The earliest stages of Huntington disease are marked by changes in gene expression that are caused in an indirect and poorly understood manner by polyglutamine expansions in the huntingtin (HTT) protein. To explore the hypothesis that DNA methylation may be altered in cells expressing mutated HTT, we use reduced representation bisulfite sequencing (RRBS) to map sites of DNA methylation in cells carrying either wild-type or mutant HTT. We find that a large fraction of the genes that change in expression in the presence of mutant huntingtin demonstrate significant changes in DNA methylation. Regions with low CpG content, which have previously been shown to undergo methylation changes in response to neuronal activity, are disproportionately affected. On the basis of the sequence of regions that change in methylation, we identify AP-1 and SOX2 as transcriptional regulators associated with DNA methylation changes, and we confirm these hypotheses using genome-wide chromatin immunoprecipitation sequencing (ChIP-Seq). Our findings suggest new mechanisms for the effects of polyglutamine-expanded HTT. These results also raise important questions about the potential effects of changes in DNA methylation on neurogenesis and cognitive decline in patients with Huntington disease.epigenomics | transcription | mRNA-Seq H untington disease (HD) is a fatal, neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin (HTT) gene, which encodes an abnormally long polyglutamine repeat in the HTT protein. In the early stages of the disease, patients are largely asymptomatic, although they may suffer from mild cognitive impairment and behavioral changes. With time, they develop severe motor dysfunction as well as more pronounced cognitive and psychiatric symptoms. Transcriptional dysregulation is a major component of the early stages of HD, before significant neuronal death. Changes in transcription have been detected in human postmortem tissue (1), mouse models (2-4), and cell culture models (5). Genes reproducibly shown to be differentially expressed across HD models are associated with processes including neurotransmission, neurotrophin receptor signaling, signal transduction, calcium ion transport, synaptic organization, chromatin remodeling, G-protein receptor-coupled signaling, and metabolism (6). The polyglutamine expanded form of HTT may have a direct role in causing these expression changes. A number of DNA-binding proteins have been shown to physically interact with either wild-type or mutant HTT. These include NRSF/REST, CBP, PGC1α, Sp1, BCL11b, p53, LXRα, polycombgroup proteins, SIN3A, and NCOR1 (6). Any effect of HTT on the subcellular localization, activity, or concentration of these proteins would be likely to directly change gene expression.Two lines of evidence suggest that alterations in HTT could also influence DNA methylation. First, histone marks are altered in HD (7-11) and SETD2, a SET domain regulator of H3K36me3, has been previously reported to interact with mutant HTT (12). Due to biochemical...

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer deaths in the United States. The deoxynucleoside analogue gemcitabine is among the most effective therapies to treat PDAC, however, nearly all patients treated with gemcitabine either fail to respond or rapidly develop resistance. One hallmark of PDAC is a striking accumulation of stromal tissue surrounding the tumor, and this accumulation of stroma can contribute to therapy resistance. To better understand how stroma limits response to therapy, we investigated cell-extrinsic mechanisms of resistance to gemcitabine. Conditioned media from pancreatic stellate cells (PSC), as well as from other fibroblasts, protected PDAC cells from gemcitabine toxicity. The protective effect of PSC-conditioned media was mediated by secretion of deoxycytidine, but not other deoxynucleosides, through equilibrative nucleoside transporters. Deoxycytidine inhibited the processing of gemcitabine in PDAC cells, thus reducing the effect of gemcitabine and other nucleoside analogues on cancer cells. These results suggest that reducing deoxycytidine production in PSCs may increase the efficacy of nucleoside analog therapies.Significance: This study provides important new insight into mechanisms that contribute to gemcitabine resistance in PDAC and suggests new avenues for improving gemcitabine efficacy.

Summary MicroRNAs (miRNAs) regulate diverse biological processes by repressing mRNAs, but their modest effects on direct targets, together with their participation in larger regulatory networks, make it challenging to delineate miRNA-mediated effects. Here, we describe an approach to characterizing miRNA-regulatory networks by systematically profiling transcriptional, post-transcriptional and epigenetic activity in a pair of isogenic murine fibroblast cell lines with and without Dicer expression. By RNA sequencing (RNA-seq) and CLIP (crosslinking followed by immunoprecipitation) sequencing (CLIP-seq), we found that most of the changes induced by global miRNA loss occur at the level of transcription. We then introduced a network modeling approach that integrated these data with epigenetic data to identify specific miRNA-regulated transcription factors that explain the impact of miRNA perturbation on gene expression. In total, we demonstrate that combining multiple genome-wide datasets spanning diverse regulatory modes enables accurate delineation of the downstream miRNA-regulated transcriptional network and establishes a model for studying similar networks in other systems.

Differentiation events contribute to phenotypic cellular heterogeneity within tumors and influence disease progression and response to therapy. Here, we dissect mechanisms controlling intratumoral heterogeneity within triple-negative basal-like breast cancers. Tumor cells expressing the cytokeratin K14 possess a differentiation state that is associated with that of normal luminal progenitors, and K14-negative cells are in a state closer to that of mature luminal cells. We show that cells can transition between these states through asymmetric divisions, which produce one K14 and one K14 daughter cell, and that these asymmetric divisions contribute to the generation of cellular heterogeneity. We identified several regulators that control the proportion of K14 cells in the population. EZH2 and Notch increase the numbers of K14 cells and their rates of symmetric divisions, and FOXA1 has an opposing effect. Our findings demonstrate that asymmetric divisions generate differentiation transitions and heterogeneity, and identify pathways that control breast cancer cellular composition.

The immense and growing repositories of transcriptional data may contain critical insights for developing new therapies. Current approaches to mining these data largely rely on binary classifications of disease vs. control, and are not able to incorporate measures of disease severity. We report an analytical approach to integrate ordinal clinical information with transcriptomics. We apply this method to public data for a large cohort of Huntington’s disease patients and controls, identifying and prioritizing phenotype-associated genes. We verify the role of a high-ranked gene in dysregulation of sphingolipid metabolism in the disease and demonstrate that inhibiting the enzyme, sphingosine-1-phosphate lyase 1 (SPL), has neuroprotective effects in Huntington’s disease models. Finally, we show that one consequence of inhibiting SPL is intracellular inhibition of histone deacetylases, thus linking our observations in sphingolipid metabolism to a well-characterized Huntington’s disease pathway. Our approach is easily applied to any data with ordinal clinical measurements, and may deepen our understanding of disease processes.