CHARGE syndrome-associated CHD7 acts at ISL1-regulated enhancers to modulate second heart field gene expression

Aims: Haploinsufficiency of the chromo-domain protein CHD7 underlies most cases of CHARGE syndrome, a multisystem birth defect including congenital heart malformation. Context specific roles for CHD7 in various stem, progenitor, and differentiated cell lineages have been reported. Previously, we showed severe defects when Chd7 is absent from cardiopharyngeal mesoderm (CPM). Here, we investigate altered gene expression in the CPM and identify specific CHD7-bound target genes with known roles in the morphogenesis of affected structures. / Methods and results: We generated conditional KO of Chd7 in CPM and analysed cardiac progenitor cells using transcriptomic and epigenomic analyses, in vivo expression analysis, and bioinformatic comparisons with existing datasets. We show CHD7 is required for correct expression of several genes established as major players in cardiac development, especially within the second heart field (SHF). We identified CHD7 binding sites in cardiac progenitor cells and found strong association with histone marks suggestive of dynamically regulated enhancers during the mesodermal to cardiac progenitor transition of mESC differentiation. Moreover, CHD7 shares a subset of its target sites with ISL1, a pioneer transcription factor in the cardiogenic gene regulatory network, including one enhancer modulating Fgf10 expression in SHF progenitor cells vs. differentiating cardiomyocytes. / Conclusion: We show that CHD7 interacts with ISL1, binds ISL1-regulated cardiac enhancers, and modulates gene expression across the mesodermal heart fields during cardiac morphogenesis

The Role of CHD7 in the Transcriptional Control of Heart Development

Chromatin remodelling provides a key mechanism for the regulation of gene expression through dynamic alterations in nucleosome occupancy at promoters and enhancers. Haploinsufficiency for the ATP-dependent chromatin remodeller chromodomain-helicase-DNA-binding protein 7 (CHD7) causes human CHARGE syndrome. CHARGE is characterised by a distinct pattern of congenital anomalies, including cardiovascular malformations, and has traditionally been considered a neurocristopathy. However, a number of the congenital heart defects associated with CHARGE cannot be attributed to disruption to the neural crest alone. This thesis therefore addresses the tissue-specific requirements and roles for CHD7 during cardiogenesis. CHD7 protein is shown to be present throughout the developing heart until E13.5. Conditional ablation of Chd7 in the early cardiogenic mesoderm results in embryonic lethality due to severe cardiovascular defects. These include haemorrhaging and oedema, major venous and arterial pole malformations, and disruption to cardiac innervation and vascularisation. To further dissect tissue-specific requirements for CHD7, cardiomyocyte-, second heart field- and endothelial-specific knockdowns were also performed. Each cross results in a milder subset of the cardiac defects observed after mesodermal ablation, indicating that CHD7 is required in multiple lineages within the cardiogenic mesoderm. Microarray analysis and validation by in situ hybridisation were used to identify genes dysregulated in the heart following mesodermal Chd7 ablation. These included components of the Semaphorin and Slit-Robo signalling pathways, which have known roles in heart development. Furthermore, aberrant expression of genes involved in calcium handling within cardiomyocytes is seen. Excitation-contraction coupling is disrupted in mutant embryonic cardiomyocytes, demonstrating relevance of the gene expression changes at the cellular level. This work reveals a requirement for CHD7 in mesodermal cardiac progenitors for both inflow and outflow tract development. Novel pathways are identified downstream of CHD7 activity in the developing heart, including the extracellular Semaphorin and Slit-Robo pathways, as well as components of the excitation-contraction coupling machinery

Trithorax group proteins: switching genes on and keeping them active

Cellular memory is provided by two counteracting groups of chromatin proteins termed Trithorax group (TrxG) and Polycomb group (PcG) proteins. TrxG proteins activate transcription and are perhaps best known because of the involvement of the TrxG protein MLL in leukaemia. However, in terms of molecular analysis, they have lived in the shadow of their more famous counterparts, the PcG proteins. Recent advances have improved our understanding of TrxG protein function and demonstrated that the heterogeneous group of TrxG proteins is of critical importance in the epigenetic regulation of the cell cycle, senescence, DNA damage and stem cell biology

Investigating the Role of the Nucleosome Remodeling Factor NURF as a Regulator of Gene Expression

