Biology subjects

van Welsem, T.

Publications and source records attributed to van Welsem, T..

7 recordsLinked to original sources

bioRxiv2025

Recruitment of Fpt1 to tRNA genes requires TFIIIB and the N-terminal TPR array of TFIIIC subunit {tau}131

Transfer-RNA genes (tDNAs) in budding yeast recruit varying amounts of Fpt1, a regulator of RNA polymerase III (RNAPIII) occupancy. Fpt1 occupancy resembles that of the general transcription factor TFIIIC but how Fpt1 is recruited to tDNAs remains unclear. Here we show that both TFIIIB and TFIIIC are required for Fpt1 binding under active as well as repressive RNAPIII conditions. Depletion of TFIIIB reduced Fpt1 occupancy without affecting TFIIIC. In contrast, TFIIIC depletion led to reduced Fpt1 and a gene-specific reduction in TFIIIB occupancy. Moreover, upon depletion of TFIIIC, Fpt1 and TFIIIB were lost to different extents. We identified the C-terminal intrinsically disordered region of Fpt1 as critical for increased Fpt1 binding under repressive conditions. Within this region, a short -helix was predicted to interact with the N-terminal tetratricopeptide repeat array of the TFIIIC subunit {tau}131, a region also known to interact with TFIIIB. Deletion of this -helix abrogated stress-induced Fpt1 recruitment to tDNAs, as did mutations in the predicted interaction surface of {tau}131, while having a milder effect on TFIIIB occupancy. Together, these findings uncovered a dual and dynamic mechanism of Fpt1 recruitment to tDNAs with independent contributions of TFIIIB and TFIIIC.

molecular biology↗

bioRxiv2025

Histone methyltransferase DOT1L differentially affects the development of dendritic cell subsets

Dendritic cells (DCs) are important orchestrators of immune responses. Their development in the bone marrow is controlled by transcription factors, but epigenetic mechanisms remain poorly understood. DOT1L is emerging as a key epigenetic regulator in immune cells. By mapping DOT1L-mediated histone H3K79 methylation in canonical DC subsets, we observed that DOT1L modified common as well as DC subset-specific genes. In vitro- or in vivo-induced deletion of Dot1l followed by in vitro cell culture resulted in a decrease in myeloid progenitors and plasmacytoid DCs (pDCs) and an increase in cDC2s, while cDC1s remained unchanged. In vitro generated Dot1l-KO DCs were unable to produce IFN upon stimulation. Moreover, transcriptomes of Dot1l-KO DC subsets exhibited enrichment of antigen presentation pathways and MHC class II surface levels were upregulated in pDCs. Mechanistically, inhibition of DOT1L linked the observed effects to its methyltransferase activity. Together, our data indicate that in DCs DOT1L differentially affects the development of canonical subsets and suppresses antigen presentation pathways.

cell biology↗

bioRxiv2025

Histone methyltransferase DOT1L maintains identity and restricts cytotoxic potential of CD8 T cells

The histone methyltransferase DOT1L is emerging as a central epigenetic regulator in immune cells. Loss of DOT1L during development of CD8 T cells in vivo leads to gain of memory-features but has also been reported to compromise CD8 T cell viability and activity. Here, we determined the cell-intrinsic role of DOT1L in mature mouse CD8 T cells. After conditional deletion of Dot1L in vitro, CD8 T cells retained in vivo proliferative capacity and anti-tumor reactivity. Moreover, Dot1L knock-out CD8 T cells showed increased antigen-specific cytotoxicity towards tumor cells in vitro. Mechanistically, loss of DOT1L resulted in an altered cell-identity program with loss of T-cell and gain of NK-cell features. These transcriptional changes were mediated by loss of DOT1L methyltransferase activity in a dose-dependent manner. Our findings show that ablation of DOT1L activity in mature CD8 T cells is well-tolerated and rewires their cell identity towards the NK-cell lineage, concomitantly enhancing intrinsic cytotoxic capacity. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/633937v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1641c55org.highwire.dtl.DTLVardef@1803510org.highwire.dtl.DTLVardef@1bedceorg.highwire.dtl.DTLVardef@199a6d5_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDifferentiation and identity of cytotoxic T cells depend on the methyltransferase DOT1L C_LIO_LIDOT1L intrinsically limits the cytotoxic activity of mature CD8 T cells C_LIO_LIDOT1L prohibits the acquisition of NK cell features in CD8 T cells C_LIO_LIDOT1L maintains CD8 T cell identity through its catalytic activity in a dose-dependent manner C_LI

immunology↗

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↗

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↗

bioRxiv2017

Decoding the chromatin proteome of a single genomic locus by DNA sequencing

Transcription, replication and repair involve interactions of specific genomic loci with many different proteins. How these interactions are orchestrated at any given location and under changing cellular conditions is largely unknown because systematically measuring protein-DNA interactions at a specific locus in the genome is challenging. To address this problem, we developed Epi-Decoder, a Tag-ChIP-Barcode-Seq technology in budding yeast to identify and quantify in an unbiased and systematic manner the proteome of an individual genomic locus. Epi-Decoder is orthogonal to proteomics approaches because it does not rely on mass spectrometry but instead takes advantage of DNA sequencing. Analysis of the proteome of a transcribed locus proximal to an origin of replication revealed more than 400 proteins. Moreover, replication stress induced changes in local chromatin-proteome composition prior to local origin firing, affecting replication proteins as well as transcription proteins. Epi-Decoder will enable the delineation of complex and dynamic protein-DNA interactions across many regions of the genome.

genomics↗