Biology subjects

Lhoumaud, P.

Publications and source records attributed to Lhoumaud, P..

7 recordsLinked to original sources

bioRxiv2026

Dual profiling of DNA modifications with enhancer features during the exit of naive pluripotency

Cis-regulatory elements, such as enhancers, play an essential role in coordinating gene expression programs during cellular transitions. As such, substantial efforts have been made to characterize enhancer elements, e.g., chromatin accessibility, transcription factor (TF) binding sites, histone post-translational modifications (PTMs), and 5-cytosine DNA methylation and hydroxymethylation (5mC and 5hmC). Elevated 5mC levels are typically correlated with the inactive enhancer state. However, whether 5mC precludes TF binding or is simply a downstream consequence of enhancer decommissioning is difficult to determine. Bulk genomics assays from cell populations fail to fully capture cellular heterogeneity, and single-cell assays often suffer from limited read counts per cell. Here, we leveraged tagmentation-based technologies to assess chromatin features with 5mC simultaneously: Methyl-ATAC and Methyl-CUT&Tag. In addition, we modified the technique to interrogate 5hmC dynamics (hM-ATAC). We employed these techniques during the exit of naive pluripotency in mouse embryonic stem cells (mESCs), which recapitulates the embryonic DNA methylation establishment program. This system withstands the complete absence of DNA methylation and demethylation machinery, allowing us to further dissect the temporal contributions of 5mC and 5hmC. Given the affordability of these techniques, we were able to obtain robust, dynamic single-molecule 5mC/5hmC information during this cellular transition for chromatin accessibility and histone marks in wild-type and mutant conditions. The sum of these data allowed for unprecedented insight into the role that 5mC turnover plays at enhancers during a key window of mammalian embryonic development.

Molecular Biology↗

bioRxiv2025

A developmentally regulated long-range enhancer-promoter contact mediates human neural development

SOX2 is a core pluripotency factor in human embryonic stem cells (hESCs), but upon differentiation to the three germ layers, its expression is preserved selectively in neuroectoderm. The mechanisms regulating SOX2 transcription in distinct developmental stages remain incompletely understood. Here, we demonstrate that a distant enhancer 550 kb from the human SOX2 locus is selectively activated in neural stem cells (NSCs) and establishes long-range contact with the SOX2 gene. CRISPR-Cas9 excision of the enhancer has no effect in hESCs but reduces SOX2 transcription in NSCs and impairs neuroectodermal differentiation and forebrain specification in teratomas and cerebral organoids. CRISPR excision of a CTCF recognition motif adjacent to the enhancer does not affect enhancer activation in neuroectoderm but reduces chromatin looping and SOX2 transcription to partially reproduce phenotypes seen with enhancer deletion. Our findings indicate that the development of the human nervous system depends on a developmentally regulated long-range contact between a distant enhancer and the SOX2 locus.

developmental biology↗

bioRxiv2024

Brain and cancer associated binding domain mutations provide insight into CTCF's relationship with chromatin and its ability to act as a chromatin organizer

Here we used a series of CTCF mutations to explore CTCFs relationship with chromatin and its contribution to gene regulation. CTCFs impact depends on the genomic context of bound sites and the unique binding properties of WT and mutant CTCF proteins. Specifically, CTCFs signal strength is linked to changes in accessibility, and the ability to block cohesin is linked to its binding stability. Multivariate modelling reveals that both CTCF and accessibility contribute independently to cohesin binding and insulation, however CTCF signal strength has a stronger effect. CTCF and chromatin have a bidirectional relationship such that at CTCF sites, accessibility is reduced in a cohesin-dependent, mutant specific fashion. In addition, each mutant alters TF binding and accessibility in an indirect manner, changes which impart the most influence on rewiring transcriptional networks and the cells ability to differentiate. Collectively, the mutant perturbations provide a rich resource for determining CTCFs site-specific effects.

molecular biology↗

bioRxiv2023

Crosstalk between chromatin and the transcription factor Shavenbaby defines transcriptional output along the Drosophila intestinal stem cell lineage.

