1,721,159 research outputs found
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The Function and Regulation of AUTS2
Full text linkThe autism susceptibility candidate 2 (AUTS2) gene is associated with multiple neurological diseases, including autism, and has been implicated as an important gene in human-specific evolution. In this thesis, I begin (chapter 1) with an introduction reviewing the literature regarding AUTS2, including its discovery, expression, association with autism and other neurological and non-neurological traits, implication in human evolution, function, regulation, and genetic pathways. Part of the research included in this introduction was performed by me and co-authors, and described in detail in the following chapters. In chapter 2, I investigate the expression, function, and regulation of auts2. auts2 is expressed primarily in the zebrafish brain. Knockdown of this gene in zebrafish leads to a smaller head size, neuronal reduction and decreased mobility. I identified twenty-three functional zebrafish enhancers, ten of which are active in the brain. Mouse enhancer assays characterized three brain enhancers that overlap an ASD-associated deletion and four enhancers that reside in regions implicated in human evolution, two of which are active in the brain. Combined, I show that AUTS2 is important for neurodevelopment and expose candidate enhancer sequences in which nucleotide variation could lead to neurological disease and human-specific traits.In chapter 3, I investigate the regulatory role and targets of Auts2. Using ChIP-seq and RNA-seq on mouse embryonic day 16.5 forebrains, we I elucidated the gene regulatory networks of Auts2. It was found that the majority of promoters bound by Auts2 belong to genes highly expressed in the developing forebrain, suggesting that Auts2 is involved in transcriptional activation. Auts2 non-promoter bound regions significantly overlap developing brain-associated enhancer marks and are located near genes involved in neurodevelopment. Auts2 marked sequences are enriched for binding site motifs of neurodevelopmental transcription factors, including Pitx3 and TCF3. I characterized ten non-coding Auts2 marked sites near critical ASD-related genes for enhancer activity in zebrafish, four of which showed positive enhancer activity. Additionally, I characterized two of the positive brain enhancers near NRXN1 and ATP2B2 in mice. The results implicate Auts2 as an active regulator of important neurodevelopmental genes and pathways and identify novel genomic regions which could be associated with ASD and other neurodevelopmental diseases. In summary, this thesis investigates the function of AUTS2. I conclude that this gene is critical for the proper development of neurons, and may act as a cofactor to positively regulate genes expressed in the forebrain involved in neurodevelopment
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Cis-regulatory elements in limb development and human congenital malformations
Full text linkRegulatory elements provide information necessary for the spatial, temporal and dosage appropriate expression of genes. Developmental genes in particular rely on cis-regulatory enhancers to control expression in the various tissues where they are active and cause defects in development. Congenital limb malformations are the second most common class of human birth defects and can be caused both by environmental and genetic factors, and identifying the causal mutation in a patient with an isolated limb malformation is often difficult. The difficulty of identification may be due, in part, to the growing number of cases with isolated limb malformations that are shown to be the result of nucleotide changes in regulatory elements. These regulatory mutations affect gene expression in the developing limb and can cause dramatic changes to patterning, leading to congenital limb malformations. There are multiple examples of mutations in an enhancer known as the zone of polarizing activity regulatory sequence (ZRS) that cause preaxial polydactyly and other malformation phenotypes. The identification of further ZRS mutations along with changes they have to transcription factor binding and resulting phenotypes can help elucidate the mechanisms by which it controls gene expression. While the genes and pathways that determine specific limb signaling centers have been described, the identification of enhancers that determine these centers has been limited. It is possible to identify enhancers that are specific to the zone of polarizing activity (ZPA) and apical ectodermal ridge (AER) signaling centers by isolating these regions. Using H3K27ac ChIP-seq on mouse E11.5 ZPA and AER fluorescently sorted cells, I identified thousands of specific signaling center enhancers. Mouse transgenic assays confirmed that several of them function as ZPA and AER enhancers. Combined, these results provide novel ZPA and AER enhancers that may play important roles in limb development. Because the ZPA and AER have critical roles in establishing the three axes and patterning the limb, changes in enhancer function can result in malformations of limb structures
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Identifying and Dissecting Regulatory Elements that Drive Drug Response and Human Evolution
Full text linkGene regulation is known to contribute to the wide diversity of biological differences between cell types, individuals, and species. Enhancers are regulatory elements that determine when, where, and how much a protein-coding gene is expressed in every tissue. They contain short motifs called transcription factor binding sites and function through chromatin remodeling and DNA looping to activate transcription of their target genes. Due to their role in activating gene expression across tissues and developmental timepoints, disruption in enhancer function can lead to disease and morphological differences between species. By characterizing enhancers we can learn how genetic changes in non-coding DNA alter gene function and ultimately use this knowledge to diagnose and treat disease.Using RNA-sequencing and chromatin immunoprecipitation (ChIP) sequencing, I identified genome-wide antibiotic-induced changes in gene expression and regulation in HepG2 cells, a human liver cell line. More specifically, I found 209 genes responsive to penicillin-streptomycin (PenStrep), a commonly used cell culture antibiotic cocktail, and 9,514 H3K27ac peaks that were PenStrep-responsive. I also performed a massively parallel reporter assay (MPRA) to quantify enhancer activity of conserved DNA elements that have rapidly evolved in humans called human accelerated regions (HARs) in human and chimpanzee iPSC-derived neural and glial progenitor cells. This method allowed us to detect novel brain enhancers with species-specific function and dissect the regulatory architecture of these enhancers. Our results showed that the cis features or sequence level changes were greater drivers of differences in enhancer activity than the trans environment, or cell species and cell stage, that these sequences were tested in. My research sheds insight on the regulatory code driving drug response to common antibiotics, as well as the uniquely human patterns in early neurodevelopment
