The specific roles of cohesin-SA1 in telomere cohesion and gene expression: implications for cancer and development

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 08-11-2012Cohesin is a protein complex originally identified for its role in sister chromatid cohesion. Increasing evidence portrays this complex as a major organizer of interphase chromatin. In vertebrate somatic cells, there are two cohesin complexes that consist of Smc1, Smc3, Rad21 and either SA1 or SA2. To explore their functional specificity and their relevance for cancer and development, we generated a mouse model deficient for SA1. Mouse embryos lacking SA1 die before birth and show developmental delay and features reminiscent of Cornelia de Lange syndrome (CdLS), a genetic disorder linked to cohesin dysfunction. SA1-­‐ heterozygous mice have shorter lifespan and earlier onset of tumourigenesis. SA1-­‐deficient cells present decreased proliferation and increased aneuploidy as a result of chromosome segregation defects. These defects are not caused by impaired centromere cohesion, which depends on cohesin-­‐SA2. Instead, we found that they arise from defective telomere replication, which requires cohesion mediated specifically by cohesin-­‐SA1. In addition, analysis of cohesin genome-­‐wide distribution reveals that SA1 is responsible for the accumulation of the complex at promoters and sites bound by the chromatin insulator CTCF. In the absence of SA1, cohesin-­‐SA2 redistributes away from those sites and transcription of several genes is altered. Among them we found genes involved in biological processes related to CdLS pathogenesis. The presence of cohesin-­‐SA1 at gene promoters positively regulates the expression of genes such as Myc or Protocadherins, a function that cannot be compensated by cohesin-­‐SA2. Moreover, the cohesin-­‐binding pattern along gene clusters is altered in the absence of SA1 and affects the transcriptional regulation of the genes within. Thus, cohesin-­‐SA1 prevents generation of aneuploidy and tumourigenesis by assuring an efficient telomere replication, while its impaired function in gene expression regulation most likely underlies the etiology of CdLS.El complejo cohesina se identificó inicialmente por su papel en cohesión, pero evidencias más recientes lo describen como organizador de la cromatina interfásica. En células somáticas de vertebrados hay dos tipos de complejos compuestos por Smc1, Smc3, Rad21 y bien SA1 o SA2. Para estudiar su especificidad funcional y relevancia en cáncer y desarrollo embrionario, generamos un modelo murino deficiente en SA1. Los embriones deficientes en SA1 no son viables, presentan un retraso en el desarrollo y características propias del síndrome de Cornelia de Lange (CdLS), asociado a una disfunción en cohesina. Los ratones SA1 heterocigotos presentan reducción en la esperanza de vida y aparición temprana de tumores espontáneos. Las células deficientes en SA1 muestran una menor capacidad proliferativa y mayor aneuploidía, como resultado de los defectos en segregación cromosómica. Estos últimos no se deben a defectos en cohesión centromérica, mediada por SA2, sino que tienen su origen en una replicación telomérica deficiente, al faltar la cohesión mediada específicamente por SA1 en esta región. Además, el análisis de la distribución genómica de cohesina revela que SA1 es responsable de la acumulación del complejo en promotores y sitios de unión del factor insulator CTCF. En ausencia de SA1, la cohesina-­‐SA2 se desplaza a otras localizaciones y se altera la transcripción de varios genes. Entre ellos se encuentran genes implicados en procesos biológicos relacionados con la patogénesis de CdLS. La presencia de SA1 en promotores regula positivamente la expresión de genes como Myc y Protocaderinas, función que no puede ser compensada por SA2. Además, las posiciones de cohesina en clústeres génicos se alteran en ausencia de SA1, lo cual afecta a su regulación transcripcional. La cohesina-­‐SA1 previene la generación de aneuploidía y tumores al garantizar una replicación telomérica eficiente. Asimismo, es probable que su papel en regulación de la expresión génica guarde relación directa con la etiología de CdLS

The contribution of cohesin-SA1 to gene expression and chromatin architecture in two murine tissues

