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Extensive Changes in DNA Methylation Are Associated with Expression of Mutant Huntingtin

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Extensive Changes in DNA Methylation Are Associated with Expression of Mutant Huntingtin Extensive changes in DNA methylation are associated with expression of mutant huntingtin Christopher W. Nga, Ferah Yildirima, Yoon Sing Yapa, Simona Dalina, Bryan J. Matthewsa, Patricio J. Veleza, Adam Labadorfa, David E. Housmanb,1, and Ernest Fraenkela,1 Departments of aBiological Engineering and bBiology, Massachusetts Institute of Technology, Cambridge, MA 02142 Contributed by David E. Housman, December 6, 2012 (sent for review November 16, 2012) The earliest stages of Huntington disease are marked by changes directly influence DNA methylation. For example, NRSF/REST in gene expression that are caused in an indirect and poorly (16) is a neuronal repressor implicated in HD (7) that recruits re- understood manner by polyglutamine expansions in the huntingtin pressive complexes containing HDACs and the DNA methylation (HTT) protein. To explore the hypothesis that DNA methylation may reader MECP2 (17), and changes to NRSF/REST binding could be altered in cells expressing mutated HTT, we use reduced affect local DNA methylation levels. Because DNA methylation in fl – representation bisulfite sequencing (RRBS) to map sites of DNA the brain in uences a wide range of cognitive functions (18 20), fi methylation in cells carrying either wild-type or mutant HTT. We these epigenetic modi cations could have long-term consequences find that a large fraction of the genes that change in expression in on HD patients. the presence of mutant huntingtin demonstrate significant changes For our analysis, we used cell lines derived from mouse striatal neurons expressing full-length HTT with either the wild-type in DNA methylation. Regions with low CpG content, which have or expanded polyglutamine repeat (5). Derived from mouse previously been shown to undergo methylation changes in re- embryos, these cells serve as a highly reproducible model for sponse to neuronal activity, are disproportionately affected. On the earliest stages of the disease. Using reduced representation the basis of the sequence of regions that change in methylation, bisulfite sequencing (RRBS), we identified extensive changes in we identify AP-1 and SOX2 as transcriptional regulators associated DNA methylation. Regions with low CpG content are overrep- fi with DNA methylation changes, and we con rm these hypotheses resented among those that change in methylation. These regions using genome-wide chromatin immunoprecipitation sequencing are largely intergenic and transition from fully methylated in wild- (ChIP-Seq). Our findings suggest new mechanisms for the effects type cells to partially methylated in cells expressing mutant HTT. of polyglutamine-expanded HTT. These results also raise important As partially methylated regions have been shown to be occupied by questions about the potential effects of changes in DNA meth- transcription factors that influence methylation (14), we analyzed the ylation on neurogenesis and cognitive decline in patients with sequences of these sites to identify several potential DNA-binding Huntington disease. proteins. Using genome-wide chromatin immunoprecipitation se- quencing (ChIP-Seq), we confirmed that the binding of SOX2 and epigenomics | transcription | mRNA-Seq the AP-1 proteins FRA-2 and JUND are associated with changes in DNA methylation resulting from expression of mutant HTT. untington disease (HD) is a fatal, neurodegenerative disor- For many years, DNA methylation was believed to be an ex- Hder caused by an expanded CAG repeat in the huntingtin tremely stable epigenetic mark relevant mostly to developmental (HTT) gene, which encodes an abnormally long polyglutamine processes and, by extension, to tumor development. However, it is repeat in the HTT protein. In the early stages of the disease, becoming increasingly clear that DNA methylation has a role in a much wider range of biological processes, including neuronal ac- patients are largely asymptomatic, although they may suffer from – mild cognitive impairment and behavioral changes. With time, tivity, learning, and memory (18 22). Our analysis reveals that they develop severe motor dysfunction as well as more prono- polyglutamine-expanded HTT causes changes in DNA meth- unced cognitive and psychiatric symptoms. Transcriptional dys- ylation at both promoter proximal and distal regulatory regions. regulation is a major component of the early stages of HD, before These results shed new light on the etiology of HD and suggest fi a possible explanation for mild cognitive impairment that occurs at signi cant neuronal death. Changes in transcription have been fi detected in human postmortem tissue (1), mouse models (2–4), the early stages of the disease, before signi cant loss of neurons. and cell culture models (5). Genes reproducibly shown to be differentially expressed across HD models are associated with Results processes including neurotransmission, neurotrophin receptor Expression