Biology subjects

Rocha, P. P.

Publications and source records attributed to Rocha, P. P..

10 recordsLinked to original sources

bioRxiv2025

Absence of posterior commissure and sub-commissural organ precedes encephalocele development in a new mouse model

Encephalocele is a congenital defect involving herniation of the meninges, with or without brain tissue, outside the skull. Although traditionally considered a neural tube defect (NTD) alongside anencephaly and open spina bifida (myelomeningocele), encephalocele typically shows well-formed brain tissue, and is likely to represent a later-arising, post-neurulation developmental anomaly, with a different pathogenic mechanism from the open NTDs. A detailed understanding of encephalocele pathogenesis requires experimental studies and, recently, we developed a new mouse model in which the genes encoding FGF3, 4 and 15 are over-expressed in the embryonic day (E) 9.5 brain. Encephalocele subsequently develops as a broad forebrain-midbrain swelling, visible from E11.5, that resolves by birth into a focal brain herniation resembling human parieto-occipital encephalocele. A structural analysis of the brain in mutant embryos reveals absence of the posterior commissure and sub-commissural organ, and at later stages the pineal gland. These structures normally develop just rostral to the forebrain-midbrain boundary, and Pax6 immuno-histochemistry demonstrates that this boundary remains intact in the mutant embryos. Histological analysis reveals a more general change in tissue composition of the neural tube roof in the forebrain-midbrain region, with diminished thickness of the neuroepithelium and increased thickness of the overlying layer, including the non-neural ectoderm (future epidermis). We conclude that the posterior commissure and sub-commissural organ, previously implicated in hydrocephalus, may also be fundamental for development of the earlier-arising brain malformation, encephalocele.

developmental biology↗

bioRxiv2024

Structural perturbation of chromatin domains with multiple developmental regulators can severely impact gene regulation and development

Chromatin domain boundaries delimited by CTCF motifs can restrict the range of enhancer action. However, disruption of domain structure often results in mild gene dysregulation and thus predicting the impact of boundary rearrangements on animal development remains challenging. Here, we tested whether structural perturbation of a chromatin domain with multiple developmental regulators can result in more acute gene dysregulation and severe developmental phenotypes. We targeted clusters of CTCF motifs in a domain of the mouse genome containing three FGF ligand genes--Fgf3, Fgf4, and Fgf15--that regulate several developmental processes. Deletion of the 23.9kb cluster that defines the centromeric boundary of this domain resulted in ectopic interactions of the FGF genes with enhancers located across the deleted boundary that are active in the developing brain. This caused strong induction of FGF expression and perinatal lethality with encephalocele and orofacial cleft phenotypes. Heterozygous boundary deletion was sufficient to cause these fully penetrant phenotypes, and strikingly, loss of a single CTCF motif within the cluster also recapitulated ectopic FGF expression and caused encephalocele. However, such phenotypic sensitivity to perturbation of domain structure did not extend to all CTCF clusters of this domain, nor to all developmental processes controlled by these three FGF genes--for example, the ability to undergo lineage specification in the blastocyst and pre-implantation development were not affected. By tracing the impact of different chromosomal rearrangements throughout mouse development, we start to uncover the determinants of phenotypic robustness and sensitivity to perturbation of chromatin boundaries. Our data show how small sequence variants at certain domain boundaries can have a surprisingly outsized effect and must be considered as potential sources of gene dysregulation during development and disease.

developmental biology↗

bioRxiv2023

Polycomb protein binding and looping mediated by Polycomb Response Elements in the ON transcriptional state

Polycomb group proteins (PcG) mediate epigenetic silencing of important developmental genes and other targets. In Drosophila, canonical PcG-target genes contain Polycomb Response Elements (PREs) that recruit PcG protein complexes including PRC2 that tri- methylates H3K27 forming large H3K27me3 domains. In the OFF transcriptional state, PREs loop with each other and this looping strengthens silencing. Here we address the question of what PcG proteins bind to PREs when canonical PcG target genes are expressed, and whether PREs loop when these genes are ON. Our data show that the answer to this question is PRE-specific but general conclusions can be made. First, within a PcG-target gene, some regulatory DNA can remain covered with H3K27me3 and PcG proteins remain bound to PREs in these regions. Second, when PREs are within H3K27ac domains, PcG- binding decreases, however, this depends on the protein and PRE. The DNA binding protein GAF, and the PcG protein Ph remain at PREs even when other PcG proteins are greatly depleted. In the ON state, PREs can still loop with each other, but also form loops with presumptive enhancers. These data support the model that, in addition to their role in PcG silencing, PREs can act as "promoter-tethering elements" mediating interactions between promoter proximal PREs and distant enhancers.

genetics↗

bioRxiv2023

Loss of Ezh2 in the medial ganglionic eminence alters interneuron fate, cell morphology and gene expression profiles