Cohesin, which in somatic vertebrate cells consists of SMC1, SMC3, RAD21 and either SA1 or SA2, mediates higher-order chromatin organization. To determine how cohesin contributes to the establishment of tissue-specific transcriptional programs, we compared genome-wide cohesin distribution, gene expression and chromatin architecture in cerebral cortex and pancreas from adult mice. More than one third of cohesin binding sites differ between the two tissues and these show reduced overlap with CCCTC-binding factor (CTCF) and are enriched at the regulatory regions of tissue-specific genes. Cohesin/CTCF sites at active enhancers and promoters contain, at least, cohesin-SA1. Analyses of chromatin contacts at the Protocadherin (Pcdh) and Regenerating islet-derived (Reg) gene clusters, mostly expressed in brain and pancreas, respectively, revealed remarkable differences that correlate with the presence of cohesin. We could not detect significant changes in the chromatin contacts at the Pcdh locus when comparing brains from wild-type and SA1 null embryos. In contrast, reduced dosage of SA1 altered the architecture of the Reg locus and decreased the expression of Reg genes in the pancreas of SA1 heterozygous mice. Given the role of Reg proteins in inflammation, such reduction may contribute to the increased incidence of pancreatic cancer observed in these animals.The Spanish Ministry of Economy and Competitiveness (MINECO) [BFU2013-48481-R to A.L.]; 'Ramon y Cajal' Contract [RYC-2010-06122 to A.C.]; Fundacion La Caixa [PhD Fellowship to S.R.]. Funding for open access charge: The Spanish Ministry of Economy and Competitiveness (MINECO) [BFU2013-48481-R]S

Epigenetics, Enhancer Function and 3D Chromatin Organization in Reprogramming to Pluripotency

Genome architecture, epigenetics and enhancer function control the fate and identity of cells. Reprogramming to induced pluripotent stem cells (iPSCs) changes the transcriptional profile and chromatin landscape of the starting somatic cell to that of the pluripotent cell in a stepwise manner. Changes in the regulatory networks are tightly regulated during normal embryonic development to determine cell fate, and similarly need to function in cell fate control during reprogramming. Switching off the somatic program and turning on the pluripotent program involves a dynamic reorganization of the epigenetic landscape, enhancer function, chromatin accessibility and 3D chromatin topology. Within this context, we will review here the current knowledge on the processes that control the establishment and maintenance of pluripotency during somatic cell reprogramming

Diet-induced rewiring of the Wnt gene regulatory network connects aberrant splicing to fatty liver and liver cancer in DIAMOND mice

Abstract Several preclinical models have been recently developed for metabolic associated fatty liver disease (MAFLD) and associated hepatocellular carcinoma (HCC) but comprehensive analysis of the regulatory and transcriptional landscapes underlying disease in these models are still missing. We investigated the regulatory and transcriptional landscape in fatty livers and liver tumours from DIAMOND mice that faithfully mimic human HCC development in the context of MAFLD. RNA-sequencing and ChIP-sequencing revealed rewiring of the Wnt/β-catenin regulatory network in DIAMOND tumours, as manifested by chromatin remodelling and associated switching in the expression of the canonical TCF/LEF downstream effectors. We identified splicing as a major mechanism leading to constitutive oncogenic activation of β-catenin in a large subset of DIAMOND tumours, a mechanism that is independent on somatic mutations in the locus and that has not been previously shown. Similar splicing events were found in a fraction of human HCC and hepatoblastoma samples

Epigenomic perturbation of novel EGFR enhancers reduces the proliferative and invasive capacity of glioblastoma and increases sensitivity to temozolomide

Background: Glioblastoma (GB) is the most aggressive of all primary brain tumours and due to its highly invasive nature, surgical resection is nearly impossible. Patients typically rely on radiotherapy with concurrent temozolomide (TMZ) treatment and face a median survival of ~ 14 months. Alterations in the Epidermal Growth Factor Receptor gene (EGFR) are common in GB tumours, but therapies targeting EGFR have not shown significant clinical efficacy. Methods: Here, we investigated the influence of the EGFR regulatory genome on GB cells and identified novel EGFR enhancers located near the GB-associated SNP rs723527. We used CRISPR/Cas9-based approaches to target the EGFR enhancer regions, generating multiple modified GB cell lines, which enabled us to study the functional response to enhancer perturbation. Results: Epigenomic perturbation of the EGFR regulatory region decreases EGFR expression and reduces the proliferative and invasive capacity of glioblastoma cells, which also undergo a metabolic reprogramming in favour of mitochondrial respiration and present increased apoptosis. Moreover, EGFR enhancer-perturbation increases the sensitivity of GB cells to TMZ, the first-choice chemotherapeutic agent to treat glioblastoma. Conclusions: Our findings demonstrate how epigenomic perturbation of EGFR enhancers can ameliorate the aggressiveness of glioblastoma cells and enhance the efficacy of TMZ treatment. This study demonstrates how CRISPR/Cas9-based perturbation of enhancers can be used to modulate the expression of key cancer genes, which can help improve the effectiveness of existing cancer treatments and potentially the prognosis of difficult-to-treat cancers such as glioblastoma

