Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChlP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 10(4) -5 x 10(5) cells). Importantly, this protocol is compatible with ChlP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues

Epigenomics-Based Identification of Major Cell Identity Regulators within Heterogeneous Cell Populations

Cellular heterogeneity within embryonic and adult tissues is involved in multiple biological and pathological processes. Here, we present a simple epigenomic strategy that allows the functional dissection of cellular heterogeneity. By integrating H3K27me3 chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) data, we demonstrate that the presence of broad H3K27me3 domains at transcriptionally active genes reflects the heterogeneous expression of major cell identity regulators. Using dorsoventral patterning of the spinal neural tube as a model, the proposed approach successfully identifies the majority of previously known dorsoventral patterning transcription factors with high sensitivity and precision. Moreover, poorly characterized patterning regulators can be similarly predicted, as shown for ZNF488, which confers p1/p2 neural progenitor identity. Finally, we show that, as our strategy is based on universal chromatin features, it can be used to functionally dissect cellular heterogeneity within various organisms and tissues, thus illustrating its potential applicability to a broad range of biological and pathological contexts

The chromatin, topological and regulatory properties of pluripotency-associated poised enhancers are conserved in vivo

Poised enhancers (PEs) represent a genetically distinct set of distal regulatory elements that control the expression of major developmental genes. Before becoming activated in differentiating cells, PEs are already bookmarked in pluripotent cells with unique chromatin and topological features that could contribute to their privileged regulatory properties. However, since PEs were originally characterized in embryonic stem cells (ESC), it is currently unknown whether PEs are functionally conserved in vivo. Here, we show that the chromatin and 3D structural features of PEs are conserved among mouse pluripotent cells both in vitro and in vivo. We also uncovered that the interactions between PEs and their target genes are globally controlled by the combined action of Polycomb, Trithorax and architectural proteins. Moreover, distal regulatory sequences located close to developmental genes and displaying the typical genetic (i.e. CpG islands) and chromatin (i.e. high accessibility and H3K27me3 levels) features of PEs are commonly found across vertebrates. These putative PEs show high sequence conservation within specific vertebrate clades, with only a few being evolutionary conserved across all vertebrates. Lastly, by genetically disrupting PEs in mouse and chicken embryos, we demonstrate that these regulatory elements play essential roles during the induction of major developmental genes in vivo. Poised enhancers (PEs) in embryonic stem cells have accessible chromatin, are bound by repressive Polycomb Group proteins, and interact with their targets prior to activation. However, whether this is recapitulated in vivo is unknown. Here the authors show PEs display these features in mouse embryos, are prevalent across vertebrates, and are required for developmental gene expression

Pathological ASXL1 Mutations and Protein Variants Impair Neural Crest Development

Summary: The neural crest (NC) gives rise to a multitude of fetal tissues, and its misregulation is implicated in congenital malformations. Here, we investigated molecular mechanisms pertaining to NC-related symptoms in Bohring-Opitz syndrome (BOS), a developmental disorder linked to mutations in the Polycomb group factor Additional sex combs-like 1 (ASXL1). Genetically edited human pluripotent stem cell lines that were differentiated to NC progenitors and then xenotransplanted into chicken embryos demonstrated an impairment of NC delamination and emigration. Molecular analysis showed that ASXL1 mutations correlated with reduced activation of the transcription factor ZIC1 and the NC gene regulatory network. These findings were supported by differentiation experiments using BOS patient-derived induced pluripotent stem cell lines. Expression of truncated ASXL1 isoforms (amino acids 1–900) recapitulated the NC phenotypes in vitro and in ovo, raising the possibility that truncated ASXL1 variants contribute to BOS pathology. Collectively, we expand the understanding of truncated ASXL1 in BOS and in the human NC. : In this study, Drukker and colleagues developed human pluripotent stem cell models for the rare congenital disorder Bohring-Opitz syndrome, which is caused by mutations in the Polycomb factor ASXL1. In these lines, they found impaired neural crest emigration in vitro and in vivo and link this phenotype to impaired activation of ZIC1. Keywords: ASXL1, neural crest, Bohring-Opitz syndrome, Polycomb, ZIC

MAPRE2 mutations result in altered human cranial neural crest migration, underlying craniofacial malformations in CSC-KT syndrome

