The Krüppel-associated box zinc finger protein (KRAB-ZFP) family diversified in mammals. The majority of human KRAB-ZFPs bind transposable elements (TEs), however, since most TEs are inactive in ...humans it is unclear whether KRAB-ZFPs emerged to suppress TEs. We demonstrate that many recently emerged murine KRAB-ZFPs also bind to TEs, including the active ETn, IAP, and L1 families. Using a CRISPR/Cas9-based engineering approach, we genetically deleted five large clusters of KRAB-ZFPs and demonstrate that target TEs are de-repressed, unleashing TE-encoded enhancers. Homozygous knockout mice lacking one of two KRAB-ZFP gene clusters on chromosome 2 and chromosome 4 were nonetheless viable. In pedigrees of chromosome 4 cluster KRAB-ZFP mutants, we identified numerous novel ETn insertions with a modest increase in mutants. Our data strongly support the current model that recent waves of retrotransposon activity drove the expansion of KRAB-ZFP genes in mice and that many KRAB-ZFPs play a redundant role restricting TE activity.
The ventricular zone (VZ) of the nervous system contains radial glia cells that were originally considered relatively homogenous in their gene expression, but a detailed characterization of ...transcriptional diversity in these VZ cells has not been reported. Here, we performed single-cell RNA sequencing to characterize transcriptional heterogeneity of neural progenitors within the VZ and subventricular zone (SVZ) of the ganglionic eminences (GEs), the source of all forebrain GABAergic neurons. By using a transgenic mouse line to enrich for VZ cells, we characterize significant transcriptional heterogeneity, both between GEs and within spatial subdomains of specific GEs. Additionally, we observe differential gene expression between E12.5 and E14.5 VZ cells, which could provide insights into temporal changes in cell fate. Together, our results reveal a previously unknown spatial and temporal genetic diversity of VZ cells in the ventral forebrain that will aid our understanding of initial fate decisions in the forebrain.
Imprinted genes occur in discrete clusters that are coordinately regulated by shared DNA elements called Imprinting Control Regions. H19 and Igf2 are linked imprinted genes that play critical roles ...in development. Loss of imprinting (LOI) at the IGF2/H19 locus on the maternal chromosome is associated with the developmental disorder Beckwith Wiedemann Syndrome (BWS) and with several cancers. Here we use comprehensive genetic and genomic analyses to follow muscle development in a mouse model of BWS to dissect the separate and shared roles for misexpression of Igf2 and H19 in the disease phenotype. We show that LOI results in defects in muscle differentiation and hypertrophy and identify primary downstream targets: Igf2 overexpression results in over-activation of MAPK signaling while loss of H19 lncRNA prevents normal down regulation of p53 activity and therefore results in reduced AKT/mTOR signaling. Moreover, we demonstrate instances where H19 and Igf2 misexpression work separately, cooperatively, and antagonistically to establish the developmental phenotype. This study thus identifies new biochemical roles for the H19 lncRNA and underscores that LOI phenotypes are multigenic so that complex interactions will contribute to disease outcomes.
Abstract
Fate-determining transcription factors (TFs) can promote lineage-restricted transcriptional programs from common progenitor states. The inner cell mass (ICM) of mouse blastocysts ...co-expresses the TFs NANOG and GATA6, which drive the bifurcation of the ICM into either the epiblast (Epi) or the primitive endoderm (PrE), respectively. Here, we induce GATA6 in embryonic stem cells–that also express NANOG–to characterize how a state of co-expression of opposing TFs resolves into divergent lineages. Surprisingly, we find that GATA6 and NANOG co-bind at the vast majority of Epi and PrE enhancers, a phenomenon we also observe in blastocysts. The co-bound state is followed by eviction and repression of Epi TFs, and quick remodeling of chromatin and enhancer-promoter contacts thus establishing the PrE lineage while repressing the Epi fate. We propose that co-binding of GATA6 and NANOG at shared enhancers maintains ICM plasticity and promotes the rapid establishment of Epi- and PrE-specific transcriptional programs.
