Biology subjects

Pantier, R.

Publications and source records attributed to Pantier, R..

9 recordsLinked to original sources

bioRxiv2026

The DNA demethylase TET1 recognises chromatin via nucleosome engagement of its N-terminal region

TET1 is a DNA demethylase critical for mammalian embryonic development, yet how it is recruited to its chromatin substrate remains poorly understood. Here we identify a previously uncharacterized N-terminal region of TET1 that directly binds nucleosomes and promotes chromatin binding in mouse embryonic stem cells. Robust chromatin association was evidenced by fluorescence recovery after photobleaching during interphase and chromosome retention throughout mitosis. A 2.47 angstroms cryo-EM structure of the TET1-nucleosome complex reveals recognition of the nucleosome acidic patch through a canonical arginine anchor centred on R75, with additional contacts extending along the H2B surface. Biochemical and cellular analyses validate this interaction and demonstrate its contribution to TET1 chromatin engagement. The region is absent from TET2 and TET3 and displays strong conservation within mammals, indicating that direct nucleosome recognition is a specialized feature of TET1. Together, these findings uncover a previously unknown chromatin-binding module in TET1 and establish a structural basis for its interaction with nucleosomes.

molecular biology↗

bioRxiv2025

Pervasive binding of the stem cell transcription factor SALL4 shapes the chromatin landscape

Mechanistic understanding of how gene activity is regulated has focussed on the roles of transcription factors at promoters and enhancers, whereas mechanisms capable of globally fine-tuning gene expression through dispersed binding across large genomic regions have received less attention. Here we provide evidence that the essential stem cell transcription factor SALL4 modulates gene expression according to DNA base composition by reading the frequency of its AT-rich target motifs. Using an acute depletion strategy, we establish that SALL4-repressed genes localise to AT-rich genomic domains with high levels of dispersed SALL4 occupancy. While SALL4 is localised within peaks and distributed broadly across the genome, explainable machine learning revealed that its occupancy across the gene body is a strong predictor of transcriptional output. We observed rapid increases in chromatin accessibility and histone acetylation independent of transcriptional activity, suggesting that SALL4 primarily acts upon chromatin, while transcriptional changes are secondary. Accordingly, preventing SALL4 from recruiting the histone deacetylase and nucleosome remodelling corepressor NuRD mimicked a Sall4-null phenotype in stem cells and animal models. Our findings reveal that SALL4s interpretation of DNA sequence optimises the global epigenome and transcriptome, a process integral to maintaining the stem cell gene expression programme.

genomics↗

bioRxiv2025

Tetramerisation governs SALL transcription factor function in development and disease

Spalt-like (SALL) proteins are C2H2 zinc-finger transcription factors important for embryogenesis, with mutations in SALL1 and SALL4 causing rare congenital disorders Townes-Brocks and Okihiro syndromes, respectively. While SALL proteins are known to associate with one another, the biological significance of the resulting complexes is unknown. Here we define a conserved glutamine-rich region that mediates SALL1/4 homo- and heterotetramerisation and find that complex formation is indispensable for DNA binding. Modelling a patient mutation that abolishes SALL4 multimerisation led to gene misregulation and, in mice, embryonic lethality, therefore phenocopying a complete Sall4 knockout. Furthermore, a common disease-causing SALL1 truncation, which retains multimerisation but lacks DNA-binding domains, sequesters SALL4 into heterotetramers that are defective in DNA binding, thereby providing a mechanistic explanation for the dominant-negative effects of many Townes-Brocks mutations. Together, our findings establish tetramerisation as a prerequisite for SALL function, linking complex formation to developmental gene regulation and human disease.

molecular biology↗

bioRxiv2024

TET knockout cells transit between pluripotent states and exhibit precocious germline entry

