Biology subjects

Smallwood, S. A.

Publications and source records attributed to Smallwood, S. A..

6 recordsLinked to original sources

bioRxiv2024

A comprehensive Schizosaccharomyces pombe atlas of physical transcription factor interactions with proteins and chromatin

Transcription factors (TFs) are key regulators of gene expression, yet many of their targets and modes of action remain unknown. In Schizosaccharomyces pombe, one-third of TFs are solely homology-predicted, with few experimentally validated. We created a comprehensive library of 89 endogenously tagged S. pombe TFs, mapping their protein and chromatin interactions using immunoprecipitation-mass spectrometry and chromatin immunoprecipitation sequencing. Our study identified protein interactors for half the TFs, with over a quarter potentially forming stable complexes. We discovered DNA binding sites for most TFs across 2,027 unique genomic regions, revealing motifs for 38 TFs and uncovering a complex regulatory network of extensive TF cross- and autoregulation. Characterization of the largest TF family revealed conserved DNA sequence preferences but diverse binding patterns, and identified a repressive heterodimer, Ntu1/Ntu2, linked to perinuclear gene localization. Our TFexplorer webtool makes all data interactively accessible, offering new insights into TF interactions and regulatory mechanisms with broad biological relevance. HIGHLIGHTSO_LIComprehensive strain library of endogenously tagged S. pombe TFs C_LIO_LIExperimentally determined atlas of TF interactions with proteins and chromatin C_LIO_LITFexplorer web application for interactive exploration of TF interactomes C_LIO_LIIdentification of repressive Nattou complex linked to perinuclear gene localization C_LI

molecular biology↗

bioRxiv2024

Preventing CpG island hypermethylation in oocytes safeguards mouse development

In mammalian somatic and male germline cells, genomes are extensively DNA methylated (DNAme). In oocytes, however, DNAme is largely limited to transcribed regions only. Regulatory CpG-island (CGI) sequences are also devoid of repressive DNAme in somatic and germ cells of both sexes. The mechanisms restricting de novo DNAme acquisition in developing oocytes, at CGIs and globally, and the relevance thereof for regulating zygotic gene expression and embryo development after fertilization are largely unknown. Here we show that the histone H3 lysine 36 dimethyl (H3K36me2) demethylases KDM2A and KDM2B prevent genome-wide accumulation of H3K36me2, thereby impeding global DNMT3A-catalyzed de novo DNAme, including at CGI gene promoters. By recruiting variant Polycomb Repressive Complex 1 (vPRC1), they further control H2A mono-ubiquitin deposition and vPRC1-dependent gene repression. Through genetic perturbations, we demonstrate that aberrant Dnmt3a-dependent DNAme established in Kdm2a/Kdm2b double mutant oocytes represses transcription from maternal loci in two-cell embryos. The lethality of Kdm2a/Kdm2b maternally deficient pre-implantation embryos is suppressed by Dnmt3a deficiency during oogenesis. Hence, KDM2A/KDM2B are essential for confining the oocyte DNA methylome, conferring competence for early embryonic development. Our research implies that the reprogramming capacity eminent to early embryos is insufficient to erase aberrant DNAme from maternal chromatin, and that early development is vulnerable to gene dosage haplo-insufficiency effects. HIGHLIGHTSDemethylation of H3K36me2 by KDM2A and KDM2B prevents aberrant de novo DNA methylation in mouse oocytes. Sequence composition and H3K4me3 modulate the probability for aberrant H3K36me2 and DNA methylation at CpG islands. Aberrant oocyte DNA methylation is not reprogrammed in early embryos and suppresses maternal gene transcription. Aberrant oocyte DNA methylation causes embryonic lethality during pre-implantation development. GRAPHICAL SUMMARY O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/595726v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@198608org.highwire.dtl.DTLVardef@1aea04dorg.highwire.dtl.DTLVardef@19e3dd7org.highwire.dtl.DTLVardef@1ee11f6_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2024

DNA methylation modulates nucleosome retention in sperm and H3K4 methylation deposition in early mouse embryos.