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Genome variation over multiple timescales and dimensions
Full text linkGenomic variation does not only include nucleotide changes, it also comprises changes in DNA shape, structure, epigenetic marks, and expression, all of which can occur over generations, cellular differentiation, the span of a few hours or a few millennia. This doctoral thesis explores the implications and opportunities presented by these multiple forms of genomic variation for genome editing, cellular differentiation, genome regulation and comparative genomics, all towards improving our understanding of genome evolution and development and benefiting human health
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Characterizing Regulatory Elements That Could Lead to Obesity Susceptibility
Full text linkHaploinsufficiency of the Single Minded homology 1 (SIM1) gene in humans and mice leads to severe obesity, suggesting that altered expression of SIM1, by way of regulatory elements such as enhancers, could predispose individuals to obesity. To identify enhancers that could regulate SIM1, we used comparative genomics coupled with zebrafish and mouse transgenic enhancer assays. Due to the dual role of Sim1 in hypothalamic development and in adult energy homeostasis, the enhancer activity of these sequences was annotated from embryonic to adult age. Of the seventeen tested sequences, two (SCE2 and SCE8) were found to have midbrain enhancer activity in zebrafish. Both SCE2 and SCE8 also exhibited embryonic hypothalamus enhancer expression in mice, and time course analysis of SCE2 activity showed overlapping expression to Sim1 from embryonic to adult age. Using a deletion series, we identified the critical region in SCE2 that is needed for hypothalamus enhancer activity. Sequencing this region in obese and lean cohorts revealed a higher prevalence of SNPs that were unique to obese individuals, with one variant reducing developmental enhancer activity in zebrafish. In summary, we have characterized two hypothalamus enhancers in the SIM1 locus and identified a set of obesity-specific SNPs within one of them, which may predispose individuals to obesity
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Genomic and Transcriptomic Examination of Functional Elements and Absent Sequences
Full text linkBackground: Genome-wide association studies have identified numerous disease-associated variants, but a vast majority are located in non-coding regions, making it challenging to understand their functional impact. This complexity necessitates new techniques to identify causal variants in non-coding regions and elucidate their specific cellular contexts and mechanisms of action. Here we present work i) examining mutations that create nullomers in the human genome to explore its potential utility in identifying pathogenic mutations and ii) a single-cell multi-omic study identifying the transcriptome and regulome of the human and mouse hypothalamus to identify regulatory regions of obesity-associated variants.Methods: (i) We generated all possible mutations of the human genome that can lead to emergence of a nullomer, and examine where in the genome they emerge. (ii) We apply single-cell RNA and ATAC sequencing to adult hypothalamus samples.
Results and Conclusions: (i) Our findings highlight CpG hypermutability and methylated cytosines as key elements leading to resurfacing of nullomers in individuals. We also showcase that nullomers can have applications in disease annotation and pathogenic variant identification. (ii) We identified regulatory elements of hypothalamus cell types and mapped obesity-associated variants to cell-type specific peaks. We validated these regions to be enhancers using CRISPR editing and CRISPRi
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Decoding Doublecortin Function Using Cellular Models and Genome Editing
Full text linkProper establishment of cortical structures during early brain development is vital to normal brain function. A key component of normal lamination of the cerebral cortex is the migration of neurons after they are born from precursor cells to their specific destination in one of the six cortical layers. Neuronal migration involves dynamic changes to microtubules and other cytoskeletal components at the tip of an extending axon or dendrite with the aid of microtubule-associated proteins (MAPs). Doublecortin (DCX) is a MAP that is highly and specifically expressed in immature, migrating neurons. Dysfunction of X-linked DCX causes lissencephaly in males, a malformation characterized by a lack of gyri in the cortex, and subcortical band heterotropia (SBH) or a "double cortex" in females. The role DCX plays in neuronal migration is not well understood and studies in mouse models have only investigated the effects of a complete knockout (KO) of Dcx, which resulted in no cortical lamination phenotype in male or female mice, in contrast to the conspicuous phenotypes observed in humans. However, documented disease-causing human DCX mutations involve a missense mutation in one of DCX’s microtubule binding domains, which has been shown to not remove DCX function entirely.Despite characterization of human-specific mutations and mouse knockout models, there remains an unmet need for elucidating the cellular and molecular mechanisms of patient-specific mutations in their native genetic context. I hypothesize that the limited phenotypes in Dcx KO mice are due to compensation by other proteins in Dcx’s absence, and not due to intrinsic species differences. In particular, I predict that the mutant phenotype observed in the cortex is due to altered binding to microtubules during neuronal migration, producing a dominant negative effect. To begin to elucidate the role of Doublecortin in regulation of neuronal migration, I have introduced a human disease-causing DCX mutation (T203R) into the endogenous mouse Dcx locus for in vivo experiments. These studies involving a disease-causing, patient-derived mutation of the endogenous Doublecortin locus in mouse contexts have yielded important insights into the biology of Doublecortin and cortical development, addressing unresolved mechanisms of patient-specific DCX mutations at the molecular and cellular level