The nucleosome remodeling factor (NURF) is an evolutionary conserved ATP-dependent chromatin remodeling factor. It was first isolated from Drosophila as a complex with enzymatic activity that once recruited to nucleosome, it slides the nucleosome to provide accessibility for transcription factors. Since then, numerous works from animal models and cell lines show the role of NURF as a regulator of gene expression. NURF interacts with H3K4me3 and sequence specific transcription factors that recruit the complex to promoter regions. Whether this is the only mechanism by which NURF regulates gene expression is not known. However, other ATP-dependent chromatin remodeling complexes are known to regulate gene expression independent from transcription initiation. In order to explore the role of NURF in regulating gene expression, we utilized two genome wide approaches to map NURF binding and NURF dependent changes in chromatin structure using ChIP-Seq and FAIRE-Seq, respectively. From these analyses, we discovered that NURF broadly localizes in the genome with preferences to gene bodies and 3’ends of genes. Also, we found that NURF maintains open chromatin regions at upstream, intron and downstream of genes. These novel findings shed light on new roles for NURF complex within genes, in addition to its classical role at promoter regions. Furthermore, we discovered the function of a previously uncharacterized domain in the NURF specific subunit BPTF. We show that the N-terminal the plant homeodomain (PHD) of BPTF directly interacts with THOC4, a protein associated with RNA-pol 2. Also, we show using ChIP analyses that this interaction recruits BPTF to gene bodies. Next, we investigated functional consequences for NURF recruitment to gene bodies using Cyclin D1 (Ccnd1) gene as a model. These analyses revealed that NURF is required for normal mRNA processing and loss of NURF induces intron retention, which results in unstable transcripts. Finally, we show that the defect in mRNA processing is not specific to the Ccnd1 gene, as we observe similar defects in four other BPTF dependent genes. Together, our work uncovered new role of mammalian NURF complex in regulating gene expression through mRNA processing

Transcription factor MITF and remodeller BRG1 define chromatin organisation at regulatory elements in melanoma cells

Microphthalmia-associated transcription factor (MITF) is the master regulator of the melanocyte lineage. To understand how MITF regulates transcription, we used tandem affinity purification and mass spectrometry to define a comprehensive MITF interactome identifying novel cofactors involved in transcription, DNA replication and repair, and chromatin organisation. We show that MITF interacts with a PBAF chromatin remodelling complex comprising BRG1 and CHD7. BRG1 is essential for melanoma cell proliferation in vitro and for normal melanocyte development in vivo. MITF and SOX10 actively recruit BRG1 to a set of MITF-associated regulatory elements (MAREs) at active enhancers. Combinations of MITF, SOX10, TFAP2A, and YY1 bind between two BRG1-occupied nucleosomes thus defining both a signature of transcription factors essential for the melanocyte lineage and a specific chromatin organisation of the regulatory elements they occupy. BRG1 also regulates the dynamics of MITF genomic occupancy. MITF-BRG1 interplay thus plays an essential role in transcription regulation in melanoma

Defining Neuronal Identity Using MicroRNA-Mediated Reprogramming

Cell fate reprogramming is transforming our understanding of the establishment and maintenance of cellular identity. In addition, reprogramming holds great promise to model diseases affecting cell types that are prohibitively difficult to study, such as human neurons. Overexpression of the brain-enriched microRNAs (miRNAs), miR-9/9* and miR-124 (miR-9/9*-124) results in reprogramming human somatic cells into neurons and has recently been used to generate specific neuronal subtypes affected in neurodegenerative disorders. However, the mechanisms governing the ability of miR-9/9*-124 to generate alternative subtypes of neurons remained unknown. In this thesis, I report that overexpressing miR-9/9*-124 triggers reconfiguration of chromatin accessibility, DNA methylation, and mRNA expression to induce a default neuronal state. MiR-9/9*-124-induced neurons (miNs) are functionally excitable and are uncommitted towards specific subtypes yet possess open chromatin at neuronal subtype-specific loci, suggesting such identity can be imparted by additional lineage-specific transcription factors. Consistently, we show ISL1 and LHX3 selectively drive conversion to a highly homogenous population of human spinal cord motor neurons. This work shows that modular synergism between miRNAs and neuronal subtype-specific transcription factors can drive lineage-specific neuronal reprogramming, thereby providing a general platform for high-efficiency generation of distinct subtypes of human neurons. Since many neurodegenerative diseases occur after development, modeling them requires reprogramming methods capable of generating functionally mature neurons. However, few robust molecular hallmarks existed to identify such neurons, or to compare efficiencies between reprogramming methods. Recent studies demonstrated that active long genes (\u3e100 kb from transcription start to end) are highly enriched in neurons, which provided an opportunity to identify neurons based on the expression of these long genes. We therefore worked to develop an R package, LONGO, to analyze gene expression based on gene length. We developed a systematic analysis of long gene expression (LGE) in RNA-seq or microarray data to enable validation of neuronal identity at the single-cell and population levels. By combining this conceptual advancement and statistical tool in a user-friendly and interactive software package, we intended to encourage and simplify further investigation into LGE, particularly as it applies to validating and improving neuronal differentiation and reprogramming methodologies. Using this tool, I found by single-cell RNA sequencing that microRNA-mediated neuronal reprogramming of human adult fibroblasts yields a homogenous population of mature neurons, and that LGE distinguishes mature from immature neurons. I found that LGE correlates with expression of neuronal subunits of the Swi/Snf-like (BAF) chromatin remodeling complex, such as ACTL6B/BAF53b. Finally, I found that the loss of a functional neuronal BAF complex, as well as chemical inhibition of topoisomerase I, decreases LGE and reduces spontaneous electrical activity. Together, these results provide mechanistic insights into microRNA-mediated neuronal reprogramming, and demonstrate a transcriptomic feature of functionally mature neurons