DNA methylation (DNAme) serves a stable gene regulatory function in somatic cells (1). In the germ line and during early embryogenesis, however, DNAme undergoes global erasure and re-establishment to support germ cell and embryonic development (2). While de novo DNAme acquisition during male germ cell development is essential for setting genomic DNA methylation imprints, other intergenerational roles for paternal DNAme in defining embryonic chromatin after fertilization are unknown. To approach this question, we reduced levels of DNAme in developing male germ cells through conditional gene deletion of the de novo DNA methyltransferases DNMT3A and DNMT3B in undifferentiated spermatogonia. We observed that DNMT3A serves a DNAme maintenance function in undifferentiated spermatogonia while DNMT3B catalyzes de novo DNAme during spermatogonial differentiation. Mutant male germ cells nevertheless completed their differentiation to sperm. Failing de novo DNAme in Dnmt3a/Dnmt3b double deficient spermatogonia is associated with increased nucleosome occupancy in mature sperm, preferentially at sites with higher CpG content, supporting the model that DNAme modulates nucleosome retention in sperm (3). To assess the impact of altered sperm chromatin in the formation of embryonic chromatin, we measured H3K4me3 occupancy at paternal and maternal alleles in 2-cell embryos using a newly developed transposon-based tagging assay for modified chromatin. Our data show that reduced DNAme in sperm renders paternal alleles permissive for H3K4me3 establishment in early embryos, independently of possible paternal inheritance of sperm born H3K4me3. Together, this study provides first evidence that paternally inherited DNAme directs chromatin formation during early embryonic development.

genetics↗

bioRxiv2023

Dynamics and plasticity of stem cells in the regenerating human colonic epithelium

The human intestinal epithelium is a tissue with rapid turnover. Its complex regenerative process and differentiation trajectories have been challenging to study due to its inaccessibility and lack of temporal sampling. To this end, we developed a workflow to culture adult stem cell-derived human intestinal organoids from single cells to maturation. Extensive characterization of our model system indicated a transient regenerative response, followed by differentiation into all mature cell lineages. This switch is accompanied by a transition between two stem cell states. High-content screening and comparison to in vivo studies revealed that an initial fetal-like state is crucial for achieving successful regeneration, while the subsequent adult-like state is vital for maintaining a balance of cell lineages and continuous support of crypt-morphogenesis. Taken together, this study highlights the extensive plasticity of the intestinal epithelium and paves the way for further studies of human intestinal regeneration and its deregulation in pathologies.

cell biology↗

bioRxiv2020

A BTB-DOMAIN TRANSCRIPTION FACTOR RECRUITS CHROMATIN REMODELERS AND A HISTONE CHAPERONE DURING THE EXIT FROM PLURIPOTENCY

Transcription factors (TFs) harboring a btb (Broad-Complex, Tramtrack and Bric a brac) domain play important roles in development and disease. They are thought to recruit transcriptional modulators to DNA through their btb domain. However, a systematic molecular understanding of this TF family is lacking. Here, we identify the zinc finger btb-TF Zbtb2 in a genetic screen for regulators of exit from pluripotency and dissect its mechanistic mode of action. We show that ZBTB2 binds the chromatin remodeler Ep400 to mediate downstream transcription. Independently, the btb domain directly interacts with the chromatin remodeller NuRD and the histone chaperone HiRA via the GATAD2A/B and UBN2 subunits, respectively. NuRD recruitment is a common feature of btb-TFs and we propose by phylogenetic analysis that this is an evolutionary ancient property. Binding to UBN2, in contrast, is specific to ZBTB2 and requires a C-terminal extension of the btb domain. This study therefore identifies a btb-domain TF that recruits chromatin modifiers and a histone chaperone during a developmental cell state transition, and defines unique and shared molecular functions of the btb-domain TF family.

molecular biology↗

bioRxiv2018

Genome-scale oscillations in DNA methylation during exit from pluripotency

Pluripotency is accompanied by the erasure of parental epigenetic memory with naive pluripotent cells exhibiting global DNA hypomethylation both in vitro and in vivo. Exit from pluripotency and priming for differentiation into somatic lineages is associated with genome-wide de novo DNA methylation. We show that during this phase, coexpression of enzymes required for DNA methylation turnover, DNMT3s and TETs, promotes cell-to-cell variability in this epigenetic mark. Using a combination of single-cell sequencing and quantitative biophysical modelling, we show that this variability is associated with coherent, genome-scale, oscillations in DNA methylation with an amplitude dependent on CpG density. Analysis of parallel single-cell transcriptional and epigenetic profiling provides evidence for oscillatory dynamics both in vitro and in vivo. These observations provide fresh insights into the emergence of epigenetic heterogeneity during early embryo development, indicating that dynamic changes in DNA methylation might influence early cell fate decisions.\n\nHighlightsO_LICo-expression of DNMT3s and TETs drive genome-scale oscillations of DNA methylation\nC_LIO_LIOscillation amplitude is greatest at a CpG density characteristic of enhancers\nC_LIO_LICell synchronisation reveals oscillation period and link with primary transcripts\nC_LIO_LIMultiomic single-cell profiling provides evidence for oscillatory dynamics in vivo\nC_LI

genomics↗