TALE and NF-Y co-occupancy marks enhancers of developmental control genes during zygotic genome activation in zebrafish [preprint]

Animal embryogenesis is initiated by maternal factors, but zygotic genome activation (ZGA) shifts control to the embryo at early blastula stages. ZGA is thought to be mediated by specialized maternally deposited transcription factors (TFs), but here we demonstrate that NF-Y and TALE – TFs with known later roles in embryogenesis – co-occupy unique genomic elements at zebrafish ZGA. We show that these elements are selectively associated with early-expressed genes involved in transcriptional regulation and possess enhancer activity in vivo. In contrast, we find that elements individually occupied by either NF-Y or TALE are associated with genes acting later in development – such that NF-Y controls a cilia gene expression program while TALE TFs control expression of hox genes. We conclude that NF-Y and TALE have a shared role at ZGA, but separate roles later during development, demonstrating that combinations of known TFs can regulate subsets of key developmental genes at vertebrate ZGA

Molecular Characterization of HOXA2 and HOXA3 Binding Properties

From MDPI via Jisc Publications RouterHistory: accepted 2021-11-30, pub-electronic 2021-12-03Publication status: PublishedFunder: SPARC (Scheme for Promotion of Academic and Research Collaboration) and UKIERI (United Kingdom India Research Initiative); Grant(s): project P657Funder: Biotechnology and Biological Sciences Research Council; Grant(s): BB/N00907X/1 and BB/T007761/1The highly conserved HOX homeodomain (HD) transcription factors (TFs) establish the identity of different body parts along the antero−posterior axis of bilaterian animals. Segment diversification and the morphogenesis of different structures is achieved by generating precise patterns of HOX expression along the antero−posterior axis and by the ability of different HOX TFs to instruct unique and specific transcriptional programs. However, HOX binding properties in vitro, characterised by the recognition of similar AT-rich binding sequences, do not account for the ability of different HOX to instruct segment-specific transcriptional programs. To address this problem, we previously compared HOXA2 and HOXA3 binding in vivo. Here, we explore if sequence motif enrichments observed in vivo are explained by binding affinities in vitro. Unexpectedly, we found that the highest enriched motif in HOXA2 peaks was not recognised by HOXA2 in vitro, highlighting the importance of investigating HOX binding in its physiological context. We also report the ability of HOXA2 and HOXA3 to heterodimerise, which may have functional consequences for the HOX patterning function in vivo

HOX paralogs selectively convert binding of ubiquitous transcription factors into tissue-specific patterns of enhancer activation [preprint]

Gene expression programs determine cell fate in embryonic development and their dysregulation results in disease. Transcription factors (TFs) control gene expression by binding to enhancers, but how TFs select and activate their target enhancers is still unclear. HOX TFs share conserved homeodomains with highly similar sequence recognition properties, yet they impart the identity of different animal body parts. To understand how HOX TFs control their specific transcriptional programs in vivo, we compared HOXA2 and HOXA3 binding profiles in the mouse embryo. HOXA2 and HOXA3 directly cooperate with TALE TFs and selectively target different subsets of a broad TALE chromatin platform. Binding of HOX and tissue-specific TFs convert low affinity TALE binding into high confidence, tissue-specific binding events, which bear the mark of active enhancers. We propose that HOX paralogs, alone and in combination with tissue-specific TFs, generate tissue-specific transcriptional outputs by modulating the activity of TALE TFs at selected enhancers

Laser Capture and Deep Sequencing Reveals the Transcriptomic Programmes Regulating the Onset of Pancreas and Liver Differentiation in Human Embryos.

To interrogate the alternative fates of pancreas and liver in the earliest stages of human organogenesis, we developed laser capture, RNA amplification, and computational analysis of deep sequencing. Pancreas-enriched gene expression was less conserved between human and mouse than for liver. The dorsal pancreatic bud was enriched for components of Notch, Wnt, BMP, and FGF signaling, almost all genes known to cause pancreatic agenesis or hypoplasia, and over 30 unexplored transcription factors. SOX9 and RORA were imputed as key regulators in pancreas compared with EP300, HNF4A, and FOXA family members in liver. Analyses implied that current in vitro human stem cell differentiation follows a dorsal rather than a ventral pancreatic program and pointed to additional factors for hepatic differentiation. In summary, we provide the transcriptional codes regulating the start of human liver and pancreas development to facilitate stem cell research and clinical interpretation without inter-species extrapolation.This project received support from the UK Medical Research Council (MRC) (R.E.J. was a clinical research training fellow; additional funding from MR/L009986/1 to N.B. and N.A.H.; and MR/J003352/1 to K.P.H.), the Academy of Medical Sciences (supported by Wellcome Trust, MRC, British Heart Foundation, Arthritis Research UK, the Royal College of Physicians and Diabetes UK) (R.E.J.), the Society for Endocrinology (R.E.J.), the Wellcome Trust (N.A.H. was a senior fellow in clinical science, 088566; additional support from grant 105610/Z/14/Z), and the British Council and JDRF (14BX15NHBG to N.A.H.)

