ChIP-seq of histone modifications and EZH2 in the striatum of four-month-old HttQ111/+ and wildtype mice

Progressive striatal gene expression changes and epigenetic alterations are a prominent feature of Huntington’s disease (HD), but direct relationships between the huntingtin (HTT) protein and chromatin remain poorly described. Here, using chromatin immunoprecipitation and sequencing (ChIP-seq), we show that HTT reproducibly occupies specific locations in the mouse genome, including thousands of genomic loci that are differentially occupied in striatal tissue from a knock-in mouse model of the HD mutation (B6.HttQ111/+) versus wildtype controls. ChIP-seq of histone modifications, generated in parallel, revealed genotype-specific colocalization of HTT with trimethylation of histone 3 lysine 27 (H3K27me3), a repressive chromatin mark. Near genes that are differentially regulated in HD, greater HTT occupancy in HttQ111/+ vs. wildtype mice predicted increased H3K27me3, reduced histone 3 lysine 4 (H3K4me3), a marker of poised and active promoters, and down-regulated gene expression. Altered huntingtin-chromatin interactions may therefore play a direct role in driving transcriptional dysregulation in HD. Fresh frozen striatal tissue from five male four-month-old HttQ111/+ and age-matched Htt+/+ mice were pooled per replicate for n = 3 samples. Further processing was performed at Active Motif (Carlsbad, CA). Tissue was fixed in 1% formaldehyde, lysed, and disrupted with a Dounce homogenizer. DNA was sonicated to an average fragment length of 300-500bp, and 25ug chromatin plus 200ng Drosophila spike-in chromatin was incubated with antibody targeting EZH2, H3K27me3, H3K4me3, or H3K27ac (Active Motif catalog numbers 39901, 39155, 399159, and 39133, respectively). Antibody against H2Av was also present in the reaction to ensure efficient pull-down of the spike-in chromatin. Complexes were captured using protein A agarose beads (Invitrogen). Illumina sequencing libraries were prepared from ChIP and Input DNA by end-polishing, dA-addition, and adaptor ligation. Libraries were quantified and sequenced on Illumina’s NextSeq 500 (75nt single end reads). Reads were aligned consecutively to the mouse (mm10) and Drosophila (dm3) genomes using the BWA algorithm (default settings). The number of mouse alignments used in the analysis was scaled to the number of Drosophila spike-in alignments. Peak-calling was performed with MACS and SICER.