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Development of a CRISPR activation-based approach for the treatment of SCN2A haploinsufficiency in Autism Spectrum Disorder
Full text linkHaploinsufficiency, having only one functional gene copy, is associated with close to 100 autism spectrum disorder (ASD) risk genes. Here, using SCN2A haploinsufficiency, a major ASD risk condition, we show that CRISPR activation (CRISPRa) of the existing functional copy at adolescent stages provides a viable therapeutic approach. First, we demonstrate the potential for a therapeutic to rescue electrophysiological deficits in mice by utilizing heterozygous Scn2a conditional knockin mice. Next, using an AAV-based CRISPRa approach, we rescue these electrophysiological deficits in Scn2a heterozygous mice and human SCN2A heterozygous excitatory neurons. Our results provide a novel therapeutic approach for numerous ASD-associated genes and also suggest that rescue Scn2a function, even in relatively mature developmental stages, could ameliorate neurodevelopmental phenotypes
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Functional Genomics Approaches for Investigating Human Evolution
Full text linkThe enumeration of human-specific genetic variants by comparative studies of ape genomes paired with advances in programmable genetic editing enables the functional interrogation of our evolutionary history. Because it is difficult to predict how millions of genomic alterations within the hominid lineage contribute to differences in phenotypes, there is a critical need for high-throughput, systematic approaches to probe genetic variation. Here, we develop a quantitative genome-scale platform for identifying the phenotypic consequences of human-specific genetic variants at a cellular and molecular level. First, we investigate the ancestral function of structural variant-sized human-specific deletions (hDels) by performing CRISPR interference (CRISPRi) genetic screens in chimpanzee induced pluripotent stem (iPS) cells. We further characterize the epigenetic state of chromatin at hDels using Omni ATAC-seq and CUT&Tag and perform single-cell CRISPRi (Perturb-seq) to identify their cis- and trans-regulatory target genes. We discover hDels removing cis-regulatory elements controlling the expression of proliferation-modifying genes including MBD3, MRPS14, and RPL26, and identify the cis-regulatory target genes of 16 nonessential hDels intersecting Omni ATAC-seq, H3K4me1, and H3K27ac. Separately, we investigate the evolution of gene regulatory networks during recent hominid evolution by performing paired CRISPRi genetic screens in human and chimpanzee iPS cells. We identify 75 genes with species-specific effects on cellular proliferation, many of which interact in biological pathways including RNA metabolism, chromatin organization, cell cycle phase transition, and UFMylation. Together, these findings underscore the importance of deletions as a source of evolutionary innovation and suggest that the regulation of essential cellular processes has evolved along the human lineage
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Transcriptional Regulation of Tracheoesophageal Fate Specification in the Mammalian Foregut
Full text linkSpecification of the trachea and esophagus from the embryonic foregut is critical for the function of the respiratory and digestive systems. Congenital birth defects associated with improper tracheoesophageal development in humans are common and have severe consequences to respiration and feeding that require immediate surgical intervention. During development, tracheoesophageal specification is dependent on proper dorsoventral patterning of the foregut endoderm tube. This involves the establishment of ventral NKX2.1 and dorsal SOX2 expression domains that are thought to promote tracheal and esophageal fate, respectively. Loss of Nkx2.1 results in failed foregut separation, and adoption of SOX2 expression throughout the common foregut tube. Similarly, loss of Sox2 results in a common foregut tube expressing NKX2.1, leading to the previous conclusion that both of these transcription factors are master regulators of tracheal and esophageal fate, respectively. However, our understanding of tracheoesophageal fate specification is limited by a lack of information on dorsoventral foregut patterning at the transcriptome level. In this study, we use genome-wide methods to understand how tracheal and esophageal lineages are specified during mouse embryonic development. We use single cell RNA-sequencing to define transcriptomic profiles of early developing mouse trachea, lung, and esophagus, and discover a multitude of previously unknown markers of these tissues. Transcriptomic analysis of Nkx2.1-/- mutant foreguts reveals that NKX2.1 loss does not result in lineage conversion to esophagus as previously hypothesized and exposes an NKX2.1-independent tracheoesophageal program. Using ChIP-seq against NKX2.1, we identify direct NKX2.1 regulatory targets and interrogate their combinatorial regulation by NKX2.1 and SOX2 in compound mouse mutant analysis. Amongst the novel targets we identify are Shh and Wnt7b, which we demonstrate are regulated by NKX2.1 to control tracheal and esophageal mesenchyme specification to cartilage and smooth muscle. Together, these data dramatically revise our understanding of how tracheal and esophageal cell types are specified during development and uncover a limited yet critical role for Nkx2.1 in this process
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