Rewiring of the promoter-enhancer interactome and regulatory landscape in glioblastoma orchestrates gene expression underlying neurogliomal synaptic communication

Chromatin organization controls transcription by modulating 3D-interactions between enhancers and promoters in the nucleus. Alterations in epigenetic states and 3D-chromatin organization result in gene expression changes contributing to cancer. Here, we map the promoter-enhancer interactome and regulatory landscape of glioblastoma, the most aggressive primary brain tumour. Our data reveals profound rewiring of promoter-enhancer interactions, chromatin accessibility and redistribution of histone marks in glioblastoma. This leads to loss of long-range regulatory interactions and overall activation of promoters, which orchestrate changes in the expression of genes associated to glutamatergic synapses, axon guidance, axonogenesis and chromatin remodelling. SMAD3 and PITX1 emerge as major transcription factors controlling genes related to synapse organization and axon guidance. Inhibition of SMAD3 and neuronal activity stimulation cooperate to promote proliferation of glioblastoma cells in co-culture with glutamatergic neurons, and in mice bearing patient-derived xenografts. Our findings provide mechanistic insight into the regulatory networks that mediate neurogliomal synaptic communication

Reduction of Nipbl impairs cohesin loading locally and affects transcription but not cohesion-dependent functions in a mouse model of Cornelia de Lange Syndrome.

Cornelia de Lange Syndrome (CdLS) is a genetic disorder linked to mutations in cohesin and its regulators. To date, it is unclear which function of cohesin is more relevant to the pathology of the syndrome. A mouse heterozygous for the gene encoding the cohesin loader Nipbl recapitulates many features of CdLS. We have carefully examined Nipbl deficient cells and here report that they have robust cohesion all along the chromosome. DNA replication, DNA repair and chromosome segregation are carried out efficiently in these cells. While bulk cohesin loading is unperturbed, binding to certain promoters such as the Protocadherin genes in brain is notably affected and alters gene expression. These results provide further support for the idea that developmental defects in CdLS are caused by deregulated transcription and not by malfunction of cohesion-related processes

Pan-AMPK activator O304 prevents gene expression changes and remobilisation of histone marks in islets of diet-induced obese mice

AMP-activated protein kinase (AMPK) has an important role in cellular energy homeostasis and has emerged as a promising target for treatment of Type 2 Diabetes (T2D) due to its beneficial effects on insulin sensitivity and glucose homeostasis. O304 is a pan-AMPK activator that has been shown to improve glucose homeostasis in both mouse models of diabetes and in human T2D subjects. Here, we describe the genome-wide transcriptional profile and chromatin landscape of pancreatic islets following O304 treatment of mice fed high-fat diet (HFD). O304 largely prevented genome-wide gene expression changes associated with HFD feeding in CBA mice and these changes were associated with remodelling of active and repressive chromatin marks. In particular, the increased expression of the β-cell stress marker Aldh1a3 in islets from HFD-mice is completely abrogated following O304 treatment, which is accompanied by loss of active chromatin marks in the promoter as well as distant non-coding regions upstream of the Aldh1a3 gene. Moreover, O304 treatment restored dysfunctional glucose homeostasis as well as expression of key markers associated with β-cell function in mice with already established obesity. Our findings provide preclinical evidence that O304 is a promising therapeutic compound not only for T2D remission but also for restoration of β-cell function following remission of T2D diabetes

Reduction of Nipbl impairs cohesin loading locally and affects transcription but not cohesion-dependent functions in a mouse model of Cornelia de Lange Syndrome.

Cornelia de Lange Syndrome (CdLS) is a genetic disorder linked to mutations in cohesin and its regulators. To date, it is unclear which function of cohesin is more relevant to the pathology of the syndrome. A mouse heterozygous for the gene encoding the cohesin loader Nipbl recapitulates many features of CdLS. We have carefully examined Nipbl deficient cells and here report that they have robust cohesion all along the chromosome. DNA replication, DNA repair and chromosome segregation are carried out efficiently in these cells. While bulk cohesin loading is unperturbed, binding to certain promoters such as the Protocadherin genes in brain is notably affected and alters gene expression. These results provide further support for the idea that developmental defects in CdLS are caused by deregulated transcription and not by malfunction of cohesion-related processes