Biology subjects

Leemans, C.

Publications and source records attributed to Leemans, C..

10 recordsLinked to original sources

bioRxiv2025

The epigenetic reader MORC3 is required for T cell development in the thymus

Cell fate decisions are orchestrated by an interplay of epigenetic factors in collaboration with transcriptional activators and repressors. The epigenetic reader MORC3 is known as a silencer of endogenous retroviruses in mouse embryonic stem cells. Here, we show that mouse MORC3 is expressed in the thymus and reveal that MORC3 loss of function leads to a severe arrest in T cell development at the DN1 stage, accompanied by an expansion of natural killer and myeloid cells. MORC3 function in thymus depends on its ATPase and its ability to bind H3K4me via the CW-type zinc finger domain. We find altered chromatin accessibility at regulatory elements of key T cell transcription factors in DN1 cells and show that re-expressing TCF1 in MORC3-deficient progenitor cells, rescues T cell development. Our work indicates a novel function for MORC3 in modulating regulatory sites essential for T cell lineage commitment at the level of chromatin.

genetics↗

bioRxiv2024

Functional maps of a genomic locus reveal confinement of an enhancer by its target gene.

Genes are often activated by enhancers located at large genomic distances. The importance of this positioning is poorly understood. By relocating promoter-reporter constructs into >1,000 alternative positions within a single locus, we dissected the positional relationship between the mouse Sox2 gene and its distal enhancer. This revealed an intricate, sharply confined activation landscape, in which the native Sox2 gene occupies an optimal position for its activation. Deletion of the gene relaxes this confinement and broadly increases reporter activity. Surprisingly, the confining effect of the Sox2 gene is partially conferred by its [~]1 kb coding region. Our local relocation approach provides high-resolution functional maps of a genomic locus and reveals that a gene can strongly constrain the realm of influence of its enhancer.

molecular biology↗

bioRxiv2024

Chromatin protein complexes involved in gene repression in lamina-associated domains

Lamina-associated domains (LADs) are large chromatin regions that are associated with the nuclear lamina (NL) and form a repressive environment for transcription. The molecular players that mediate gene repression in LADs are currently unknown. Here we performed FACS-based whole-genome genetic screens in human cells using LAD-integrated fluorescent reporters to identify such regulators. Surprisingly, the screen identified very few NL proteins, but revealed roles for dozens of known chromatin regulators. Among these are the negative elongation factor (NELF) complex and interacting factors involved in RNA polymerase pausing, suggesting that regulation of transcription elongation is a mechanism to repress transcription in LADs. Furthermore, the chromatin remodeler complex BAF and the activation complex Mediator can work both as activators and repressors in LADs, depending on the local context and possibly rewiring of heterochromatin. Our data clearly emphasize that the fundamental regulatory steps of the transcription process and chromatin remodeling factors, rather than interaction with NL proteins, play a major role in the regulation of transcription within LADs. HIGHLIGHTSO_LIHaploid genetic screens identify proteins that control gene activity in LADs C_LIO_LIChromatin proteins rather than NL proteins control repression in LADs C_LIO_LIRegulators of elongation contribute to repression of transcription in LADs C_LIO_LIBAF and Mediator can both repress and activate transcription in LADs C_LI

molecular biology↗

bioRxiv2024

Predicting the DNA binding specificity of mutated transcription factors using family-level biophysically interpretable machine learning

Sequence-specific interactions of transcription factors (TFs) with genomic DNA underlie many cellular processes. High-throughput in vitro binding assays coupled with machine learning have made it possible to accurately define such molecular recognition in a biophysically interpretable way for hundreds of TFs across many structural families, providing new avenues for predicting how the sequence preference of a TF is impacted by disease-associated mutations in its DNA binding domain. We developed a method based on a reference-free tetrahedral representation of variation in base preference within a given structural family that can be used to accurately predict the effect of mutations in the protein sequence of the TF. Using the basic helix-loop-helix (bHLH) and homeodomain families as test cases, our results demonstrate the feasibility of accurately predicting the shifts ({Delta}{Delta}{Delta}G/RT) in binding free energy associated with TF mutants by leveraging high-quality DNA binding models for sets of homologous wild-type TFs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/577115v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@3a6674org.highwire.dtl.DTLVardef@1c4de4org.highwire.dtl.DTLVardef@3dcd6forg.highwire.dtl.DTLVardef@17a63c5_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

genomics↗

bioRxiv2024

Genome - nuclear lamina interactions are multivalent and cooperative

Lamina-associated domains (LADs) are megabase-sized genomic regions that interact with the nuclear lamina (NL). It is not yet understood how their interactions with the NL are encoded in their DNA. Here, we designed an efficient LAD "scrambling" approach, based on transposon-mediated local hopping of loxP recombination sites, to generate series of large deletions and inversions that span LADs and flanking sequences. Mapping of NL interactions in these rearrangements revealed that a single LAD contacts the NL through multiple regions that act cooperatively or redundantly; some have more affinity for the NL than others and can pull neighbouring sequences to the NL. Genes drawn towards the NL showed often, but not always, reduced expression and increased H3K9me3 levels. Furthermore, neighbouring LADs can cooperatively interact with the NL when placed close enough to each other. These results elucidate principles that govern the positioning of megabase-sized genomic regions inside the cell nucleus. HighlightsO_LIEfficient generation of LAD rearrangements by transposon hopping of loxP elements. C_LIO_LINL interactions are multivalent, with some subregions being more potent tethers than others. C_LIO_LILADs can cooperate to promote their association to the NL. C_LIO_LIChanges in NL association are often accompanied by changes in gene activity and H3K9me3. C_LI

molecular biology↗

bioRxiv2022

Widespread chromatin context-dependencies ofDNA double-strand break repair proteins

