Biology subjects

van den Brand, T.

Publications and source records attributed to van den Brand, T..

7 recordsLinked to original sources

bioRxiv2023

A chromatin-associated regulator of RNA Polymerase III assembly at tRNA genes revealed by locus-specific proteomics

Transcription of tRNA genes by RNA Polymerase III (RNAPIII) is tightly regulated by signaling cascades in response to nutrient availability. The emerging notion of differential tRNA gene regulation implies the existence of additional regulatory mechanisms. However, tRNA gene-specific regulatory factors have not been described. For that reason, we decoded the proteome of a single native tRNA gene locus in yeast. We observed dynamic reprogramming of the core RNAPIII transcription machinery upon nutrient perturbation. In addition, we identified Fpt1, a protein of unknown function. Fpt1 uniquely occupied tRNA genes but its occupancy varied and correlated with the efficiency of RNAPIII eviction upon nutrient perturbation. Decoding the proteome of a tRNA gene in the absence of Fpt1 revealed that Fpt1 promotes eviction of RNAPIII. Cells without Fpt1 also showed impaired shutdown of ribosome biogenesis genes upon nutrient perturbation. Our findings provide support for a chromatin-associated mechanism required for RNAPIII eviction from tRNA genes and for tuning an integrated physiological response to changing metabolic demands. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/534528v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1b2ef53org.highwire.dtl.DTLVardef@7e6234org.highwire.dtl.DTLVardef@1614b5borg.highwire.dtl.DTLVardef@9a4933_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2022

Identifying cross-lineage dependencies of cell-type specific regulators in gastruloids

Correct gene expression levels in space and time are crucial for normal development. Advances in genomics enable the inference of gene regulatory programs that are active during development. However, this approach cannot capture the complex multicellular interactions that occur during embryogenesis. Compared to model organisms such as fruit flies and zebrafish, the growth of mammalian embryos in utero further complicates the analysis of cell-cell communication during development. However, in vitro models of mammalian development such as gastruloids can overcome this limitation. Using time-resolved single-cell chromatin accessibility analysis, we have delineated the regulatory landscape during gastruloid development and thereby identified the critical drivers of developmental transitions. We observed that gastruloids develop from pluripotent cells driven by the transcription factor (TF) dimer OCT4-SOX2 and differentiate along two main branches. A mesoderm branch characterized by the TF MSGN1 and a spinal cord branch characterized by CDX1, 2, 4 (CDX). Consistent with our lineage reconstruction, {Delta}CDX gastruloids fail to form spinal cord. Conversely, Msgn1 ablation inhibits the development of paraxial mesoderm, as expected. However, this also abolished spinal cord cells, which is surprising given that MSGN1 is not associated with differentiation along this branch. Therefore, formation of paraxial mesoderm is required for spinal cord development. To validate this, we generated chimeric gastruloids using {Delta}MSGN1 and wildtype cells, which formed both spinal cord and paraxial mesoderm. Strikingly, {Delta}MSGN1 cells specifically contributed to spinal cord, suggesting that cell-cell interactions between paraxial mesoderm and spinal cord are necessary for the formation of the latter. Our work has important implications for the study of cell-cell communication in development and how gene regulatory programs are functionally executed to form complex multicellular developmental structures.

systems biology↗

bioRxiv2022

Pioneer activity distinguishes activating from non-activating pluripotency transcription factor binding sites

Genome-wide transcriptional activity involves the binding of many transcription factors to thousands of sites in the genome. Determining which sites are directly driving transcription remains a challenge. Here we use acute protein depletion of the pioneer transcription factors OCT4 and SOX2 to establish their functionality in maintaining chromatin accessibility. We show that thousands of accessible sites are lost within an hour of protein depletion, indicating rapid turnover of these sites in the absence of pioneer factors. To understand the relationship with transcription we performed nascent transcription analysis and found that open chromatin sites that are maintained by SOX2 are highly predictive of gene expression, in contrast to SOX2 binding sites that do not maintain accessibility. We use CRISPR-Cas9 genome editing in the Klf2 locus to functionally validate a predicted regulatory element. We conclude that the regulatory activity of SOX2 is exerted largely at sites where it maintains accessibility and that other binding sites are largely dispensable for gene regulation.

