An understanding of how heterozygous loss-of-function mutations in autism spectrum disorder (ASD) risk genes, such as TBR1, contribute to ASD remains elusive. Conditional Tbr1 deletion during late ...mouse gestation in cortical layer 6 neurons (Tbr1layer6 mutants) provides novel insights into its function, including dendritic patterning, synaptogenesis, and cell-intrinsic physiology. These phenotypes occur in heterozygotes, providing insights into mechanisms that may underlie ASD pathophysiology. Restoring expression of Wnt7b largely rescues the synaptic deficit in Tbr1layer6 mutant neurons. Furthermore, Tbr1layer6 heterozygotes have increased anxiety-like behavior, a phenotype seen ASD. Integrating TBR1 chromatin immunoprecipitation sequencing (ChIP-seq) and RNA sequencing (RNA-seq) data from layer 6 neurons and activity of TBR1-bound candidate enhancers provides evidence for how TBR1 regulates layer 6 properties. Moreover, several putative TBR1 targets are ASD risk genes, placing TBR1 in a central position both for ASD risk and for regulating transcriptional circuits that control multiple steps in layer 6 development essential for the assembly of neural circuits.
•Tbr1 specifies layer 6 dendritic patterning and cell-intrinsic physiology•Tbr1 promotes synapse numbers through Wnt7b•Tbr1 heterozygotes provide insight into ASD pathophysiology•TBR1 directly regulates transcriptional circuits that controls ASD risk genes
TBR1 directly regulates transcriptional circuits in heterozygous mutant mice that specify layer 6 identity and synapse number. As TBR1 is an ASD risk gene, our results provide insights into mechanisms that underlie ASD pathophysiology.
The chromatin remodeling gene CHD8 represents a central node in neurodevelopmental gene networks implicated in autism. We examined the impact of germline heterozygous frameshift Chd8 mutation on ...neurodevelopment in mice. Chd8
mice displayed normal social interactions with no repetitive behaviors but exhibited cognitive impairment correlated with increased regional brain volume, validating that phenotypes of Chd8
mice overlap pathology reported in humans with CHD8 mutations. We applied network analysis to characterize neurodevelopmental gene expression, revealing widespread transcriptional changes in Chd8
mice across pathways disrupted in neurodevelopmental disorders, including neurogenesis, synaptic processes and neuroimmune signaling. We identified a co-expression module with peak expression in early brain development featuring dysregulation of RNA processing, chromatin remodeling and cell-cycle genes enriched for promoter binding by Chd8, and we validated increased neuronal proliferation and developmental splicing perturbation in Chd8
mice. This integrative analysis offers an initial picture of the consequences of Chd8 haploinsufficiency for brain development.
Deleterious genetic variants in POGZ, which encodes the chromatin regulator Pogo Transposable Element with ZNF Domain protein, are strongly associated with autism spectrum disorder (ASD). Although it ...is a high-confidence ASD risk gene, the neurodevelopmental functions of POGZ remain unclear. Here we reveal the genomic binding of POGZ in the developing forebrain at euchromatic loci and gene regulatory elements (REs). We profile chromatin accessibility and gene expression in Pogz−/− mice and show that POGZ promotes the active chromatin state and transcription of clustered synaptic genes. We further demonstrate that POGZ forms a nuclear complex and co-occupies loci with ADNP, another high-confidence ASD risk gene, and provide evidence that POGZ regulates other neurodevelopmental disorder risk genes as well. Our results reveal a neurodevelopmental function of an ASD risk gene and identify molecular targets that may elucidate its function in ASD.
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•POGZ binds enhancers and promoters at euchromatic loci in the developing brain.•POGZ promotes neuronal gene expression and chromatin accessibility.•POGZ forms a nuclear complex with ADNP; Adnp expression is reduced in Pogz+/−.•POGZ bound sites are enriched for other autism risk genes and transposable elements.
Markenscoff-Papadimitriou et al. map POGZ occupancy in developing human and mouse brain tissue and find POGZ binds euchromatic loci at promoters and enhancers. Analyses of Pogz−/− mice reveal POGZ specifically promotes the chromatin accessibility and expression of clustered synapse genes, and biochemical analyses show POGZ co-occupies loci with ADNP.
DLX transcription factors (TFs) are master regulators of the developing vertebrate brain, driving forebrain GABAergic neuronal differentiation. Ablation of Dlx1&2 alters expression of genes that are ...critical for forebrain GABAergic development. We integrated epigenomic and transcriptomic analyses, complemented with in situ hybridization (ISH), and in vivo and in vitro studies of regulatory element (RE) function. This revealed the DLX-organized gene regulatory network at genomic, cellular, and spatial levels in mouse embryonic basal ganglia. DLX TFs perform dual activating and repressing functions; the consequences of their binding were determined by the sequence and genomic context of target loci. Our results reveal and, in part, explain the paradox of widespread DLX binding contrasted with a limited subset of target loci that are sensitive at the epigenomic and transcriptomic level to Dlx1&2 ablation. The regulatory properties identified here for DLX TFs suggest general mechanisms by which TFs orchestrate dynamic expression programs underlying neurodevelopment.