DNA double-strand breaks are repaired by multiple pathways, including non-homologous end-joining (NHEJ) and microhomology-mediated end-joining (MMEJ). The balance of these pathways is dependent on the local chromatin context, but the underlying mechanisms are poorly understood. By combining knockout screening with a dual MMEJ:NHEJ reporter inserted in 19 different chromatin environments, we identified dozens of DNA repair proteins that modulate pathway balance dependent on the local chromatin state. Proteins that favor NHEJ mostly synergize with euchromatin, while proteins that favor MMEJ generally synergize with distinct types of heterochromatin. BRCA2 is an example of the former, which is corroborated by chromatin-dependent shifts in mutation patterns of BRCA2-/- cancer genomes. These results uncover a complex network of proteins that regulate MMEJ:NHEJ balance in a chromatin context-dependent manner. ONE SENTENCE SUMMARYA multiplexed screen reveals how dozens of proteins sense the local chromatin context to tune the balance between two DNA repair pathways.

molecular biology↗

bioRxiv2020

Impact of chromatin context on Cas9-induced DNA double-strand break repair pathway balance

DNA double-strand break (DSB) repair is mediated by multiple pathways, including classical non-homologous end-joining pathway (NHEJ) and several homology-driven repair pathways. This is particularly important for Cas9-mediated genome editing, where the outcome critically depends on the pathway that repairs the break. It is thought that the local chromatin context affects the pathway choice, but the underlying principles are poorly understood. Using a newly developed multiplexed reporter assay in combination with Cas9 cutting, we systematically measured the relative activities of three DSB repair pathways as function of chromatin context in >1,000 genomic locations. This revealed that NHEJ is broadly biased towards euchromatin, while microhomology-mediated end-joining (MMEJ) is more efficient in specific heterochromatin contexts. In H3K27me3-marked heterochromatin, inhibition of the H3K27 methyltransferase EZH2 shifts the balance towards NHEJ. Single-strand templated repair (SSTR), often used for precise CRISPR editing, competes with MMEJ, and this competition is weakly associated with chromatin context. These results provide insight into the impact of chromatin on DSB repair pathway balance, and guidance for the design of Cas9-mediated genome editing experiments.

molecular biology↗

bioRxiv2019

Local rewiring of genome - nuclear lamina interactions by transcription.

Transcriptionally inactive genes are often positioned at the nuclear lamina (NL), as part of large lamina-associated domains (LADs). Activation of such genes is often accompanied by repositioning towards the nuclear interior. How this process works and how it impacts flanking chromosomal regions is poorly understood. We addressed these questions by systematic manipulation of gene activity and detailed analysis of NL interactions. Activation of genes inside LADs typically causes detachment of the entire transcription unit but rarely more than 50-100 kb of flanking DNA, even when multiple neighboring genes are activated. The degree of detachment depends on the expression level and the length of the activated gene. Loss of NL interactions coincides with a switch from late to early replication timing, but the latter can involve longer stretches of DNA. These findings show how NL interactions can be shaped locally by transcription and point to a remarkable flexibility of interphase chromosomes.

genomics↗

bioRxiv2018

Promoter-intrinsic and local chromatin features determine gene repression in lamina-associated domains

It is largely unclear whether genes that are naturally embedded in lamina associated domains (LADs) are inactive due to their chromatin environment, or whether LADs are merely secondary to the lack of transcription. We show that hundreds of human promoters become active when moved from their native LAD position to a neutral context in the same cells, indicating that LADs form a repressive environment. Another set of promoters inside LADs is able to \"escape\" repression, although their transcription elongation is attenuated. By inserting reporters into thousands of genomic locations, we demonstrate that these escaper promoters are intrinsically less sensitive to LAD repression. This is not simply explained by promoter strength, but by the interplay between promoter sequence and local chromatin features that vary strongly across LADs. Enhancers also differ in their sensitivity to LAD chromatin. This work provides a general framework for the systematic understanding of gene regulation by repressive chromatin.\n\nHighlightsO_LITwo promoter transplantation strategies elucidate the regulatory role of LAD chromatin\nC_LIO_LILADs are generally repressive, but also highly heterogeneous\nC_LIO_LILADs can impede both promoter activity and transcription elongation\nC_LIO_LIPromoters vary intrinsically in their sensitivity to LAD repression\nC_LI

genomics↗

bioRxiv2017

Easy quantification of template-directed CRISPR/Cas9 editing

Template-directed CRISPR/Cas9 editing is a powerful tool for introducing subtle mutations in genomes. However, the success rate of incorporation of the desired mutations at the target site is difficult to predict and therefore must be empirically determined. Here, we adapted the widely used TIDE method for quantification of templated editing events, including point mutations. The resulting TIDER method is a rapid, cheap and accessible tool for testing and optimization of template-directed genome editing strategies.

genetics↗