A spatio-temporally constrained gene regulatory network directed by PBX1/2 acquires limb patterning specificity via HAND2.

A lingering question in developmental biology has centered on how transcription factors with widespread distribution in vertebrate embryos can perform tissue-specific functions. Here, using the murine hindlimb as a model, we investigate the elusive mechanisms whereby PBX TALE homeoproteins, viewed primarily as HOX cofactors, attain context-specific developmental roles despite ubiquitous presence in the embryo. We first demonstrate that mesenchymal-specific loss of PBX1/2 or the transcriptional regulator HAND2 generates similar limb phenotypes. By combining tissue-specific and temporally controlled mutagenesis with multi-omics approaches, we reconstruct a gene regulatory network (GRN) at organismal-level resolution that is collaboratively directed by PBX1/2 and HAND2 interactions in subsets of posterior hindlimb mesenchymal cells. Genome-wide profiling of PBX1 binding across multiple embryonic tissues further reveals that HAND2 interacts with subsets of PBX-bound regions to regulate limb-specific GRNs. Our research elucidates fundamental principles by which promiscuous transcription factors cooperate with cofactors that display domain-restricted localization to instruct tissue-specific developmental programs

Genome-wide occupancy links Hoxa2 to Wnt–β-catenin signaling in mouse embryonic development

The regulation of gene expression is central to developmental programs and largely depends on the binding of sequence-specific transcription factors with cis-regulatory elements in the genome. Hox transcription factors specify the spatial coordinates of the body axis in all animals with bilateral symmetry, but a detailed knowledge of their molecular function in instructing cell fates is lacking. Here, we used chromatin immunoprecipitation with massively parallel sequencing (ChIP-seq) to identify Hoxa2 genomic locations in a time and space when it is actively instructing embryonic development in mouse. Our data reveals that Hoxa2 has large genome coverage and potentially regulates thousands of genes. Sequence analysis of Hoxa2-bound regions identifies high occurrence of two main classes of motifs, corresponding to Hox and Pbx–Hox recognition sequences. Examination of the binding targets of Hoxa2 faithfully captures the processes regulated by Hoxa2 during embryonic development; in addition, it uncovers a large cluster of potential targets involved in the Wnt-signaling pathway. In vivo examination of canonical Wnt–β-catenin signaling reveals activity specifically in Hoxa2 domain of expression, and this is undetectable in Hoxa2 mutant embryos. The comprehensive mapping of Hoxa2-binding sites provides a framework to study Hox regulatory networks in vertebrate developmental processes

Osteopontin is a novel downstream target of SOX9 with diagnostic implications for progression of liver fibrosis in humans

Osteopontin (OPN) is an important component of the extracellular matrix (ECM), which promotes liver fibrosis and has been described as a biomarker for its severity. Previously, we have demonstrated that Sex-determining region Y-box 9 (SOX9) is ectopically expressed during activation of hepatic stellate cells (HSC) when it is responsible for the production of type 1 collagen, which causes scar formation in liver fibrosis. Here, we demonstrate that SOX9 regulates OPN. During normal development and in the mature liver, SOX9 and OPN are coexpressed in the biliary duct. In rodent and human models of fibrosis, both proteins were increased and colocalized to fibrotic regions in vivo and in culture-activated HSCs. SOX9 bound a conserved upstream region of the OPN gene, and abrogation of Sox9 in HSCs significantly decreased OPN production. Hedgehog (Hh) signaling has previously been shown to regulate OPN expression directly by glioblastoma (GLI) 1. Our data indicate that in models of liver fibrosis, Hh signaling more likely acts through SOX9 to modulate OPN. In contrast to Gli2 and Gli3, Gli1 is sparse in HSCs and is not increased upon activation. Furthermore, reduction of GLI2, but not GLI3, decreased the expression of both SOX9 and OPN, whereas overexpressing SOX9 or constitutively active GLI2 could rescue the antagonistic effects of cyclopamine on OPN expression. Conclusion: These data reinforce SOX9, downstream of Hh signaling, as a core factor mediating the expression of ECM components involved in liver fibrosis. Understanding the role and regulation of SOX9 during liver fibrosis will provide insight into its potential modulation as an antifibrotic therapy or as a means of identifying potential ECM targets, similar to OPN, as biomarkers of fibrosis. (HEPATOLOGY 2012;56:1108–1116

Dynamic changes in the epigenomic landscape regulate human organogenesis and link to developmental disorders

