Biology subjects

Shchuka, V. M.

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

6 recordsLinked to original sources

bioRxiv2023

SOX4 exerts contrasting regulatory effects on labor-associated gene promoters in myometrial cells

The uterine muscular layer, or myometrium, undergoes profound changes in global gene expression during its progression from a quiescent state during pregnancy to a contractile state at the onset of labor. In this study, we investigate the role of SOX family transcription factors in myometrial cells and provide evidence for the role of SOX4 in regulating labor-associated genes. We show that Sox4 has elevated expression in the murine myometrium during a term laboring process and in two mouse models of preterm labor. Additionally, SOX4 differentially affects labor-associated gene promoter activity in cooperation with activator protein 1 (AP-1) dimers. SOX4 exerted no effect on the Gja1 promoter; a JUND-specific activation effect at the Fos promoter; a positive activation effect on the Mmp11 promoter with the AP-1 dimers; and surprisingly, we noted that the reporter expression of the Ptgs2 promoter in the presence of JUND and FOSL2 was repressed by the addition of SOX4. Our data indicate SOX4 may play a diverse role in regulating gene expression in the laboring myometrium in cooperation with AP-1 factors. This study enhances our current understanding of the regulatory network that governs the transcriptional changes associated with the onset of labor and highlights a new molecular player that may contribute to the labor transcriptional program.

molecular biology↗

bioRxiv2023

A Proximal Sox2 Enhancer Cluster is Required for the Anterior Regional Identity of Neural Progenitors

Embryonic development depends on spatially and temporally orchestrated gene regulatory networks. Expressed in neural stem and progenitor cells (NSPCs), the transcription factor sex-determining region Y box 2 (Sox2) is critical for embryogenesis and stem cell maintenance in neural development. Whereas Sox2 is regulated by a distal cluster of enhancers in embryonic stem cells (ESCs), enhancers closer to the gene have been implicated in Sox2 transcriptional regulation in the neural lineage. Using functional genomics data, and deletion analysis we show that a downstream enhancer cluster regulates Sox2 transcription in NSPCs derived from mouse ESCs. By generating allelic mutants using CRISPR-Cas9 mediated deletions, we show that this proximal enhancer cluster, termed Sox2 regulatory regions 2-18 (SRR2-18), is a cis regulator of Sox2 transcription during neural differentiation. Transcriptome analyses demonstrate that loss of even one copy of SRR2-18 disrupts the region-specific identity of NSPCs. Biallelic deletion of this Sox2 neural enhancer cluster causes reduced SOX2 protein, less frequent interaction with transcriptional machinery, and leads to perturbed chromatin accessibility genome-wide further affecting the expression of neurodevelopmental and anterior-posterior regionalization genes. Furthermore, homozygous NSPC deletants exhibit self-renewal defects and impaired differentiation into cell types found in the brain. Altogether, our data define a cis-regulatory enhancer cluster controlling Sox2 transcription in NSPCs and highlight the sensitivity of neural differentiation processes to decreased Sox2 transcription, which influences their differentiation into posterior neural fates, specifically the caudal neural tube.

genetics↗

bioRxiv2023

Competition between transcription and loop extrusion modulates promoter and enhancer dynamics

The spatiotemporal configuration of genes with distal regulatory elements, and the impact of chromatin mobility on transcription, remain unclear. Loop extrusion is an attractive model for bringing genetic elements together, but how this functionally interacts with transcription is also largely unknown. We combine live tracking of genomic loci and nascent transcripts with molecular dynamics simulations to assess the 4D arrangement of the Sox2 gene and its enhancer, in response to a battery of perturbations. We find that alterations in chromatin mobility, not promoter-enhancer distance, is more informative about transcriptional status. Active elements display more constrained mobility, consistent with confinement within specialized nuclear sites, and alterations in enhancer mobility distinguish poised from transcribing alleles. Strikingly, we find that whereas loop extrusion and transcription factor-mediated clustering contribute to promoter-enhancer proximity, they have antagonistic effects on chromatin dynamics. This provides an experimental framework for the underappreciated role of chromatin dynamics in genome regulation.