TET1, TET2 and TET3 are DNA demethylases with critical roles in development and differentiation. To assess the contributions of TET proteins to cell function during early development, single and compound knockouts of Tet genes in mouse pluripotent embryonic stem cells (ESCs) were generated. Here we show that TET proteins are not required to transit between naive, formative and primed pluripotency. Moreover, ESCs with double-knockouts of Tet1 and Tet2 or triple-knockouts of Tet1, Tet2 and Tet3 are phenotypically indistinguishable. These TET-deficient ESCs exhibit differentiate defects; they fail to activate somatic gene expression and retain expression of pluripotency transcription factors. Therefore, TET1 and TET2, but not TET3 act redundantly to facilitate somatic differentiation. Importantly however, TET-deficient ESCs can differentiate into primordial germ cell-like cells (PGCLCs), and do so at high efficiency in the presence or absence of PGC-promoting cytokines. Moreover, acquisition of a PGCLC transcriptional programme occurs more rapidly in TET-deficient cells. These results establish that TET proteins act at the juncture between somatic and germline fates: without TET proteins, epiblast cell differentiation defaults to the germline.

developmental biology↗

bioRxiv2023

MeCP2 binds to methylated DNA independently of phase separation and heterochromatin organisation

Correlative evidence has suggested that DNA methylation promotes the formation of transcriptionally silent heterochromatin. Accordingly, the methyl-CpG binding domain protein MeCP2 is often portrayed as a constituent of heterochromatin. This interpretation has been reinforced by the use of mouse cells as an experimental system for studying the mammalian epigenome, as heterochromatin, DNA methylation and MeCP2 colocalise in prominent foci. The findings presented here revise this view. We show that focal localisation of MeCP2 in mice is independent of heterochromatin, as DNA methylation-dependent MeCP2 foci persist even when the signature heterochromatin histone mark H3K9me3 is absent and heterochromatin protein HP1 is diffuse. Contrary to the proposal that MeCP2 forms condensates at mouse heterochromatic foci via liquid-liquid phase transition, the short methyl-CpG binding domain, which lacks the disordered domains thought to be required for condensation, is sufficient to target foci in mouse cells. Importantly, we find that the formation of MeCP2 foci in mice is highly atypical, as they are indetectable in 14 out of 16 other mammalian species, including humans. Notably, MeCP2 foci are absent in Mus spretus which can interbreed with Mus musculus but lacks its highly methylated pericentric satellite DNA repeats. We conclude that MeCP2 has no intrinsic tendency to form nuclear condensates and its localisation is independent of heterochromatin formation. Instead, the distribution of MeCP2 in the nucleus is primarily determined by global DNA methylation patterns and is typically euchromatic.

cell biology↗

bioRxiv2022

Structural analysis of SALL4 zinc-finger domain reveals a link between AT-rich DNA binding and Okihiro syndrome

Spalt-like 4 (SALL4) maintains vertebrate embryonic stem cell identity and is required for the development of multiple organs, including limbs. Mutations in SALL4 are associated with Okihiro syndrome and SALL4 is also a known target of thalidomide. SALL4 protein has a distinct preference for AT-rich sequences, recognised by a pair of zinc fingers at the C-terminus. However, unlike many characterised zinc finger proteins, SALL4 shows flexible recognition with many different combinations of AT-rich sequences being targeted. SALL4 interacts with the NuRD corepressor complex which potentially mediates repression of AT-rich genes. We present a crystal structure of SALL4 C-terminal zinc fingers with an AT-rich DNA sequence, which shows that SALL4 uses small hydrophobic and polar side chains to provide flexible recognition in the major groove. Missense mutations reported in patients that lie within the C-terminal zinc fingers reduced overall binding to DNA but not the preference for AT-rich sequences. Furthermore, these mutations altered association of SALL4 with AT-rich genomic sites, providing evidence that these mutations are likely pathogenic.