Circumferential skin creases (CSC-KT) is a rare polymalformative syndrome characterised by intellectual disability associated with skin creases on the limbs, and very characteristic craniofacial malformations. Previously, heterozygous and homozygous mutations in MAPRE2 were found to be causal for this disease. MAPRE2 encodes for a member of evolutionary conserved microtubule plus end tracking proteins, the end binding (EB) family. Unlike MAPRE1 and MAPRE3, MAPRE2 is not required for the persistent growth and stabilization of microtubules, but plays a role in other cellular processes such as mitotic progression and regulation of cell adhesion. The mutations identified in MAPRE2 all reside within the calponin homology domain, responsible to track and interact with the plus-end tip of growing microtubules, and previous data showed that altered dosage of MAPRE2 resulted in abnormal branchial arch patterning in zebrafish. In this study, we developed patient derived induced pluripotent stem cell lines for MAPRE2, together with isogenic controls, using CRISPR/Cas9 technology, and differentiated them towards neural crest cells with cranial identity. We show that changes in MAPRE2 lead to alterations in neural crest migration in vitro but also in vivo, following xenotransplantation of neural crest progenitors into developing chicken embryos. In addition, we provide evidence that changes in focal adhesion might underlie the altered cell motility of the MAPRE2 mutant cranial neural crest cells. Our data provide evidence that MAPRE2 is involved in cellular migration of cranial neural crest and offers critical insights into the mechanism underlying the craniofacial dysmorphisms and cleft palate present in CSC-KT patients. This adds the CSC-KT disorder to the growing list of neurocristopathies

Modelling the pathological long-range regulatory effects of human structural variation with patient-specific hiPSCs

The pathological consequences of structural variants disrupting 3D genome organization can be difficult to elucidate in vivo due to differences in gene dosage sensitivity between mice and humans. This is illustrated by branchiooculofacial syndrome (BOFS), a rare congenital disorder caused by heterozygous mutations within TFAP2A, a neural crest regulator for which humans, but not mice, are haploinsufficient. Here, we present a BOFS patient carrying a heterozygous inversion with one breakpoint located within a topologically associating domain (TAD) containing enhancers essential for TFAP2A expression in human neural crest cells (hNCCs). Using patient-specific hiPSCs, we show that, although the inversion shuffles the TFAP2A hNCC enhancers with novel genes within the same TAD, this does not result in enhancer adoption. Instead, the inversion disconnects one TFAP2A allele from its cognate enhancers, leading to monoallelic and haploinsufficient TFAP2A expression in patient hNCCs. Our work illustrates the power of hiPSC differentiation to unveil long-range pathomechanisms

Overarching control of autophagy and DNA damage response by CHD6 revealed by modeling a rare human pathology

Members of the chromodomain-helicase-DNA binding (CHD) protein family are chromatin remodelers implicated in human pathologies, with CHD6 being one of its least studied members. We discovered a de novo CHD6 missense mutation in a patient clinically presenting the rare Hallermann-Streiff syndrome (HSS). We used genome editing to generate isogenic iPSC lines and model HSS in relevant cell types. By combining genomics with functional in vivo and in vitro assays, we show that CHD6 binds a cohort of autophagy and stress response genes across cell types. The HSS mutation affects CHD6 protein folding and impairs its ability to recruit co-remodelers in response to DNA damage or autophagy stimulation. This leads to accumulation of DNA damage burden and senescence-like phenotypes. We therefore uncovered a molecular mechanism explaining HSS onset via chromatin control of autophagic flux and genotoxic stress surveillance. Members of the CHD chromatin remodeler family are implicated in human pathologies, however CHD6 remained poorly studied. Here, the authors show that CHD6 binds to and regulates autophagy and stress response genes across cell types. They identify a clinical mutation that affects its ability to recruit cofactors, leading to impaired autophagy induction and DNA repair

yylncT Defines a Class of Divergently Transcribed lncRNAs and Safeguards the T-mediated Mesodermal Commitment of Human PSCs

Human protein-coding genes are often accompanied by divergently transcribed non-coding RNAs whose functions, especially in cell fate decisions, are poorly understood. Using an hESC-based cardiac differentiation model, we define a class of divergent lncRNAs, termed yin yang lncRNAs (yylncRNAs), that mirror the cell-type-specific expression pattern of their protein-coding counterparts. yylncRNAs are preferentially encoded from the genomic loci of key developmental cell fate regulators. Most yylncRNAs are spliced polyadenylated transcripts showing comparable expression patterns in vivo in mouse and in human embryos. Signifying their developmental function, the key mesoderm specifier BRACHYURY (T) is accompanied by yylncT, which localizes to the active T locus during mesoderm commitment. yylncT binds the de novo DNA methyltransferase DNMT3B, and its transcript is required for activation of the T locus, with yylncT depletion specifically abolishing mesodermal commitment. Collectively, we report a lncRNA-mediated regulatory layer safeguarding embryonic cell fate transitions