The transcription factor Shavenbaby (Svb), the only member of the OvoL family in Drosophila, controls intestinal stem cell differentiation. Post-translational modification of Svb produces two protein isoforms, Svb-ACT and Svb-REP, which promote intestinal stem cell renewal or differentiation, respectively. Using engineered cell lines, we express either isoform to define their mode of action, and develop an unbiased method to identify Svb target genes in intestinal cells. Within a given cell type, Svb-ACT and Svb-REP antagonistically regulate the expression of a set of target genes, binding specific enhancers whose accessibility is constrained by. During intestinal differentiation, the set of target genes progressively changes, together with chromatin accessibility. Moreover, Svb-REP binding stabilizes three-dimensional enhancer-promoter loops, while influencing the local chromatin landscape to repress target genes. We propose that SvbACT-to-REP switch promotes enterocyte differentiation of intestinal stem cells through direct gene regulation and chromatin remodeling.

genomics↗

bioRxiv2019

A B-cell acute lymphoblastic leukemia regulatory network defines novel therapeutic targets in IGH-CRLF2 patients

Although genetic alterations are initial drivers of disease, aberrantly activated transcriptional regulatory programs are often responsible for the maintenance and progression of cancer. CRLF2-overexpression in B-ALL patients leads to activation of JAK-STAT, PI3K and ERK/MAPK signaling pathways and is associated with poor outcome. Although inhibitors of these pathways are available, there remains the issue of treatment-associated toxicities, thus it is important to identify new therapeutic targets. Using a network inference approach, we reconstructed a B-ALL specific transcriptional regulatory network to evaluate the impact of CRLF2-overexpression on downstream regulatory interactions. Comparing RNA-seq from CRLF2-High and other B-ALL patients (CRLF2-Low), we defined a CRLF2-High gene signature. Patient-specific chromatin accessibility was interrogated to identify altered putative regulatory elements that could be linked to transcriptional changes. To delineate these regulatory interactions, a B-ALL cancer-specific regulatory network was inferred using 868 B-ALL patient samples from the NCI TARGET database coupled with priors generated from ATAC-seq peak TF-motif analysis. CRISPRi, siRNA knockdown and ChIP-seq of nine TFs involved in the inferred network were analyzed to validate predicted TF-gene regulatory interactions. In this study, a B-ALL specific regulatory network was constructed using ATAC-seq derived priors. Inferred interactions were used to identify differential patient-specific transcription factor activities predicted to control CRLF2-High deregulated genes, thereby enabling identification of new potential therapeutic targets.

genomics↗

bioRxiv2019

NSD2 overexpression drives clustered chromatin and transcriptional changes in insulated domains

CTCF and cohesin play a key role in organizing chromatin into TAD structures. Disruption of a single CTCF binding site is sufficient to change chromosomal interactions leading to alterations in chromatin modifications and gene regulation. However, the extent to which alterations in chromatin modifications can disrupt 3D chromosome organization leading to transcriptional changes is unknown. In multiple myeloma a 4;14 translocation induces overexpression of the histone methyltransferase, NSD2 resulting in expansion of H3K36me2 and shrinkage of antagonistic H3K27me3 domains. Using isogenic cell lines producing high and low levels of NSD2, we find oncogene activation is linked to alterations in H3K27ac and CTCF within H3K36me2 enriched chromatin. A linear regression model reveals that changes in both CTCF and/or H3K27ac significantly increase the probability that a gene sharing the same insulated domain will be differentially expressed. These results identify a bidirectional relationship between 2D chromatin and 3D genome organization in gene regulation.

genomics↗

bioRxiv2019

EpiMethylTag simultaneously detects ATAC-seq or ChIP-seq signals with DNA methylation

Activation of regulatory elements is thought to be inversely correlated with DNA methylation levels. However, it is difficult to determine whether DNA methylation is compatible with chromatin accessibility or transcription factor (TF) binding if assays are performed separately. We developed a low input, low sequencing depth method, EpiMethylTag that combines ATAC-seq or ChIP-seq (M-ATAC or M-ChIP) with bisulfite conversion, to simultaneously examine accessibility/TF binding and methylation on the same DNA.

genomics↗