Biology subjects

Braccioli, L.

Publications and source records attributed to Braccioli, L..

6 recordsLinked to original sources

bioRxiv2026

A dual role for CTCF in development

CTCF is an essential DNA binding protein whose absence leads to embryonic lethality. CTCF is primarily known for its role in 3D genome organization where its N-terminal domain interacts with cohesin to anchor chromatin loops. How CTCF facilitates proper embryonic development remains unclear, necessitating temporal control to resolve its stage-specific functions. By combining gastruloids, an in vitro model of embryonic development, with a degron system to rapidly deplete CTCF at defined timepoints, we show that early CTCF depletion impairs early gastruloid morphogenesis. Surprisingly, ATAC-seq and time-resolved RNA-seq revealed that differentiation was unaffected. CTCF binding is strongly enriched at promoters of downregulated genes. Re-expression of a CTCF variant with an N-terminal truncation, incapable of looping, was sufficient to rescue the expression of CTCF-promoter bound genes and the defects in morphogenesis. However, extended culture (up to 168 hours) of gastruloids reconstituted with N-terminal truncated CTCF led to their collapse. Our work shows that CTCF has a dual function in early mammalian development: at early stages CTCF regulates developmentally important genes through promoter binding, while at later stages its looping function is required for correct development. HighlightsO_LICTCF is essential for gastruloid morphogenesis but dispensable for cell differentiation C_LIO_LICTCF activates genes through promoter binding C_LIO_LICTCF promoter target regulation drives in vitro gastrulation C_LIO_LIPost-gastrulation development in vitro is driven by CTCFs looping function C_LI

genomics↗

bioRxiv2026

Effects of CTCF on the regulatory landscape of the mouse Sox2 locus

Regulatory elements that control gene transcription are scattered along the genome, yet it is still poorly understood how their precise positioning affects the regulatory landscape. We previously reported a method to relocate a regulatory element to thousands of positions in a genomic locus and probe the position-dependent impact on gene regulation. Here, we apply this approach to systematically query the role and position-dependence of CTCF binding sites (CBSs), which are thought to modulate communication between distal regulatory elements. We focused on the mouse Sox2 locus in embryonic stem cells, where the Sox2 gene is activated by a potent distal enhancer. First, we found that a CBS that is naturally located immediately upstream of the Sox2 promoter endows this promoter with strong orientation-dependent activation, anywhere within the gene-enhancer interval. Second, throughout this interval, insertion of CBSs alone consistently reduces Sox2 expression in an orientation-dependent manner, suggesting a polarity in the loop extrusion process. Third, two native CBSs located within and downstream of the enhancer subtly help to confine the activation realm of the enhancer. Together, these results illuminate the interplay between CBSs and the detailed regulatory landscape of a genomic locus.

molecular biology↗

bioRxiv2024

Synergy between cis-regulatory elements can render cohesin dispensable for distal enhancer function

Enhancers are critical genetic elements controlling transcription from promoters, but the mechanisms by which they convey regulatory information across large genomic distances remain elusive. Here, we engineered pluripotent stem cells in which cohesin loop extrusion can be inducibly disrupted without causing confounding cell cycle defects. While evident, transcriptional dysregulation was cell-type specific, and not all loci with distal enhancers depend equally on cohesin extrusion. Using comparative genome editing, we demonstrate that enhancer-promoter communication across as little as 20 kilobases can rely on cohesin. However, promoter-proximal regulatory elements can support long-range, cohesin-independent enhancer action - either upon disabling extrusion or across strong CTCF insulators. Finally, transcriptional dynamics and the emergence of new embryonic cell types in response to differentiation cues remained largely robust to disrupting cohesin extrusion. Beyond establishing novel experimental strategies to study cohesin functions in enhancer biology, our work provides mechanistic insight accounting for both cell type- and genomic context-specificity.

molecular biology↗

bioRxiv2024

ZNF143 is a transcriptional regulator of nuclear-encoded mitochondrial genes that acts independently of looping and CTCF