Enhancer of zeste homolog 2 (Ezh2) is responsible for trimethylation of histone 3 at lysine 27 (H3K27me3), resulting in gene repression. Here, we explore the role of Ezh2 in forebrain GABAergic interneuron development. Loss of Ezh2 increases somatostatin-expressing (SST+) and decreases parvalbumin-expressing (PV+) interneurons in multiple brain regions. We also observe fewer MGE-derived interneurons in the first postnatal week, indicating reduced interneuron production. Intrinsic electrophysiological properties in SST+ and PV+ interneurons are normal, but PV+ interneurons display increased axonal complexity in Ezh2 mutant mice. Single cell multiome analysis revealed differential gene expression patterns in the embryonic MGE that are predictive of these cell fate changes. Lastly, CUT&Tag analysis revealed differential H3K27me3 levels at specific genomic loci, with some genes displaying a relative increase in H3K27me3 indicating they may be resistant to epigenetic modifications. Thus, loss of Ezh2 in the MGE alters interneuron fate, morphology, and gene expression and regulation.

neuroscience↗

bioRxiv2022

A therapeutically targetable NOTCH1-SIRT1-KAT7 axis in T-cell Leukemia

T-cell Acute Lymphoblastic Leukemia (T-ALL) is a NOTCH1-driven disease in need of novel therapies. Here, we identify a NOTCH1-SIRT1-KAT7 link as a therapeutic vulnerability in T-ALL, in which SIRT1 is overexpressed downstream of a novel NOTCH1-bound enhancer. SIRT1 loss impairs leukemia generation, while SIRT1 overexpression accelerates leukemia and confers resistance to NOTCH1 inhibition in a deacetylase-dependent manner. Moreover, secondary SIRT1 loss extends survival and synergizes with NOTCH1 inhibition. Global acetyl-proteomics upon SIRT1 loss uncovered hyperacetylation of KAT7 and BRD1, subunits of a histone acetyltransferase complex targeting H4K12. Metabolic and gene expression profiling revealed a metabolic crisis together with a transcriptional signature resembling KAT7 deletion. Consistently, SIRT1 loss resulted in reduced H4K12ac, and overexpression of a non-acetylatable KAT7 mutant partly rescued SIRT1 loss-induced proliferation defects. The newly unveiled NOTCH1-SIRT1-KAT7 axis uncovers novel therapeutic targets in T-ALL and reveals a circular feedback mechanism balancing deacetylase/acetyltransferase activation with potentially broad relevance in cancer. Statement of significanceWe identified a novel axis in T-ALL whereby NOTCH1 activates SIRT1 through an enhancer region, and SIRT1 deacetylates and activates KAT7. Targeting SIRT1 shows antileukemic effects, partly mediated by KAT7 inactivation. Our results identify novel therapeutic targets and uncover a rheostat mechanism between deacetylase/acetyltransferase activities with potentially broader cancer relevance.

cancer biology↗

bioRxiv2022

High affinity enhancer-promoter interactions can bypass CTCF/cohesin-mediated insulation and contribute to phenotypic robustness

Transcriptional control by distal enhancers is an integral feature of gene regulation. To understand how enhancer-promoter interactions arise and assess the impact of disrupting 3D chromatin structure on gene expression, we generated an allelic series of mouse mutants that perturb the physical structure of the Sox2 locus. We show that in the epiblast and in neuronal tissues, CTCF-mediated loops are neither required for the interaction of the Sox2 promoter with distal enhancers, nor for its expression. Insertion of various combinations of CTCF motifs between Sox2 and its distal enhancers generated ectopic loops with varying degrees of insulation that directly correlated with reduced transcriptional output. Yet, even the mutants exhibiting the strongest insulation, with six CTCF motifs in divergent orientation, could not fully abolish activation by distal enhancers, and failed to disrupt implantation and neurogenesis. In contrast, cells of the anterior foregut were more susceptible to chromatin structure disruption with no detectable SOX2 expression in mutants with the strongest CTCF-mediated boundaries. These animals phenocopied loss of SOX2 in the anterior foregut, failed to separate trachea from esophagus and died perinatally. We propose that baseline transcription levels and enhancer density may influence the tissue-specific ability of distal enhancers to overcome physical barriers and maintain faithful gene expression. Our work suggests that high affinity enhancer-promoter interactions that can overcome chromosomal structural perturbations, play an essential role in maintaining phenotypic robustness.

developmental biology↗

bioRxiv2021

SOX2-phosphorylation toggles a bistable differentiation-switch in squamous cell carcinoma

The fate choice between stem cell self-renewal and differentiation is regulated by bistable transcriptional networks, which are balanced in homeostasis and imbalanced in tumors. Yet, how stem cells switch from self-renewal to differentiation remains a conundrum. Here, we discover a molecular mechanism that allows stem cell-like tumor propagating cells (TPCs) in squamous cell carcinomas (SCCs) to switch from a mutually exclusive SOX2-PITX1-TP63 self-renewal circuit to a KLF4 driven differentiation program, dependent on the relative occupancy of a novel Klf4-regulatory enhancer cluster (Klf4EC944) by SOX2 or KLF4, respectively. We find SOX2 occupies this site in TPCs to inhibit Klf4 transcription, but upon phosphorylation SOX2 becomes evicted from Klf4EC944, allowing residual KLF4 to occupy this site instead, boost the expression of KLF4 and its downstream targets, and differentiate self-renewing TPCs into post-mitotic SCC cells. This mechanism allows SOX2 to promote self-renewal and tumor formation, while preserving the differentiation potential in SCC cells. Our data suggest that stochastic cell fate decisions depend on the effective concentration of enzymatically regulated transcription factors. The surprising specificity by which SOX2-phosphorylation governs the bistable Klf4EC944 network-switch in SCCs reveals a conceptual framework for the identification of similar switches in other stem cell and cancer types and their potential development into cell type specific differentiation therapies for diseases in which tissue homeostasis has gone awry.

