Biology subjects

Chervova, A.

Publications and source records attributed to Chervova, A..

12 recordsLinked to original sources

bioRxiv2026

Embryonic lympho-epithelial cell interactions play an essential role in the establishment of adult T cell tolerance

Thymic seeding progenitors (TSP) colonize the embryonic thymus in two successive waves, with the first wave uniquely contributing to lymphoid tissue inducer and invariant {gamma}{delta} T cells that drive primordial medullary thymic epithelial cell (mTEC) maturation. Although recognized during thymic organogenesis, their long-term impact on T cell selection and immune tolerance remains unclear. We show that selective depletion of first-wave TSP alters thymic architecture and TEC composition. This results in reduced mTEC numbers, failure of medullary islets to expand and coalesce, altered dendritic cell composition, and delayed regulatory T cell development. Single-cell transcriptional analysis revealed a decrease in the most immature and expanding compartments and signs of accelerated maturation. It also revealed abnormal TEC differentiation trajectories, expansion of post-Aire and mimetic TEC subsets, and altered expression of tissue-restricted antigen-coding genes. Functionally, first-wave TSP depletion resulted in impaired thymic recovery after injury, and age-associated autoimmunity, with elevated anti-nuclear antibodies and lymphocytic infiltration in peripheral tissues. Thus, first-wave TSP imprint long-lasting control of thymic function and immune tolerance. One sentence summaryEarly embryonic thymic seeding progenitors program thymic epithelial maturation and regulatory T cell development, ensuring long-term immune tolerance and protection from age-associated autoimmunity.

immunology↗

bioRxiv2025

Nuclear Argonaute HRDE-1 Sustains Chromatin-Independent Transcriptional Silencing and Epigenetic Inheritance

Epigenetic inheritance of transcriptional silencing is traditionally attributed to chromatin-based mechanisms in which Argonaute-small RNA complexes recruit histone-modifying enzymes. Here we show that, in Caenorhabditis elegans, the nuclear Argonaute HRDE-1 maintains germline transcriptional repression independently of canonical heterochromatin marks. Using inducible, germline-specific HRDE-1 depletion combined with nuclear sorting, CUT&Tag, and nascent transcription profiling, we identified endogenous genes that become transcriptionally activated upon loss of HRDE-1 despite retaining H3K9me3 and H3K23me3. HRDE-1 directly restrains RNA polymerase II (Pol II) while simultaneously promoting polyUG-dependent amplification of antisense 22G-RNAs in perinuclear condensates, thereby coupling nuclear transcriptional repression to small RNA biogenesis. HRDE-1 loss causes progressive erosion of 22G-RNAs and delays silencing re-establishment, revealing a transgenerational feedback loop in which small RNAs, and not chromatin modifications, constitute the primary heritable signal. These findings redefine nuclear Argonautes as active drivers of RNA-based epigenetic inheritance and broaden our understanding of how small RNA pathways maintain transcriptional silencing across generations.

molecular biology↗

bioRxiv2025

DNA supercoiling modulates eukaryotic transcription in a gene-orientation dependent manner

DNA supercoiling is an intrinsic consequence of transcription that must be resolved to maintain proper gene expression. How DNA supercoiling shapes transcription dynamics in chromatinized animal genomes remains unclear. Here, we acutely depleted topoisomerases I and II in Caenorhabditis elegans and applied nascent transcription profiling, nuclear and total RNA-seq, histone modification mapping, and long-read sequencing to capture the immediate transcriptional and chromatin responses to topological stress. We show that the genomic context influences the effect of supercoiling on transcription initiation, elongation and coordinated expression of adjacent genes. The impact of supercoiling on transcription initiation is not uniformly repressive but instead depends on the orientation and proximity of neighboring genes. We find that negative supercoiling promotes coordinated expression of divergent gene pairs, while positive supercoiling inhibits and uncouples expression of convergent genes. DNA supercoiling hinders transcription elongation globally resulting in reduced production of longer transcripts and overall shortening of poly(A) tails. While DNA supercoiling generated by neighboring transcription impacts initiation, elongation defects are driven by local supercoiling generated by the genes own transcription. These elongation effects are not accompanied by global changes in elongation-associated histone modifications but coincide with modest reductions in promoter and enhancer marks. Our findings reveal a directional mechanism by which genome architecture shapes transcriptional responses to DNA supercoiling, uncovering a DNA topological basis for coordinated gene expression in a multicellular organism.