Phenotyping the potential antagonistic knock-out of the chromatin remodeler EZH2 and CHD7 in neural stem cells and the adult brain

CHARGE syndrome describes a combination of severe developmental defects including, among other things, a delayed or retarded mental development, sometimes combined with a variety of psychological symptoms. Especially, the molecular mechanisms behind this brain phenotype is little understood, and cannot be treated effectively up to now. This syndrome is caused by a heterozygous mutation in the Chromodomain helicase protein 7 (chd7) gene, which encodes an epigenetically active protein (CHD7) mediating nucleosome sliding, and associated with open and active chromatin (H3K27ac, Feng et al., 2017). Another epigenetically active protein fulfilling an opposite task is EZH2 (the catalytic part of the polycomb repressive complex 2 (PRC2)) which silences gene expression by methylating histon 3 lysin 27. Both EZH2 and CHD7 are expressed in overlapping brain regions, and a knock-out of the respective protein has opposing effects on neuronal branching (Feng et al., 2013 and Liu, Y.). With these hints it was hypothesized that EZH2 might counteract CHD7 deficiency and could be beneficial for the CHARGE condition. In the frame of this thesis I was able to generate and analyze a CHARGE mouse (CMVCre;CHD7+/fl) that displayed a variety of CHARGE related symptoms. Heterozygous chd7 mutant mice had a reduced body weight, were significantly more active, developed sever eye malformations with narrowed palpebral fissures, significantly smaller eye diameter, a thinner retina and inner nuclear layer, and partially completely dysfunctional eyes. Furthermore, the cerebellum of CHARGE mice showed a missing anterior lobe, and a disorganized purkinje cell layer with less primary branches. Also CHARGE mice have less neurogenesis ind the hippocampus. Following the main hypothesis a genetic rescue (CMVCre;CHD7+/fl;EZH2+/fl) could restore; body and brain size, the strong eye malformations observed under CHARGE conditions, the amount of neural stem cells in the dentate gyrus, and the cerebella purkinje cell layer. Moreover, an RNA sequencing of the cerebellum tissue revealed that the gene expression of many genes in the double knock-out was indeed rescued. Furthermore, a homozygous inducible double knock-out of chd7 and ezh2 (NesCreERT2;CHD7fl/fl;EZH2fl/fl) rescued the neural stem cell pool in comparison to a chd7 single knock-out. Finally, these findings encourage me to use a very potent EZH2 inhibitor to treat neural stem cells in vitro and in vivo, thus attempting a novel CHD7 deficient treatment

Doctor of Philosophy

dissertationCells of the early vertebrate embryo are distinct in their ability to commit into any cell lineage. How the embryo acquires this remarkable plasticity from two terminally differentiated gametes remains largely unknown. The plasticity in early embryo relies on achieving a unique transcriptome, which is regulated at multiple levels - including chromatin accessibility at developmental enhancers and genes. To understand the global landscape of chromatin accessibility during early embryogenesis, we utilized zebrafish embryos and explored three aspects of chromatin regulation. We first focused on the ATPase subunits (Brg1 and Brm) of SWI/SNF complexes, which are important regulators of chromatin accessibility and gene expression in all eukaryotes. To understand where they act in the genome, we profiled the occupancy of Brg1 and Brm by ChIP-seq at three early embryonic stages around the major onset of zygotic genome activation. We observed the occupancy of Brg1 and Brm during early embryogenesis is highly dynamic. The promoters of key pluripotency factors and other developmental transcription factors are robustly occupied by Brg1 and Brm. Interestingly, Brg1, but not Brm, is highly correlated with active histone modifications. However, only Brm commonly occupies gene bodies, which is dependent on transcription elongation. This work suggests SWI/SNF complexes might play important roles during early embryogenesis, and also reveals distinct roles of Brg1 and Brm in early zebrafish development. We then profiled the global landscape of accessible chromatin by ATAC-seq at three embryonic stages, as well as one differentiated tissue, adult liver. The data suggest chromatin accessibility increases during early embryogenesis. Here, 60% of open chromatin regions reside at genic regions and are highly enriched at promoters. Furthermore, many interesting candidate transcription factors are revealed based on motif analyses. Finally, ATAC-seq fragments with length of 120-220bp, together with MNase-seq date are used to profile nucleosome positioning. Our data determines nucleosome positioning during early embryogenesis, also discovered many interesting sequence characteristics involved in nucleosome positioning at various gene features. In summary, this work has extensively investigated the dynamics of chromatin landscape and the role of chromatin remodelers during early zebrafish development, which allow the comprehensive understanding of the regulation during early embryogenesis