cell biology↗

bioRxiv2022

MYB and ELF3 differentially modulate labor-inducing gene expression in myometrial cells

Spontaneous uterine contractions are initiated when smooth muscle cells (SMCs) within the uterine muscle, or myometrium, transition from a functionally dormant to an actively contractile phenotype at the end of the pregnancy period. We know that this process is accompanied by gestational time point-specific differences in the SMC transcriptome, which can be modulated by the Activator protein 1 (AP-1), Nuclear factor kappa beta (Nf-{kappa}{beta}), Estrogen receptor (ER), and Progesterone receptor (PR) transcription factors. Less is known, however, about the additional proteins that might assist these factors in conferring the transcriptional changes observed at labor onset. Here, we present functional evidence for the roles of two proteins previously understudied in the SMC context - MYB and ELF3 - which can contribute to the regulation of labor-driving gene transcription. We show that the MYB and ELF3 genes exhibit elevated transcript expression levels in mouse and human myometrial tissues during spontaneous term labor. The expression of both genes was also significantly increased in mouse myometrium during preterm labor induced by the progesterone antagonist, mifepristone (RU486), but not during infection-simulating preterm labor induced by intrauterine infusion of Lipopolysaccharide (LPS). Furthermore, both MYB and ELF3 proteins affect labor-driving gene promoter activity, although in surprisingly opposing ways: Gja1 and Fos promoter activation increases in the presence of MYB and decreases in the presence of ELF3. Collectively, our study adds to the current understanding of the transcription factor network that defines the transcriptomes of SMCs during late gestation and implicates two new players in the control of labor timing.

molecular biology↗

bioRxiv2022

Transcriptional regulation and chromatin architecture maintenance are decoupled modular functions at the Sox2 locus

How distal regulatory elements control gene transcription and chromatin topology is not clearly defined, yet these processes are closely linked in lineage specification during development. Through allele-specific genome editing and chromatin interaction analyses of the Sox2 locus in mouse embryonic stem cells, we found a striking disconnection between transcriptional control and chromatin architecture. We trace nearly all Sox2 transcriptional activation to a small number of key transcription factor binding sites, whose deletions have no effect on promoter-enhancer interaction frequencies or topological domain organization. Local chromatin architecture maintenance, including at the topologically associating domain (TAD) boundary downstream of the Sox2 enhancer, is widely distributed over multiple transcription factor-bound regions and maintained in a CTCF-independent manner. Furthermore, disruption of promoter-enhancer interactions by ectopic chromatin loop formation has no effect on Sox2 expression. These findings indicate that many transcription factors are involved in modulating chromatin architecture independently of CTCF.

genetics↗

bioRxiv2020

A flexible repertoire of transcription factor binding sites and diversity threshold determines enhancer activity in embryonic stem cells

Transcriptional enhancers are critical for development, phenotype evolution and often mutated in disease contexts; however, even in well-studied cell types, the sequence code conferring enhancer activity remains unknown. We found genomic regions with conserved binding of multiple transcription factors in mouse and human embryonic stem cells (ESCs) contain on average 12.6 conserved transcription factor binding sites (TFBS). These TFBS are a diverse repertoire of 70 different sequences representing the binding sites of both known and novel ESC regulators. Remarkably, using a diverse set of TFBS from this repertoire was sufficient to construct short synthetic enhancers with activity comparable to native enhancers. Site directed mutagenesis of conserved TFBS in endogenous enhancers or TFBS deletion from synthetic sequences revealed a requirement for more than ten different TFBS. Furthermore, specific TFBS, including the OCT4:SOX2 co-motif, are dispensable, despite co-binding the OCT4, SOX2 and NANOG master regulators of pluripotency. These findings reveal a TFBS diversity threshold overrides the need for optimized regulatory grammar and individual TFBS that bind specific master regulators.

genomics↗