molecular biology↗

bioRxiv2024

Molecular and epistatic interactions between pioneer transcription factors shape nucleosome dynamics and cell differentiation

Pioneer transcription factors (TF) bind nucleosome-embedded DNA motifs to activate new regulatory elements and promote differentiation. However, the complexity, binding dependencies and temporal effects of their action remain unclear. Here, we dissect how the pioneer TF GATA6 triggers Primitive Endoderm (PrE) differentiation from pluripotent cells. We show that transient GATA6 binding exploits accessible regions to decommission active enhancers and promote pluripotency gene silencing. Simultaneously, GATA6 targets closed chromatin and initiates an extensive remodeling culminating in the establishment of fragile nucleosomes flanked by ordered nucleosome arrays and increased accessibility. This is directly enhanced by rapidly expressed PrE TFs (SOX17) and by pluripotency TFs repurposed for differentiation (OCT4/SOX2). Furthermore, GATA6 mediates the replacement of essential nuclear receptors for PrE differentiation, from ESRRB to ESRRA. Therefore, pioneer TFs orchestrate a complex gene regulatory network involving many if not all available pioneer TFs, including those required to support the original identity of differentiating cells.

molecular biology↗

bioRxiv2023

MYC and MAX drive the reactivation of the genome after mitosis

Shortly after cell division, a robust wave of hyper-transcription reactivates the genome1-3. This phenomenon is particularly pronounced in pluripotent cells4, which necessitate rapid transcrip-tome reactivation to maintain their undifferentiated state and prevent premature differentiation. While recent work has illuminated how specific groups of genes are reactivated4-8, the mechanisms enabling the global, efficient and accurate post-mitotic reactivation of the genome remain unknown. Here we elucidate the direct involvement of the MYC/MAX transcription factors in the post-mitotic reactivation of pluripotent mouse embryonic stem cells. While MYC undergoes extensive phosphorylation and largely dissociates from its DNA binding sites during mitosis, we report that MAX remains bound to its targets, preferentially at promoters, and facilitates early recruitment of MYC following mitosis. Through the application of MYC/MAX heterodimerization inhibitors, we demonstrate their indispensable role in sustaining hyper-transcription in ES cells, including during the critical transition from mitosis to G1 phase. Our findings uncover a novel role for MAX in mitotic book-marking, highlighting its pivotal role in post-mitotic MYC recruitment and the re-establishment of high global transcription levels. These findings hold significant implications for medically relevant contexts, particularly when cell proliferation is of paramount importance9. We anticipate that the study of mitotic bookmarking by MYC and MAX and of the effects of anticancer drugs targeting MYC/MAX interactions in such process10-12 will be relevant for our understanding of cancer and its potential treatments.

molecular biology↗

bioRxiv2023

Nr5a2 is essential for morula development

Early embryogenesis is driven by transcription factors (TFs) that first activate the zygotic genome and then specify the lineages constituting the blastocyst. While the TFs specifying the blastocysts lineages are well characterised, those playing earlier roles are ill-defined. Using mouse models of the TF Nr5a2, we show that Nr5a2-/- embryos arrest at the early morula stage and exhibit overt phenotypical problems such as altered lineage specification, frequent mitotic failure and substantial chromosome segregation defects. Transcriptomic profiling shows that NR5A2 is a master regulator required for appropriate expression of thousands of genes at the 8-cells stage, including lineage-specifying TFs and genes involved in mitosis, telomere maintenance and DNA repair. We conclude that NR5A2 coordinates proliferation, genome stability and lineage specification to ensure proper morula development.

developmental biology↗

bioRxiv2022

Mitotic bookmarking redundancy by nuclear receptors mediates robust post-mitotic reactivation of the pluripotency network.