molecular biology↗

bioRxiv2021

Hi-C Analyses with GENOVA: a case study with cohesin variants

Conformation capture-approaches like Hi-C can elucidate chromosome structure at a genome-wide scale. Hi-C datasets are large and require specialised software. Here, we present GENOVA: a user-friendly software package to analyse and visualise conformation capture data. GENOVA is an R-package that includes the most common Hi-C analyses, such as compartment and insulation score analysis. It can create annotated heatmaps to visualise the contact frequency at a specific locus and aggregate Hi-C signal over user-specified genomic regions such as ChIP-seq data. Finally, our package supports output from the major mapping-pipelines. We demonstrate the capabilities of GENOVA by analysing Hi-C data from HAP1 cell lines in which the cohesin-subunits SA1 and SA2 were knocked out. We find that {Delta}SA1 cells gain intra-TAD interactions and increase compartmentalisation. {Delta}SA2 cells have longer loops and a less compartmentalised genome. These results suggest that cohesinSA1 forms longer loops, while cohesinSA2 plays a role in forming and maintaining intra-TAD interactions. Our data supports the model that the genome is provided structure in 3D by the counter-balancing of loop formation on one hand, and compartmentalization on the other hand. By differentially controlling loops, cohesinSA1 and cohesinSA2 therefore also affect nuclear compartmentalization. We show that GENOVA is an easy to use R-package, that allows researchers to explore Hi-C data in great detail.

genomics↗

bioRxiv2019

The histone methyltransferase DOT1L prevents antigen-independent differentiation and safeguards epigenetic identity of CD8+ T cells

Cytotoxic T-cell differentiation is guided by epigenome adaptations but how epigenetic mechanisms control lymphocyte development has not been well defined. Here we show that the histone methyltransferase DOT1L, which marks the nucleosome core on active genes, safeguards normal differentiation of CD8+ T cells. T-cell specific ablation of Dot1L resulted in loss of naive CD8+ T cells and premature differentiation towards a memory-like state, independent of antigen exposure and in a cell-intrinsic manner. Without DOT1L, the memory-like CD8+ cells fail to acquire full effector functions in vitro and in vivo. Mechanistically, DOT1L controlled T-cell differentiation and function by ensuring normal T-cell receptor density and signaling, and by maintaining epigenetic identity, in part by indirectly supporting the repression of developmentally-regulated genes. Through our study DOT1L is emerging as a central player in physiology of CD8+ T cells, acting as a barrier to prevent premature differentiation and supporting the licensing of the full effector potential of cytotoxic T cells.

immunology↗

bioRxiv2019

Histone methyltransferase DOT1L controls state-specific identity during B cell differentiation

Differentiation of naive peripheral B cells into terminally differentiated plasma cells is characterized by epigenetic alterations, yet the epigenetic mechanisms that control B cell fate remain unclear. Here we identified a central role for the histone H3K79 methyltransferase DOT1L in controlling B cell differentiation. Murine B cells lacking Dot1L failed to establish germinal centers (GC) and normal humoral immune responses in vivo. In vitro, activated B cells showed aberrant differentiation and prematurely acquired plasma cell features. Mechanistically, combined epigenomics and transcriptomics analysis revealed that DOT1L promotes expression of a pro-proliferative, pro-GC program. In addition, DOT1L supports the repression of an anti-proliferative, plasma cell differentiation program by maintaining expression of the H3K27 methyltransferase Ezh2, the catalytic component of Polycomb Repressor Complex 2 (PRC2). Our findings show that DOT1L is a central modulator of the core transcriptional and epigenetic landscape in B cells, establishing an epigenetic barrier that warrants B cell naivety and GC B cell differentiation.

immunology↗

bioRxiv2019

WAPL maintains dynamic cohesin to preserve lineage specific distal gene regulation

The cohesin complex plays essential roles in sister chromatin cohesin, chromosome organization and gene expression. The role of cohesin in gene regulation is incompletely understood. Here, we report that the cohesin release factor WAPL is crucial for maintaining a pool of dynamic cohesin bound to regions that are associated with lineage specific genes in mouse embryonic stem cells. These regulatory regions are enriched for active enhancer marks and transcription factor binding sites, but largely devoid of CTCF binding sites. Stabilization of cohesin, which leads to a loss of dynamic cohesin from these regions, does not affect transcription factor binding or active enhancer marks, but does result in changes in promoter-enhancer interactions and downregulation of genes. Acute cohesin depletion can phenocopy the effect of WAPL depletion, showing that cohesin plays a crucial role in maintaining expression of lineage specific genes. The binding of dynamic cohesin to chromatin is dependent on the pluripotency transcription factor OCT4, but not NANOG. Finally, dynamic cohesin binding sites are also found in differentiated cells, suggesting that they represent a general regulatory principle. We propose that cohesin dynamically binding to regulatory sites creates a favorable spatial environment in which promoters and enhancers can communicate to ensure proper gene expression.\n\nHIGHLIGHTSO_LIThe cohesin release factor WAPL is crucial for maintaining a pluripotency-specific phenotype.\nC_LIO_LIDynamic cohesin is enriched at lineage specific loci and overlaps with binding sites of pluripotency transcription factors.\nC_LIO_LIExpression of lineage specific genes is maintained by dynamic cohesin binding through the formation of promoter-enhancer associated self-interaction domains.\nC_LIO_LICTCF-independent cohesin binding to chromatin is controlled by the pioneer factor OCT4.\nC_LI

genomics↗