Biology subjects

Wheeler, V.

Publications and source records attributed to Wheeler, V..

3 recordsLinked to original sources

bioRxiv2018

A Patient-Derived Cellular Model for Huntington Disease Reveals Phenotypes at Clinically Relevant CAG Lengths

The huntingtin protein participates in several cellular processes that are disrupted when the polyglutamine tract is expanded beyond a threshold of 37 CAG DNA repeats in Huntingtons disease (HD). Cellular biology approaches to understand these functional disruptions in HD have primarily focused on cell lines with synthetically long CAG length alleles that clinically represent outliers in this disease and a more severe form of HD that lacks age-onset. Patient-derived fibroblasts are limited to a finite number of passages before succumbing to cellular senescence. We used human telomerase reverse transcriptase (hTERT) to immortalize fibroblasts taken from individuals of varying age, sex, disease onset and CAG repeat length, which we have termed TruHD cells. TruHD cells display classic HD phenotypes of altered morphology, size and growth rate, increased sensitivity to oxidative stress, aberrant ADP/ATP ratios and hypophosphorylated huntingtin protein. We additionally observed dysregulated ROS-dependent huntingtin localization to nuclear speckles in HD cells. We report the generation and characterization of a human, clinically relevant cellular model for investigating disease mechanisms in HD at the single cell level, which, unlike transformed cell lines, maintains TP53 function critical for huntingtin transcriptional regulation and genomic integrity.

cell biology↗

bioRxiv2017

Amplification-free, CRISPR-Cas9 Targeted Enrichment and SMRT Sequencing of Repeat-Expansion Disease Causative Genomic Regions

Targeted sequencing has proven to be an economical means of obtaining sequence information for one or more defined regions of a larger genome. However, most target enrichment methods require amplification. Some genomic regions, such as those with extreme GC content and repetitive sequences, are recalcitrant to faithful amplification. Yet, many human genetic disorders are caused by repeat expansions, including difficult to sequence tandem repeats.\n\nWe have developed a novel, amplification-free enrichment technique that employs the CRISPR-Cas9 system for specific targeting multiple genomic loci. This method, in conjunction with long reads generated through Single Molecule, Real-Time (SMRT) sequencing and unbiased coverage, enables enrichment and sequencing of complex genomic regions that cannot be investigated with other technologies. Using human genomic DNA samples, we demonstrate successful targeting of causative loci for Huntingtons disease (HTT; CAG repeat), Fragile X syndrome (FMR1; CGG repeat), amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (C9orf72; GGGGCC repeat), and spinocerebellar ataxia type 10 (SCA10) (ATXN10; variable ATTCT repeat). The method, amenable to multiplexing across multiple genomic loci, uses an amplification-free approach that facilitates the isolation of hundreds of individual on-target molecules in a single SMRT Cell and accurate sequencing through long repeat stretches, regardless of extreme GC percent or sequence complexity content. Our novel targeted sequencing method opens new doors to genomic analyses independent of PCR amplification that will facilitate the study of repeat expansion disorders.

genomics↗

bioRxiv2016

Genome-scale transcriptional regulatory network models for the mouse and human striatum predict roles for SMAD3 and other transcription factors in Huntington’s disease

Transcriptional changes occur presymptomatically and throughout Huntingtons Disease (HD), motivating the study of transcriptional regulatory networks (TRNs) in HD. We reconstructed a genome-scale model for the target genes of 718 TFs in the mouse striatum by integrating a model of the genomic binding sites with transcriptome profiling of striatal tissue from HD mouse models. We identified 48 differentially expressed TF-target gene modules associated with age- and Htt allele-dependent gene expression changes in the mouse striatum, and replicated many of these associations in independent transcriptomic and proteomic datasets. Strikingly, many of these predicted target genes were also differentially expressed in striatal tissue from human disease. We experimentally validated a key model prediction that SMAD3 regulates HD-related gene expression changes using chromatin immunoprecipitation and deep sequencing (ChIP-seq) of mouse striatum. We found Htt allele-dependent changes in the genomic occupancy of SMAD3 and confirmed our models prediction that many SMAD3 target genes are down-regulated early in HD. Importantly, our study provides a mouse and human striatal-specific TRN and prioritizes a hierarchy of transcription factor drivers in HD.

systems biology↗