cancer biology↗

bioRxiv2021

Rapid redistribution and extensive binding of NANOG and GATA6 at shared regulatory elements underlie specification of divergent cell fates

Establishment of divergent cell types from a common progenitor requires transcription factors (TFs) to promote lineage-restricted transcriptional programs while suppressing alternative fates. In the mouse blastocyst, cells of the inner cell mass (ICM) coexpress NANOG and GATA6, two TFs that drive the bifurcation of these progenitors into either the epiblast (Epi) or the primitive endoderm (PrE), respectively. Here, using in vitro differentiation, we describe the molecular mechanisms of how GATA6 quickly induces the PrE fate while repressing the Epi lineage. GATA6 functions as a pioneer TF by inducing nucleosome repositioning at regulatory elements controlling PrE genes, making them accessible for deposition of active histone marks and leading to rewiring of chromatin interactions and ultimately transcriptional activation. GATA6 also binds most regulatory elements of Epi genes followed by eviction of the Epispecific TFs NANOG and SOX2, loss of active histone marks, and reduction in chromatin accessibility that culminates in transcriptional repression. Unexpectedly, evicted NANOG and SOX2 transiently bind PrE regulatory elements occupied by GATA6. Our study shows that GATA6 binds and modulate the same regulatory elements as Epi TFs, a phenomenon we also validated in blastocysts. We propose that the ability of PrE and Epi-specific TFs to extensively bind and regulate the same gene networks contributes to ICM plasticity and allows rapid cell lineage specification by coordinating both activation

developmental biology↗

bioRxiv2021

Single cell chromatin accessibility reveals regulatory elements and developmental trajectories in the embryonic forebrain

A comprehensive characterization of epigemonic organization in the embryonic mouse forebrain will enhance our understanding of neurodevelopment and provide insight into mechanisms of neurological disease. We collected single-cell chromatin accessibility profiles from four distinct neurogenic regions of the embryonic mouse forebrain using single nuclei ATAC-Seq (snATAC-Seq). We identified thousands of differentially accessible peaks, many restricted to distinct progenitor cell types or brain regions. We integrated snATAC-Seq and single cell transcriptome data to characterize changes of chromatin accessibility at enhancers and promoters with associated transcript abundance. Multi-modal integration of histone modifications (CUT&Tag and CUT&RUN), promoter-enhancer interactions (Capture-C) and high-order chromatin structure (Hi-C) extended these initial observations. This dataset reveals a diverse chromatin landscape with region-specific regulatory mechanisms and genomic interactions in distinct neurogenic regions of the embryonic mouse brain and represents an extensive public resource of a ground truth epigenomic landscape at this critical stage of neurogenesis.

neuroscience↗

bioRxiv2020

Regulation of human development by ubiquitin chain editing of chromatin remodelers

Embryonic development occurs through commitment of pluripotent stem cells to differentiation programs that require highly coordinated changes in gene expression. Chromatin remodeling of gene regulatory elements is a critical component of how such changes are achieved. While many factors controlling chromatin dynamics are known, mechanisms of how different chromatin regulators are orchestrated during development are not well understood. Here, we describe LINKED (LINKage-specific-deubiquitylation-deficiency-induced Embryonic Defects) syndrome, a novel multiple congenital anomaly disorder caused by hypomorphic hemizygous missense variants in the deubiquitylase OTUD5/DUBA. Studying LINKED mutations in vitro, in mouse, and in models of neuroectodermal differentiation of human pluripotent stem cells, we uncover a novel regulatory circuit that coordinates chromatin remodeling pathways during early differentiation. We show that the K48-linkage-specific deubiquitylation activity of OTUD5 is essential for murine and human development and, if reduced, leads to aberrant cell-fate specification. OTUD5 controls differentiation through preventing the degradation of multiple chromatin regulators including ARID1A/B and HDAC2, mutation of which underlie developmental syndromes that exhibit phenotypic overlap with LINKED patients. Accordingly, loss of OTUD5 during early differentiation leads to less accessible chromatin at neural and neural crest enhancers and thus aberrant rewiring of gene expression networks. Our work identifies a novel mechanistic link between phenotypically related developmental disorders and an essential function for linkagespecific ubiquitin editing of substrate groups (i.e. chromatin remodeling complexes) during early cellfate decisions - a regulatory concept, we predict to be a general feature of embryonic development.

genetics↗