Search

Haploid cell lines are a valuable research tool with broad applicability for genetic assays. As such the fully haploid human cell line, eHAP1, has been used in a wide array of studies. However, the absence of a corresponding reference genome sequence for this cell line has limited the potential for more widespread applications to experiments dependent on available sequence, like capture-clone methodologies. We generated ~15× coverage Nanopore long reads from ten GridION flowcells and utilized this data to assemble a de novo draft genome using minimap and miniasm and subsequently polished using Racon. This assembly was further polished using previously generated, low-coverage, Illumina short reads with Pilon and ntEdit. This resulted in a hybrid eHAP1 assembly with >90% complete BUSCO scores. We further assessed the eHAP1 long read data for structural variants using Sniffles and identify a variety of rearrangements, including a previously established Philadelphia translocation. Finally, we demonstrate how some of these variants overlap open chromatin regions, potentially impacting regulatory regions. By integrating both long and short reads, we generated a high-quality reference assembly for eHAP1 cells. The union of long and short reads demonstrates the utility in combining sequencing platforms to generate a high-quality reference genome de novo solely from low coverage data. We expect the resulting eHAP1 genome assembly to provide a useful resource to enable novel experimental applications in this important model cell line. • Generation of a reference genome for the human haploid cell line, eHAP1 • Long but noisy Nanopore sequencing combined with accurate but short Illumina sequencing yields a polished reference genome • Structural variants, including the Philadelphia translocation, can be identified using exclusively long reads [ABSTRACT FROM AUTHOR]

Background: Identifying functional non-coding variation is critical for defining the genetic contributions to human disease. While single-nucleotide polymorphisms (SNPs) within cis-acting transcriptional regulatory elements have been implicated in disease pathogenesis, not all cell types have been assessed and functional validations have been limited. In particular, the cells of the peripheral nervous system have been excluded from genome-wide efforts to link non-coding SNPs to altered gene function. Addressing this gap is essential for defining the genetic architecture of diseases that affect the peripheral nerve. We developed a computational pipeline to identify SNPs that affect regulatory function (rSNPs) and evaluated our predictions on a set of 144 regions in Schwann cells, motor neurons, and muscle cells. Results: We identified 28 regions that display regulatory activity in at least one cell type and 13 SNPs that affect regulatory function. We then tailored our pipeline to one peripheral nerve cell type by incorporating SOX10 ChIP-Seq data; SOX10 is essential for Schwann cells. We prioritized 22 putative SOX10 response elements harboring a SNP and rapidly validated two rSNPs. We then selected one of these elements for further characterization to assess the biological relevance of our approach. Deletion of the element from the genome of cultured Schwann cells—followed by differential gene expression studies—revealed Tubb2b as a candidate target gene. Studying the enhancer in developing mouse embryos revealed activity in SOX10-positive cells including the dorsal root ganglia and melanoblasts. Conclusions: Our efforts provide insight into the utility of employing strict conservation for rSNP discovery. This strategy, combined with functional analyses, can yield candidate target genes. In support of this, our efforts suggest that investigating the role of Tubb2b in SOX10-positive cells may reveal novel biology within these cell populations. [ABSTRACT FROM AUTHOR]

Background: The transcription factor SOX10 is essential for all stages of Schwann cell development including myelination. SOX10 cooperates with other transcription factors to activate the expression of key myelin genes in Schwann cells and is therefore a context-dependent, pro-myelination transcription factor. As such, the identification of genes regulated by SOX10 will provide insight into Schwann cell biology and related diseases. While genome-wide studies have successfully revealed SOX10 target genes, these efforts mainly focused on myelinating stages of Schwann cell development. We propose that less-biased approaches will reveal novel functions of SOX10 outside of myelination. Results: We developed a stringent, computational-based screen for genome-wide identification of SOX10 response elements. Experimental validation of a pilot set of predicted binding sites in multiple systems revealed that SOX10 directly regulates a previously unreported alternative promoter at SOX6, which encodes a transcription factor that inhibits glial cell differentiation. We further explored the utility of our computational approach by combining it with DNase-seq analysis in cultured Schwann cells and previously published SOX10 ChIP-seq data from rat sciatic nerve. Remarkably, this analysis enriched for genomic segments that map to loci involved in the negative regulation of gliogenesis including SOX5, SOX6, NOTCH1, HMGA2, HES1, MYCN, ID4, and ID2. Functional studies in Schwann cells revealed that: (1) all eight loci are expressed prior to myelination and down-regulated subsequent to myelination; (2) seven of the eight loci harbor validated SOX10 binding sites; and (3) seven of the eight loci are down-regulated upon repressing SOX10 function. Conclusions: Our computational strategy revealed a putative novel function for SOX10 in Schwann cells, which suggests a model where SOX10 activates the expression of genes that inhibit myelination during non-myelinating stages of Schwann cell development. Importantly, the computational and functional datasets we present here will be valuable for the study of transcriptional regulation, SOX protein function, and glial cell biology [ABSTRACT FROM AUTHOR]