Mitotic bookmarking transcription factors (TFs) are thought to mediate rapid and accurate post-mitotic gene reactivation. However, the loss of individual bookmarking TFs often leads to the deregulation of only a small proportion of their mitotic targets, raising doubts on the significance and importance of their bookmarking function. Here, we used targeted proteomics of the mitotic bookmarking TF ESRRB, an orphan nuclear receptor, to discover an unexpected redundancy among members of the protein superfamily of nuclear receptors. Focusing on the nuclear receptor NR5A2, which together with ES-RRB is essential for mouse pluripotency, we demonstrate conjoint bookmarking activity of both factors on promoters and enhancers of a large fraction of active genes, particularly the most rapidly and strongly reactivated ones. Upon fast and simultaneous degradation of both factors during mitotic exit, hundreds of mitotic targets of ESRRB/NR5A2, including key players of the pluripotency network, display attenuated transcriptional reactivation. We propose that redundancy in mitotic bookmarking TFs, especially by nuclear receptors, confers robustness to the reestablishment of gene regulatory networks after mitosis.

molecular biology↗

bioRxiv2022

Mitotic bookmarking by CTCF controls selected genes during the fast post-mitotic genome reactivation of ES cells

Mitosis leads to a global downregulation of transcription that then needs to be efficiently restored. In somatic cells, this is mediated by a transient hyper-active state that first leads to the reactivation of genes necessary to rebuild the interphasic cell and then of those executing specific cell functions. Here, we hypothesized that cells displaying rapid cell cycles may display accelerated gene reactivation dynamics. To test this, we focused on mouse Embryonic Stem (ES) cells, which have a short cell cycle and spend a minor time in G1. Compared to previous studies, we observed a uniquely fast global reactivation, which displays little specificity towards housekeeping versus cell identity genes. Such lack of specificity may enable the restoration of the entirety of regulatory functions before the onset of DNA replication. Genes displaying the fastest reactivation dynamics are associated with binding of CTCF, a transcription factor that largely maintains binding to its targets on DNA during mitosis. Nevertheless, we show that the post-mitotic global burst is robust and largely insensitive to CTCF depletion. There are, however, around 350 genes that respond to CTCF depletion rapidly after mitotic exit. Remarkably, these are characterised by promoter-proximal mitotic bookmarking by CTCF. We propose that the structure of the cell cycle imposes distinct constrains to post-mitotic gene reactivation dynamics in different cell types, via mechanisms that are yet to be identified but that can be modulated by mitotic bookmarking factors.

genomics↗

bioRxiv2021

In-vitro cellular reprogramming to model gonad development and its disorders

During embryonic development, mutually antagonistic signaling cascades determine the fate of the bipotential gonad towards a testicular or ovarian identity. Errors in this process result in human Disorders of Sex Development (DSDs), where there is discordance between chromosomal, gonadal, and anatomical sex. The absence of an appropriate, accessible in-vitro system is a major obstacle in understanding mechanisms of sex-determination/DSDs. Here, we describe protocols for differentiation of mouse and human pluripotent cells towards gonadal progenitors. Transcriptomic analysis reveals that the in-vitro-derived murine gonadal cells are equivalent to E11.5 in-vivo progenitors. Using similar conditions, Sertoli-like cells derived from 46,XY human induced pluripotent stem cells (hiPSCs) exhibit sustained expression of testis-specific genes, secrete AMH, migrate and form tubular structures. The cells derived from a 46,XY DSD female hiPSCs, carrying a NR5A1 variant, show aberrant gene expression and absence of tubule formation. CRISPR/Cas9-mediated correction of the variant rescued the phenotype. This is a robust tool to understand mechanisms of sex-determination and model DSDs.