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•DLX proteins modulate expression of GABAergic neuronal differentiation genes•DLXs drive a complex regulatory network by transcriptional activation and inhibition•Genomic and epigenomic context and sequence predict DLX effect on gene expression•DLX regulatory wiring may reflect general mechanisms implicated in neurodevelopment
Lindtner et al. reveal the regulatory wiring organized by DLX transcription factors in forebrain GABAergic neuronal specification, by integrating functional genomic, epigenomic, and genetic data on a transgenic mouse model. This network determines key sequence-encoded regulatory elements and implicates a combination of histone modifications and biophysical interactions.
The packaging of DNA into chromatin determines the transcriptional potential of cells and is central to eukaryotic gene regulation. Case sequencing studies have revealed mutations to proteins that ...regulate chromatin state, known as chromatin remodeling factors, with causal roles in neurodevelopmental disorders. Chromodomain helicase DNA binding protein 8 (
) encodes a chromatin remodeling factor with among the highest
loss-of-function mutation rates in patients with autism spectrum disorder (ASD). However, mechanisms associated with
pathology have yet to be elucidated. We analyzed published transcriptomic data across
and
knockdown and knockout models and CHD8 binding across published ChIP-seq datasets to identify convergent mechanisms of gene regulation by CHD8. Differentially expressed genes (DEGs) across models varied, but overlap was observed between downregulated genes involved in neuronal development and function, cell cycle, chromatin dynamics, and RNA processing, and between upregulated genes involved in metabolism and immune response. Considering the variability in transcriptional changes and the cells and tissues represented across ChIP-seq analysis, we found a surprisingly consistent set of high-affinity CHD8 genomic interactions. CHD8 was enriched near promoters of genes involved in basic cell functions and gene regulation. Overlap between high-affinity CHD8 targets and DEGs shows that reduced dosage of
directly relates to decreased expression of cell cycle, chromatin organization, and RNA processing genes, but only in a subset of studies. This meta-analysis verifies CHD8 as a master regulator of gene expression and reveals a consistent set of high-affinity CHD8 targets across human, mouse, and rat
and
studies. These conserved regulatory targets include many genes that are also implicated in ASD. Our findings suggest a model where perturbation to dosage-sensitive CHD8 genomic interactions with a highly-conserved set of regulatory targets leads to model-specific downstream transcriptional impacts.
Genes with multiple co-active promoters appear common in brain, yet little is known about functional requirements for these potentially redundant genomic regulatory elements. SCN1A, which encodes the ...Na
1.1 sodium channel alpha subunit, is one such gene with two co-active promoters. Mutations in SCN1A are associated with epilepsy, including Dravet syndrome (DS). The majority of DS patients harbor coding mutations causing SCN1A haploinsufficiency; however, putative causal non-coding promoter mutations have been identified.
To determine the functional role of one of these potentially redundant Scn1a promoters, we focused on the non-coding Scn1a 1b regulatory region, previously described as a non-canonical alternative transcriptional start site. We generated a transgenic mouse line with deletion of the extended evolutionarily conserved 1b non-coding interval and characterized changes in gene and protein expression, and assessed seizure activity and alterations in behavior.
Mice harboring a deletion of the 1b non-coding interval exhibited surprisingly severe reductions of Scn1a and Na
1.1 expression throughout the brain. This was accompanied by electroencephalographic and thermal-evoked seizures, and behavioral deficits.
This work contributes to functional dissection of the regulatory wiring of a major epilepsy risk gene, SCN1A. We identified the 1b region as a critical disease-relevant regulatory element and provide evidence that non-canonical and seemingly redundant promoters can have essential function.
Traumatic brain injury (TBI) causes acute and lasting impacts on the brain, driving pathology along anatomical, cellular, and behavioral dimensions. Rodent models offer an opportunity to study the ...temporal progression of disease from injury to recovery. Transcriptomic and epigenomic analysis were applied to evaluate gene expression in ipsilateral hippocampus at 1 and 14 days after sham (
n
= 2 and 4, respectively per time point) and moderate lateral fluid percussion injury (
n
= 4 per time point). This enabled the identification of dynamic changes and differential gene expression (differentially expressed genes; DEGs) modules linked to underlying epigenetic response. We observed acute signatures associated with cell death, astrocytosis, and neurotransmission that largely recovered by 2 weeks. Inflammation and immune signatures segregated into upregulated modules with distinct expression trajectories and functions. Whereas most down-regulated genes recovered by 14 days, two modules with delayed and persistent changes were associated with cholesterol metabolism, amyloid beta clearance, and neurodegeneration. Differential expression was paralleled by changes in histone H3 lysine residue 4 trimethylation at the promoters of DEGs at 1 day post-TBI, with the strongest changes observed for inflammation and immune response genes. These results demonstrate how integrated genomics analysis in the pre-clinical setting has the potential to identify stage-specific biomarkers for injury and/or recovery. Though limited in scope here, our general strategy has the potential to capture pathological signatures over time and evaluate treatment efficacy at the systems level.