To overcome the ethical and technical limitations of in vivo human disease models, the broader scientific community frequently employs model organism-derived cell lines to investigate disease mechanisms, pathways, and therapeutic strategies. Despite the widespread use of certain in vitro models, many still lack contemporary genomic analysis supporting their use as a proxy for the affected human cells and tissues. Consequently, it is imperative to determine how accurately and effectively any proposed biological surrogate may reflect the biological processes it is assumed to model. One such cellular surrogate of human disease is the established mouse neural precursor cell line, SN4741, which has been used to elucidate mechanisms of neurotoxicity in Parkinson disease for over 25 years. Here, we are using a combination of classic and contemporary genomic techniques – karyotyping, RT-qPCR, single cell RNA-seq, bulk RNA-seq, and ATAC-seq – to characterize the transcriptional landscape, chromatin landscape, and genomic architecture of this cell line, and evaluate its suitability as a proxy for midbrain dopaminergic neurons in the study of Parkinson disease. We find that SN4741 cells possess an unstable triploidy and consistently exhibits low expression of dopaminergic neuron markers across assays, even when the cell line is shifted to the non-permissive temperature that drives differentiation. The transcriptional signatures of SN4741 cells suggest that they are maintained in an undifferentiated state at the permissive temperature and differentiate into immature neurons at the non-permissive temperature; however, they may not be dopaminergic neuron precursors, as previously suggested. Additionally, the chromatin landscapes of SN4741 cells, in both the differentiated and undifferentiated states, are not concordant with the open chromatin profiles of ex vivo, mouse E15.5 forebrain- or midbrain-derived dopaminergic neurons. Overall, our data suggest that SN4741 cells may reflect early aspects of neuronal differentiation but are likely not a suitable proxy for dopaminergic neurons as previously thought. The implications of this study extend broadly, illuminating the need for robust biological and genomic rationale underpinning the use of in vitro models of molecular processes. [ABSTRACT FROM AUTHOR]

The progressive loss of midbrain (MB) dopaminergic (DA) neurons defines the motor features of Parkinson disease (PD), and modulation of risk by common variants in PD has been well established through genome-wide association studies (GWASs). We acquired open chromatin signatures of purified embryonic mouse MB DA neurons because we anticipated that a fraction of PD-associated genetic variation might mediate the variants' effects within this neuronal population. Correlation with >2,300 putative enhancers assayed in mice revealed enrichment for MB cis-regulatory elements (CREs), and these data were reinforced by transgenic analyses of six additional sequences in zebrafish and mice. One CRE, within intron 4 of the familial PD gene SNCA , directed reporter expression in catecholaminergic neurons from transgenic mice and zebrafish. Sequencing of this CRE in 986 individuals with PD and 992 controls revealed two common variants associated with elevated PD risk. To assess potential mechanisms of action, we screened >16,000 proteins for DNA binding capacity and identified a subset whose binding is impacted by these enhancer variants. Additional genotyping across the SNCA locus identified a single PD-associated haplotype, containing the minor alleles of both of the aforementioned PD-risk variants. Our work posits a model for how common variation at SNCA might modulate PD risk and highlights the value of cell-context-dependent guided searches for functional non-coding variation. [ABSTRACT FROM AUTHOR]

Genetic variation modulating risk of sporadic Parkinson disease (PD) has been primarily explored through genome-wide association studies (GWASs). However, like many other common genetic diseases, the impacted genes remain largely unknown. Here, we used single- cell RNA-seq to characterize dopaminergic (DA) neuron populations in the mouse brain at embryonic and early postnatal time points. These data facilitated unbiased identification of DA neuron subpopulations through their unique transcriptional profiles, including a postnatal neuroblast population and substantia nigra (SN) DA neurons. We use these population-specific data to develop a scoring system to prioritize candidate genes in all 49 GWAS intervals implicated in PD risk, including genes with known PD associations and many with extensive supporting literature. As proof of principle, we confirm that the nigrostriatal pathway is compromised in Cplxl-null mice. Ultimately, this systematic approach establishes biologically pertinent candidates and testable hypotheses for sporadic PD, informing a new era of PD genetic research. [ABSTRACT FROM AUTHOR]