developmental biology↗

bioRxiv2021

OCT4 activates a Suv39h1-repressive antisense lncRNA to couple histone H3 Lysine 9 methylation to pluripotency

Histone H3 Lysine 9 (H3K9) methylation, a characteristic mark of heterochromatin, is progressively implemented during development to contribute to cell fate restriction as differentiation proceeds. For instance, in pluripotent mouse Embryonic Stem (ES) cells the global levels of H3K9 methylation are rather low and increase only upon differentiation. Conversely, H3K9 methylation represents an epigenetic barrier for reprogramming somatic cells back to pluripotency. How global H3K9 methylation levels are coupled with the acquisition and loss of pluripotency remains largely unknown. Here, we identify SUV39H1, a major H3K9 di- and tri-methylase, as an indirect target of the pluripotency network of Transcription Factors (TFs). We find that pluripotency TFs, principally OCT4, activate the expression of an uncharacterized antisense long non-coding RNA to Suv39h1, which we name Suv39h1as. In turn, Suv39h1as downregulates Suv39h1 transcription in cis via a mechanism involving the modulation of the chromatin status of the locus. The targeted deletion of the Suv39h1as promoter region triggers increased SUV39H1 expression and H3K9me2 and H3K9me3 levels, leading to accelerated and more efficient commitment into differentiation. We report, therefore, a simple genetic circuitry coupling the global levels of H3K9 methylation to pluripotency in mouse ES cells.

molecular biology↗

bioRxiv2021

Analysis of gene expression and mutation data points on contribution of transcription to the mutagenesis by APOBEC enzymes

Since the discovery of the role of the APOBEC enzymes in human cancers, the mechanisms of this type of mutagenesis remain little understood. Theoretically, targeting of single-stranded DNA by the APOBEC enzymes could occur during cellular processes leading to the unwinding of DNA double-stranded structure. Some evidence points to the importance of replication in the APOBEC mutagenesis, while the role of transcription is still underexplored. Here, we analyzed gene expression and whole genome sequencing data from five types of human cancers with substantial APOBEC activity to estimate the involvement of transcription in the APOBEC mutagenesis and compare its impact with that of replication. Using the TCN motif as the mutation signature of the APOBEC enzymes, we observed a correlation of active APOBEC mutagenesis with gene expression, confirmed the increase of APOBEC-induced mutations in early-replicating regions, and estimated the relative impact of transcription and replication on the APOBEC mutagenesis, which turned out to be approximately equal in transcribed regions. We also found that the known effect of higher density of APOBEC-induced mutations on the lagging strand was highest in middle-replicating regions, and observed higher APOBEC mutation density on the sense strand, the latter bias positively correlated with the gene expression level. Bullet points- The APOBEC mutagenesis rate is higher in actively expressed genes - The APOBEC mutation density is higher on the sense strand - The lagging/leading strand ratio of the APOBEC mutational density is highest in middle-replicating regions

cancer biology↗

bioRxiv2020

The combined action of Esrrb and Nr5a2 is essential for naive pluripotency.

The maintenance of pluripotency in mouse embryonic stem cells (ESCs) is governed by the action of an interconnected network of transcription factors. Among them, only Oct4 and Sox2 have been shown to be strictly required for the self-renewal of ESCs and pluripotency, particularly in culture conditions where differentiation cues are chemically inhibited. Here, we report that the conjunct activity of two orphan nuclear receptors, Esrrb and Nr5a2, parallels the importance of that of Oct4 and Sox2 in naive ESCs. By occupying a large common set of regulatory elements, these two factors control the binding of Oct4, Sox2 and Nanog to DNA. Consequently, in their absence the pluripotency network collapses and the transcriptome is substantially deregulated, leading to the differentiation of ESCs. Altogether, this work identifies orphan nuclear receptors, previously thought to be performing supportive functions, as a new set of core regulators of naive pluripotency.

developmental biology↗