We uncovered a transcription factor (TF) network that regulates cortical regional patterning in radial glial stem cells. Screening the expression of hundreds of TFs in the developing mouse cortex ...identified 38 TFs that are expressed in gradients in the ventricular zone (VZ). We tested whether their cortical expression was altered in mutant mice with known patterning defects (
, and
), which enabled us to define a cortical regionalization TF network (CRTFN). To identify genomic programming underlying this network, we performed TF ChIP-seq and chromatin-looping conformation to identify enhancer-gene interactions. To map enhancers involved in regional patterning of cortical progenitors, we performed assays for epigenomic marks and DNA accessibility in VZ cells purified from wild-type and patterning mutant mice. This integrated approach has identified a CRTFN and VZ enhancers involved in cortical regional patterning in the mouse.
Enhancers integrate transcription factor signaling pathways that drive cell fate specification in the developing brain. We paired enhancer labeling and single-cell RNA-sequencing (scRNA-seq) to ...delineate and distinguish specification of neuronal lineages in mouse medial, lateral, and caudal ganglionic eminences (MGE, LGE, and CGE) at embryonic day (E)11.5. We show that scRNA-seq clustering using transcription factors improves resolution of regional and developmental populations, and that enhancer activities identify specific and overlapping GE-derived neuronal populations. First, we mapped the activities of seven evolutionarily conserved brain enhancers at single-cell resolution in vivo, finding that the selected enhancers had diverse activities in specific progenitor and neuronal populations across the GEs. We then applied enhancer-based labeling, scRNA-seq, and analysis of in situ hybridization data to distinguish transcriptionally distinct and spatially defined subtypes of MGE-derived GABAergic and cholinergic projection neurons and interneurons. Our results map developmental origins and specification paths underlying neurogenesis in the embryonic basal ganglia and showcase the power of scRNA-seq combined with enhancer-based labeling to resolve the complex paths of neuronal specification underlying mouse brain development.
Understanding biology through the use of genomic and epigenomic data presents significant challenges and opportunities. The vast amount of data that the modern high-throughput techniques have been ...generating has allowed significant advances across many areas, but the data have been largely underexplored due to challenges associated with high dimensionality, massive dataset size, and separating signal from noise. The efforts for the elucidation of complex biological systems, e.g. in development, have greatly benefited from omics data and strategies, but have been held back by the lack of sufficient computational methods to capture the whole wealth in the data. In this Dissertation research, my focus was to develop and apply computational statistical methods to more fully capture the value of transcriptomic and, most intensively, epigenomic data. To understand the gene regulatory control of critical processes leading to the patterning and specification of subpallial embryonic brain, I combined extensive published and novel transcriptomic, epigenomic, and chromosomal conformation data using innovative approaches and methods. In the first project, I computationally characterized the binding of the DLX family of transcription factors (TFs) to embryonic mouse primordial basal ganglia, and modeled the function of such binding based on TF binding and histone modification experimental data on a transgenic model generated by our research partners at UCSF to understand the mechanisms by which DLX control proliferation, migration and differentiation in early subpallial development. The second project moved the focus to the broader outstanding question in genetics, how the combinatorial binding of TFs coordinate to regulate gene expression. Addressing this question will be critical for understanding the regulation of complex processes in early brain development. There are limitations to what can be learned about combinatorial regulation from the reductionist single gene or single TF approach that has been the focus of genetics research. I integrated binding data for twelve TFs in embryonic mouse basal ganglia, and characterized combinatorial binding patterns in relation to associated gene expression, evolutionary conservation, and chromosomal topological features. Finally, I modeled the context-dependency of the mechanism of genomic control. In the final chapter, I used the same modeling approaches to a third project that studied the transcriptomic signatures following progression of pathophysiology in a rat model of traumatic brain injury. In this study, I was able to identify the gene networks involved in the clinical outcomes of healing, but also dysfunctional pathways that might lead to neurodegenerative diseases. These findings identified many genes and biological pathways associated with recovery following TBI, generating a new systems level understanding and revealing targets for future research and clinical investigation. In conclusion, the findings and developments explored in the research object of this Dissertation underscores the importance of robust methods and innovative approaches to explore the full potential of the genomic data we generate and in parallel revealed gene expression wiring of complex processes relevant to brain development and disorders.
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