From Springer Nature via Jisc Publications RouterHistory: received 2019-10-04, accepted 2020-06-18, registration 2020-06-24, pub-electronic 2020-08-06, online 2020-08-06, collection 2020-12Publication status: PublishedFunder: RCUK | Medical Research Council (MRC); doi: https://doi.org/10.13039/501100000265; Grant(s): CRTF, PhD studentship, MR/J003352/1, MR/L009986/1, MR/L009986/1, MR/S036121/1, MR/000638/1Funder: Academy of Medical Sciences; doi: https://doi.org/10.13039/501100000691; Grant(s): Lecturer starter grantFunder: Wellcome Trust (Wellcome); doi: https://doi.org/10.13039/100004440; Grant(s): 088566, 097820, 105610Abstract: How the genome activates or silences transcriptional programmes governs organ formation. Little is known in human embryos undermining our ability to benchmark the fidelity of stem cell differentiation or cell programming, or interpret the pathogenicity of noncoding variation. Here, we study histone modifications across thirteen tissues during human organogenesis. We integrate the data with transcription to build an overview of how the human genome differentially regulates alternative organ fates including by repression. Promoters from nearly 20,000 genes partition into discrete states. Key developmental gene sets are actively repressed outside of the appropriate organ without obvious bivalency. Candidate enhancers, functional in zebrafish, allow imputation of tissue-specific and shared patterns of transcription factor binding. Overlaying more than 700 noncoding mutations from patients with developmental disorders allows correlation to unanticipated target genes. Taken together, the data provide a comprehensive genomic framework for investigating normal and abnormal human development

E-Cadherin Acts as a Regulator of Transcripts Associated with a Wide Range of Cellular Processes in Mouse Embryonic Stem Cells

We have recently shown that expression of the cell adhesion molecule E-cadherin is required for LIF-dependent pluripotency of mouse embryonic stem (ES) cells.In this study, we have assessed global transcript expression in E-cadherin null (Ecad-/-) ES cells cultured in either the presence or absence of LIF and compared these to the parental cell line wtD3.We show that LIF has little effect on the transcript profile of Ecad-/- ES cells, with statistically significant transcript alterations observed only for Sp8 and Stat3. Comparison of Ecad-/- and wtD3 ES cells cultured in LIF demonstrated significant alterations in the transcript profile, with effects not only confined to cell adhesion and motility but also affecting, for example, primary metabolic processes, catabolism and genes associated with apoptosis. Ecad-/- ES cells share similar, although not identical, gene expression profiles to epiblast-derived pluripotent stem cells, suggesting that E-cadherin expression may inhibit inner cell mass to epiblast transition. We further show that Ecad-/- ES cells maintain a functional β-catenin pool that is able to induce β-catenin/TCF-mediated transactivation but, contrary to previous findings, do not display endogenous β-catenin/TCF-mediated transactivation. We conclude that loss of E-cadherin in mouse ES cells leads to significant transcript alterations independently of β-catenin/TCF transactivation

Epimorphin alters the inhibitory effects of SOX9 on Mmp13 in activated hepatic stellate cells.

Liver fibrosis is a major cause of morbidity and mortality. It is characterised by excessive extracellular matrix (ECM) deposition from activated hepatic stellate cells (HSCs). Although potentially reversible, treatment remains limited. Understanding how ECM influences the pathogenesis of the disease may provide insight into novel therapeutic targets for the disease. The extracellular protein Epimorphin (EPIM) has been implicated in tissue repair mechanisms in several tissues, partially, through its ability to manipulate proteases. In this study, we have identified that EPIM modulates the ECM environment produced by activated hepatic stellate cells (HSCs), in part, through down-regulation of pro-fibrotic Sex-determining region Y-box 9 (SOX9).Influence of EPIM on ECM was investigated in cultured primary rat HSCs. Activated HSCs were treated with recombinant EPIM or SOX9 siRNA. Core fibrotic factors were evaluated by immunoblotting, qPCR and chromatin immunoprecipitation (ChIP).During HSC activation EPIM became significantly decreased in contrast to pro-fibrotic markers SOX9, Collagen type 1 (COL1), and α-Smooth muscle actin (α-SMA). Treatment of activated HSCs with recombinant EPIM caused a reduction in α-SMA, SOX9, COL1 and Osteopontin (OPN), while increasing expression of the collagenase matrix metalloproteinase 13 (MMP13). Sox9 abrogation in activated HSCs increased EPIM and MMP13 expression.These data provide evidence for EPIM and SOX9 functioning by mutual negative feedback to regulate attributes of the quiescent or activated state of HSCs. Further understanding of EPIM's role may lead to opportunities to modulate SOX9 as a therapeutic avenue for liver fibrosis