biochemistry↗

bioRxiv2020

SALL4 controls cell fate in response to DNA base composition

SO_SCPLOWUMMARYC_SCPLOWMammalian genomes contain long domains with distinct average compositions of A/T versus G/C base pairs. In a screen for proteins that might interpret base composition by binding to AT-rich motifs, we identified the stem cell factor SALL4 which contains multiple zinc-fingers. Mutation of the domain responsible for AT binding drastically reduced SALL4 genome occupancy and prematurely up-regulated genes in proportion to their AT content. Inactivation of this single AT-binding zinc-finger cluster mimicked defects seen in Sall4-null cells, including precocious differentiation of embryonic stem cells and embryonic lethality in mice. In contrast, deletion of two other zinc-finger clusters was phenotypically neutral. Our data indicate that loss of pluripotency is triggered by down-regulation of SALL4, leading to de-repression of a set of AT-rich genes that promotes neuronal differentiation. We conclude that base composition is not merely a passive by-product of genome evolution, but constitutes a signal that aids control of cell fate. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/179481v2_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1ec4eb9org.highwire.dtl.DTLVardef@1246a36org.highwire.dtl.DTLVardef@1b9134dorg.highwire.dtl.DTLVardef@1a24904_HPS_FORMAT_FIGEXP M_FIG C_FIG

genomics↗

bioRxiv2020

TET1 interacts directly with NANOG via independent regions containing hydrophobic and aromatic residues

The DNA demethylase TET1 is highly expressed in embryonic stem cells. Knockout experiments indicate that TET1 is important for lineage commitment, and paradoxically, also for reprogramming to naïve pluripotency. TET1 binds to promoters through a CXXC domain which recognises unmethylated CpG dinucleotides. TET1 also binds to enhancers, presumably via interactions with partner proteins. The transcription factor NANOG interacts with TET1 and is predominantly localised at enhancers in ESCs. Therefore, NANOG may contribute to TET1 biological activity in pluripotent cells. However, the regions of TET1 involved in protein-protein interactions are mostly unknown. Here, we characterise the physical interaction between TET1 and NANOG using embryonic stem cells and bacterial expression systems. TET1 and NANOG interact through multiple binding sites that act independently. Critically, mutating conserved hydrophobic and aromatic residues within TET1 and NANOG abolishes the interaction. Comparative ChIP-seq analysis identifies genomic loci bound by both TET1 and NANOG, that correspond predominantly to pluripotency enhancers. Importantly, around half of NANOG transcriptional target genes are associated with TET1-NANOG co-bound sites. These results indicate a mechanism by which TET1 protein is targeted to specific sites of action at enhancers by direct interaction with a transcription factor.HighlightsNANOG and TET1 have regulatory roles in maintaining and reprogramming pluripotencyTET1 and NANOG interact via multiple independent binding regionsTET1 and NANOG interactions are mediated by aromatic and hydrophobic residuesTET1 residues that bind NANOG are highly conserved in mammalsCo-localisation of TET1 and NANOG on chromatin is enriched at NANOG target genesCompeting Interest StatementThe authors have declared no competing interest.AbbreviationsDSBHdouble stranded beta helix domainESCembryonic stem cellOGTO-linked N-acetylglucosamine transferaseTETTen-eleven-translocationWRtryptophan-repeatView Full Text

biochemistry↗

bioRxiv2017

Distinct SoxB1 networks are required for naïve and primed pluripotency

Deletion of Sox2 from embryonic stem cells (ESCs) causes trophectodermal differentiation. While this can be prevented by enforced expression of the related SOXB1 proteins, SOX1 or SOX3, the roles of SOXB1 proteins in epiblast stem cell (EpiSC) pluripotency are unknown. Here we show that Sox2 can be deleted from EpiSCs with impunity. This is due to a shift in the balance of SoxB1 expression in EpiSCs, which have decreased Sox2 and increased Sox3 compared to ESCs. Consistent with functional redundancy, Sox3 can also be deleted from EpiSCs without eliminating self-renewal. However, deletion of both Sox2 and Sox3 prevents self-renewal. The overall SOXB1 levels in ESCs affect differentiation choices: neural differentiation of Sox2 heterozygous ESCs is compromised, while increased SOXB1 levels divert the ESC to EpiSC transition towards neural differentiation. Therefore, optimal SOXB1 levels are critical for each pluripotent state and for cell fate decisions during exit from naive pluripotency.

cell biology↗