutiny, we identify that the expression of early epithelial genes PERP and ERBB3 are differentially regulated in SNAI1-induced sequential EMT changes. Mechanistically, we show that changes in PERP and ERBB3 transcript levels could be correlated to the selective enrichment loss of RAD21, a cohesin component, at the distal enhancer sites of PERP and ERBB3, which precedes that of the proximal promoter-associated sites. Furthermore, the RAD21 enrichment at the distal enhancer sites is dependent on GRHL2 expression. In a nutshell, the alteration of GRHL2-associated RAD21 enrichment in epithelial genes is crucial to redefine the transition of cellular states along the EMT spectrum.

Characterization of the major human STAG3 variants using some proteomics and bioinformatics assays

BackgroundSTAG3 is the meiotic component of cohesin and a member of the Cancer Testis Antigen (CTA) family. This gene has been found to be overexpressed in many types of cancer, and recently, its variants have been implicated in other disorders and many human diseases. Therefore, this study aimed to analyze the major variants of STAG3. Western blot (WB) and immunoprecipitation (IP) assays were performed using two different anti-STAG3 antibodies that targeted the relevant protein in MCF-7, T-47D, MDA-MB-468, and MDA-MB-231 breast cancer cells with Jurkat and MCF-10A cells as positive and negative controls, respectively. In silico analyses were searched to study the major isoforms.ResultsWB and IP assays revealed two abundant polypeptides < 191 kDa and ~ 75 kDa in size. Specific bioinformatics tools successfully determined the three-dimensional (3-D) structure, the subcellular localization, and the secondary structures of the isoforms. Furthermore, some of the physicochemical properties of the STAG3 proteins were also determined.ConclusionsThe results of this study revealed the power of applying the biological techniques (WB and IP) with the bioinformatics assays and the possibility of their exploitation in understanding diseased genes. Exploring the major variants of STAG3 at the protein level could help decipher some disorders associated with their occurrence, along with designing drugs effective at least for some relevant diseases.