Biology subjects

Neguembor, M. V.

Publications and source records attributed to Neguembor, M. V..

4 recordsLinked to original sources

bioRxiv2023

The Hippo kinase LATS1 controls CTCF chromatin occupancy and the hormonal response of three-dimensionally grown breast cancer cells

The cancer epigenome has been studied in cells cultured in 2D monolayers on plastic surfaces, but recent studies highlight the impact of the extracellular matrix (ECM) and the 3D environment on multiple cellular functions. Here, we report the physical, biochemical and genomic differences between T47D breast cancer cells cultured in 2D monolayer and as 3D spheroids in Matrigel. Cells within 3D spheroids exhibit a rounder nucleus with less accessible, more compacted chromatin, and altered expression of over 2,000 genes, the majority of which become repressed. Hi-C analysis reveals that cells grown in 3D exhibit enrichment in regions belonging to the B compartment, decrease on chromatin bound CTCF and increased fusion of Topologically Associating Domains (TADs). Upregulation of the Hippo pathway in 3D spheroids results in the activation of the LATS1 kinase, which promotes phosphorylation and displacement of CTCF from DNA, likely responsible for the observed TAD fusions. Cells grown in 3D exhibit higher progesterone receptor (PR) binding to chromatin, leading to an increase in the number hormone-regulated genes. This effect is in part mediated by the LATS1 activation, which favors cytoplasmic retention of YAP and CTCF removal.

genomics↗

bioRxiv2023

Active Transcription and Epigenetic Reactions Synergistically RegulateMeso-Scale Genomic Organization

In interphase nuclei, chromatin is organized into interspersed dense domains with characteristic sizes, both in the nuclear interior and periphery. However, the quantitative impact of transcription and histone modifications on the size and distribution of these domains remains unclear. Here, we introduce a mesoscale theoretical model that investigates the relationship between heterochromatic domain sizes and loop extrusion rates from these domains. The model considers chromatin-chromatin and chromatin-lamina interactions, methylation and acetylation kinetics, and diffusion of epigenetic marks and nucleoplasm. Our model generates testable predictions that help reveal the biophysics underlying chromatin organization in the presence of transcription-driven loop extrusion. This process is kinetically captured through the conversion of heterochromatin to euchromatin in response to RNAPII activity. We discovered that a balance between diffusive and reactive fluxes governs the steady-state sizes of heterochromatin domains. Using theory and simulations, we predicted that a loss of transcription results in increased chromatin compaction and larger heterochromatin domain sizes. To validate our predictions, we employed complementary super-resolution and nano-imaging techniques on five different cell lines with impaired transcription. We quantitatively assessed how domain sizes scale with loop extrusion rates at the hetero-euchromatin interfaces. Our analysis of previously obtained super-resolution images of nuclei revealed that excessive loop extrusion leads to smaller heterochromatin domains. The model successfully recapitulated these observations, explaining how transcription loss can counteract the effects of cohesin overloading. As the general biophysical mechanisms regulating heterochromatin domain sizes are independent of cell type, our findings have significant implications for understanding the role of transcription in global genome organization.

biophysics↗

bioRxiv2023

SMARCAD1 and TOPBP1 contribute to heterochromatin maintenance at the transition from the 2C-like to the pluripotent state

Chromocenters are established after the 2-cell (2C) stage during mouse embryonic development, but the factors that mediate chromocenter formation remain largely unknown. To identify regulators of 2C heterochromatin establishment, we generated an inducible system to convert embryonic stem cells (ESCs) to 2C-like cells. This conversion is marked by a global reorganization and dispersion of H3K9me3-heterochromatin foci, which are then reversibly formed upon re-entry into pluripotency. By profiling the chromatin-bound proteome (chromatome) through genome capture of ESCs transitioning to 2C-like cells, we uncover chromatin regulators involved in de novo heterochromatin formation. We identified TOPBP1 and investigated its binding partner SMARCAD1. SMARCAD1 and TOPBP1 associate with H3K9me3-heterochromatin in ESCs. Interestingly, the nuclear localization of SMARCAD1 is lost in 2C-like cells. SMARCAD1 or TOPBP1 depletion in mouse embryos leads to developmental arrest, reduction of H3K9me3, and remodeling of heterochromatin foci. Collectively, our findings contribute to comprehending the maintenance of chromocenters during early development.

developmental biology↗

bioRxiv2021

STAG proteins promote cohesin ring loading at R-loops.

Most studies of cohesin function consider the Stromalin Antigen (STAG/SA) proteins as core complex members given their ubiquitous interaction with the cohesin ring. Here, we provide functional data to support the notion that the SA subunit is not a mere passenger in this structure, but instead plays a key role in the localization of cohesin to diverse biological processes and promotes loading of the complex at these sites. We show that in cells acutely depleted for RAD21, SA proteins remain bound to chromatin, cluster in 3D and interact with CTCF, as well as with a wide range of RNA binding proteins involved in multiple RNA processing mechanisms. Accordingly, SA proteins interact with RNA and are localised to R-loops where they contribute to R-loop regulation. Our results place SA1 within R-loop domains upstream of the cohesin complex and reveal a role for SA1 in cohesin loading which is independent of NIPBL, the canonical cohesin loader. We propose that SA1 takes advantage of structural R-loop platforms to link cohesin loading and chromatin structure with diverse functions. Since SA proteins are pan-cancer targets, and R-loops play an increasingly prevalent role in cancer biology, our results have important implications for the mechanistic understanding of SA proteins in cancer and disease.

cell biology↗