Gene expression is orchestrated by transcription factors, which function within the context of a three-dimensional genome. Zinc finger protein 143 (ZNF143/ZFP143) is a transcription factor that has been implicated in both gene activation and chromatin looping. To study the direct consequences of ZNF143/ZFP143 loss, we generated a ZNF143/ZFP143 degron line. Our results show that ZNF143/ZFP143 depletion has no effect on chromatin looping. Systematic analysis of ZNF143/ZFP143 occupancy data revealed that a commonly used antibody cross-reacts with CTCF, leading to its incorrect association with chromatin loops. Nevertheless, ZNF143/ZFP143 specifically activates nuclear-encoded mitochondrial genes and its loss leads to severe mitochondrial dysfunction. Using an in vitro embryo model, we find that ZNF143/ZFP143 is an essential regulator of organismal development. Our results establish ZNF143/ZFP143 as a conserved transcriptional regulator of cell proliferation and differentiation by safeguarding mitochondrial activity. HighlightsO_LIAcute degradation of ZFP143 leads to rapid and specific loss of gene transcription. C_LIO_LIMolecular consequences of ZFP143 loss are inconsistent with a role in chromatin looping. C_LIO_LIZFP143 is a conserved transcriptional regulator of nuclear-encoded mitochondrial proteins. C_LIO_LIZFP143 regulation of mitochondrial homeostasis is critical for multicellular organismal development. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/583864v1_ufig1.gif" ALT="Figure 1"> View larger version (59K): org.highwire.dtl.DTLVardef@1033b53org.highwire.dtl.DTLVardef@12bd81aorg.highwire.dtl.DTLVardef@178a813org.highwire.dtl.DTLVardef@62b7bf_HPS_FORMAT_FIGEXP M_FIG C_FIG

systems biology↗

bioRxiv2022

Identifying cross-lineage dependencies of cell-type specific regulators in gastruloids

Correct gene expression levels in space and time are crucial for normal development. Advances in genomics enable the inference of gene regulatory programs that are active during development. However, this approach cannot capture the complex multicellular interactions that occur during embryogenesis. Compared to model organisms such as fruit flies and zebrafish, the growth of mammalian embryos in utero further complicates the analysis of cell-cell communication during development. However, in vitro models of mammalian development such as gastruloids can overcome this limitation. Using time-resolved single-cell chromatin accessibility analysis, we have delineated the regulatory landscape during gastruloid development and thereby identified the critical drivers of developmental transitions. We observed that gastruloids develop from pluripotent cells driven by the transcription factor (TF) dimer OCT4-SOX2 and differentiate along two main branches. A mesoderm branch characterized by the TF MSGN1 and a spinal cord branch characterized by CDX1, 2, 4 (CDX). Consistent with our lineage reconstruction, {Delta}CDX gastruloids fail to form spinal cord. Conversely, Msgn1 ablation inhibits the development of paraxial mesoderm, as expected. However, this also abolished spinal cord cells, which is surprising given that MSGN1 is not associated with differentiation along this branch. Therefore, formation of paraxial mesoderm is required for spinal cord development. To validate this, we generated chimeric gastruloids using {Delta}MSGN1 and wildtype cells, which formed both spinal cord and paraxial mesoderm. Strikingly, {Delta}MSGN1 cells specifically contributed to spinal cord, suggesting that cell-cell interactions between paraxial mesoderm and spinal cord are necessary for the formation of the latter. Our work has important implications for the study of cell-cell communication in development and how gene regulatory programs are functionally executed to form complex multicellular developmental structures.

systems biology↗

bioRxiv2019

WAPL maintains dynamic cohesin to preserve lineage specific distal gene regulation

The cohesin complex plays essential roles in sister chromatin cohesin, chromosome organization and gene expression. The role of cohesin in gene regulation is incompletely understood. Here, we report that the cohesin release factor WAPL is crucial for maintaining a pool of dynamic cohesin bound to regions that are associated with lineage specific genes in mouse embryonic stem cells. These regulatory regions are enriched for active enhancer marks and transcription factor binding sites, but largely devoid of CTCF binding sites. Stabilization of cohesin, which leads to a loss of dynamic cohesin from these regions, does not affect transcription factor binding or active enhancer marks, but does result in changes in promoter-enhancer interactions and downregulation of genes. Acute cohesin depletion can phenocopy the effect of WAPL depletion, showing that cohesin plays a crucial role in maintaining expression of lineage specific genes. The binding of dynamic cohesin to chromatin is dependent on the pluripotency transcription factor OCT4, but not NANOG. Finally, dynamic cohesin binding sites are also found in differentiated cells, suggesting that they represent a general regulatory principle. We propose that cohesin dynamically binding to regulatory sites creates a favorable spatial environment in which promoters and enhancers can communicate to ensure proper gene expression.\n\nHIGHLIGHTSO_LIThe cohesin release factor WAPL is crucial for maintaining a pluripotency-specific phenotype.\nC_LIO_LIDynamic cohesin is enriched at lineage specific loci and overlaps with binding sites of pluripotency transcription factors.\nC_LIO_LIExpression of lineage specific genes is maintained by dynamic cohesin binding through the formation of promoter-enhancer associated self-interaction domains.\nC_LIO_LICTCF-independent cohesin binding to chromatin is controlled by the pioneer factor OCT4.\nC_LI

genomics↗