Polycomb group response elements (PREs) in Drosophila are DNA-elements that recruit Polycomb proteins (PcG) to chromatin and regulate gene expression. PREs are easily recognizable in the Drosophila ...genome as strong peaks of PcG-protein binding over discrete DNA fragments; many small but statistically significant PcG peaks are also observed in PcG domains. Surprisingly, in vivo deletion of the four characterized strong PREs from the PcG regulated invected-engrailed (inv-en) gene complex did not disrupt the formation of the H3K27me3 domain and did not affect inv-en expression in embryos or larvae suggesting the presence of redundant PcG recruitment mechanism. Further, the 3D-structure of the inv-en domain was only minimally altered by the deletion of the strong PREs. A reporter construct containing a 7.5kb en fragment that contains three weak peaks but no large PcG peaks forms an H3K27me3 domain and is PcG-regulated. Our data suggests a model for the recruitment of PcG-complexes to Drosophila genes via interactions with multiple, weak PREs spread throughout an H3K27me3 domain.
A comprehensive characterization of epigenomic organization in the embryonic mouse forebrain will enhance our understanding of neurodevelopment and provide insight into mechanisms of neurological ...disease. Here 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.
Maternal loss of imprinting (LOI) at the
H19/IGF2
locus results in biallelic
IGF2
and reduced
H19
expression and is associated with Beckwith–-Wiedemann syndrome (BWS). We use mouse models for LOI to ...understand the relative importance of
Igf2
and
H19
mis-expression in BWS phenotypes. Here we focus on cardiovascular phenotypes and show that neonatal cardiomegaly is exclusively dependent on increased
Igf2
. Circulating IGF2 binds cardiomyocyte receptors to hyperactivate mTOR signaling, resulting in cellular hyperplasia and hypertrophy. These
Igf2
-dependent phenotypes are transient: cardiac size returns to normal once
Igf2
expression is suppressed postnatally. However, reduced
H19
expression is sufficient to cause progressive heart pathologies including fibrosis and reduced ventricular function. In the heart,
H19
expression is primarily in endothelial cells (ECs) and regulates EC differentiation both in vivo and in vitro. Finally, we establish novel mouse models to show that cardiac phenotypes depend on
H19
lncRNA interactions with
Mirlet7
microRNAs.
The terminal differentiation of osteoblasts and subsequent formation of bone marks an important phase in palate development that leads to the separation of the oral and nasal cavities. While the ...morphogenetic events preceding palatal osteogenesis are well explored, major gaps remain in our understanding of the molecular mechanisms driving the formation of this bony union of the fusing palate. Through bulk, single-nucleus, and spatially resolved RNA-sequencing analyses of the developing secondary palate, we identify a shift in transcriptional programming between embryonic days 14.5 and 15.5 pinpointing the onset of osteogenesis. We define spatially restricted expression patterns of key osteogenic marker genes that are differentially expressed between these developmental timepoints. Finally, we identify genes in the palate highly expressed by palate nasal epithelial cells, also enriched within palatal osteogenic mesenchymal cells. This investigation provides a relevant framework to advance palate-specific diagnostic and therapeutic biomarker discovery.
Polycomb group (PcG) proteins are responsible for maintaining the silenced transcriptional state of many developmentally regulated genes. PcG proteins are organized into multiprotein complexes that ...are recruited to DNA via cis-acting elements known as “Polycomb response elements” (PREs). In Drosophila, PREs consist of binding sites for many different DNA-binding proteins, some known and others unknown. Identification of these DNA-binding proteins is crucial to understanding the mechanism of PcG recruitment to PREs. We report here the identification of Combgap (Cg), a sequence-specific DNA-binding protein that is involved in recruitment of PcG proteins. Cg can bind directly to PREs via GTGT motifs and colocalizes with the PcG proteins Pleiohomeotic (Pho) and Polyhomeotic (Ph) at the majority of PREs in the genome. In addition, Cg colocalizes with Ph at a number of targets independent of Pho. Loss of Cg leads to decreased recruitment of Ph at only a subset of sites; some of these sites are binding sites for other Polycomb repressive complex 1 (PRC1) components, others are not. Our data suggest that Cg can recruit Ph in the absence of PRC1 and illustrate the diversity and redundancy of PcG protein recruitment mechanisms.
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