of Mutant huntingtin Is Associated with Large-Scale Changes signaling, signal transduction, calcium ion transport, synaptic in DNA Methylation. On the basis of the importance of changes in organization, chromatin remodeling, G-protein receptor-coupled DNA methylation to development (23–26) and neuronal activity signaling, and metabolism (6). The polyglutamine expanded form (22), we sought to determine whether mutant HTT might perturb of HTT may have a direct role in causing these expression changes. this epigenetic mark. To examine this hypothesis, we used RRBS A number of DNA-binding proteins have been shown to physically to measure DNA methylation at base pair resolution in striatal interact with either wild-type or mutant HTT. These include cells carrying polyglutamine-expanded HTT (STHdhQ111/Q111) NRSF/REST, CBP, PGC1α, Sp1, BCL11b, p53, LXRα, polycomb- and wild-type cells (STHdhQ7/Q7) (see Dataset S1 for full list of group proteins, SIN3A, and NCOR1 (6). Any effect of HTT on the datasets in this study). We measured the degree of methylation 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 Author contributions: C.W.N., F.Y., D.E.H., and E.F. designed research; C.W.N., Y.S.Y., S.D., influence DNA methylation. First, histone marks are altered in B.J.M., and P.J.V. performed research; C.W.N., A.L., D.E.H., and E.F. analyzed data; and HD (7–11) and SETD2, a SET domain regulator of H3K36me3, C.W.N., F.Y., and E.F. wrote the paper. has been previously reported to interact with mutant HTT (12). The authors declare no conflict of interest. Due to biochemical interactions between DNA methyltransferases Data deposition: The data reported in this paper have been deposited in the Gene Ex- and SET domain histone methyltransferases (13), changes in re- pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE43433). pressive histone marks could result in changes in DNA methyla- 1To whom correspondence may be addressed. E-mail: [email protected] or tion. Second, recent findings show that DNA-bound transcription [email protected]. factors can alter DNA methylation patterns (14, 15), suggesting This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. that the effects of HTT on DNA-binding proteins could also 1073/pnas.1221292110/-/DCSupplemental. 2354–2359 | PNAS | February 5, 2013 | vol. 110 | no. 6 www.pnas.org/cgi/doi/10.1073/pnas.1221292110 Downloaded by guest on October 2, 2021 at 97,006 cytosines in both cell lines. The data were highly re- methylated regions (LMRs)]. During development, transcription producible between biological replicate experiments (R > 0.97; factors binding near FMRs can cause partial loss of DNA meth- Materials and Methods and Fig. S1A). Overall, 61,940 bases de- ylation and conversion to LMRs. creased in methylation in STHdhQ111 relative to STHdhQ7, and We therefore asked whether similar changes occur in response to 33,974 bases increased in methylation, using a multiple-hypoth- mutant HTT. Overall, sites that do not change in methylation be- esis corrected P-value cutoff of 1E-5 and difference in meth- tween mutant and wild-type cells are predominantly fully methyl- ylation cutoff of 15%. We confirmed these findings by sequen- ated or unmethylated, whereas those that change in methylation cing immunoprecipitated methylated DNA from both cell lines tend to be partially methylated (Fig. S2 C–E). To explore the effects (MeDIP-Seq) and by qPCR of specific loci (Fig. S1 B–E). We of mutant HTT in more detail, we plotted the fractional methyla- then concatenated the cytosines that were close in spatial prox- tion of genomic regions in wild-type cells vs. cells with mutant HTT imity to one another into 154,239 methylation regions (Mate- (Fig. 1 C and D). Many methylation changes lie close to the di- rials and Methods). Of these, 21,191 regions decreased in agonal, indicating a relatively small change in the fraction of methylationinSTHdhQ111relative to STHdhQ7, and 10,413 methylation. However, 4,804 CpG-poor regions that are fully regions increased in methylation, using a multiple-hypothesis methylated in wild-type cells are only partially methylated (10–80% corrected P-value cutoff of 1E-5 (see Fig. S2B for additional methylation) in the mutant cells. In addition, 10,396 CpG-poor thresholds) and difference in methylation cutoff of 15%. regions go from fully methylated in cells with mutant HTT to only As expected, methylated regions could be divided into those that partially methylated in wild-type cells. CpG-rich regions tend to occur in regions rich in the sequence cytosine followed by guanine show smaller changes in fractional methylation. (CpG) and those with low levels of CpGs. Differentially methyl- ated regions (DMRs) between the cell lines were predominantly Relationship of Changes in DNA Methylation and Transcription. To CpG-poor. Approximately 84% of sites increasing or decreasing determine whether changes in DNA methylation were associ- in methylation between the cell lines were CpG-poor, compared ated with changes in gene expression, we measured transcript with 70% of the regions that did not change in methylation (Fig.
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