Evidence of Extensive Alternative Splicing in Post Mortem Human Brain HTT Transcription by mRNA Sequencing - Fig 1

<p>A) Read pileup of the superset reads showing coverage in exons and specific introns. Canonical HTT gene model is in blue. B) Relative contribution of reads from each disease dataset to the superset binned into exon (top) and intron (bottom) features. Each bar is the average number of covered bases per base position in the given feature divided by the number of samples in the corresponding condition. The canonical gene model lies between the bar charts. High intronic coverage is apparent in introns 9, 10, 12, 41, 49, and 58, highlighted in grey. None of the features are obviously biased toward any of the conditions. The rightward skew of counts is indicative of the poly-A selection method used in library prep.</p

Splicing events detected in reads of both novel and previously reported splice forms.

<p>Grey areas indicate overall aligned read coverage in the region, black areas are the spliced reads that contribute to the splicing events. Blue lines indicate detected splicing events with a minimum of 10 supporting reads. The blue track across the top of all plots is the canonical HTT splice form, with red boxes indicating the position in the gene region displayed. Splicing patterns shown in B, E, and F support the spliceforms HTT-d13, HTT-41b, and HTT-d46 reported in Ruzo et al. In C, the skipped exon 28 is consistent with an isoform identified in mouse and human [Hughes JMB 2014] but, in these data, is only seen in a splice pattern where exon 27 is also skipped.</p

Detected Alternative Splice Events.

<p>Twelve of the 25 alternative splicing (AS) events detected using the superset reads, as depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141298#pone.0141298.g002" target="_blank">Fig 2</a>. All AS forms are much less abundant than the canonical splice form. Read Support column lists the number of junction reads supporting the splice junction, with the percentage of total junction reads involved in this event listed in parentheses. The remaining events are included as processed data file GSE71191_all_merged_HTT.bed.gz in the GEO accession GSE71191.</p><p>Detected Alternative Splice Events.</p

Evaluation of Parkinson Disease Risk Variants as Expression-QTLs

<div><p>The recent Parkinson Disease GWAS Consortium meta-analysis and replication study reports association at several previously confirmed risk loci <em>SNCA</em>, <em>MAPT</em>, <em>GAK/DGKQ</em>, and <em>HLA</em> and identified a novel risk locus at <em>RIT2</em>. To further explore functional consequences of these associations, we investigated modification of gene expression in prefrontal cortex brain samples of pathologically confirmed PD cases (N = 26) and controls (N = 24) by 67 associated SNPs in these 5 loci. Association between the eSNPs and expression was evaluated using a 2-degrees of freedom test of both association and difference in association between cases and controls, adjusted for relevant covariates. SNPs at each of the 5 loci were tested for <em>cis</em>-acting effects on all probes within 250 kb of each locus. <em>Trans</em>-effects of the SNPs on the 39,122 probes passing all QC on the microarray were also examined. From the analysis of <em>cis</em>-acting SNP effects, several SNPs in the <em>MAPT</em> region show significant association to multiple nearby probes, including two strongly correlated probes targeting the gene <em>LOC644246</em> and the duplicated genes <em>LRRC37A</em> and <em>LRRC37A2</em>, and a third uncorrelated probe targeting the gene <em>DCAKD</em>. Significant <em>cis</em>-associations were also observed between SNPs and two probes targeting genes in the HLA region on chromosome 6. Expanding the association study to examine <em>trans</em> effects revealed an additional 23 SNP-probe associations reaching statistical significance (p<2.8×10⁻⁸) including SNPs from the <em>SNCA, MAPT</em> and <em>RIT2</em> regions. These findings provide additional context for the interpretation of PD associated SNPs identified in recent GWAS as well as potential insight into the mechanisms underlying the observed SNP associations.</p> </div

<i>Trans</i>-Acting SNP effects on gene expression.

<p><i>Trans</i>-Acting SNP effects on gene expression.</p

<i>Cis</i>-Acting SNP effects on gene expression for probes within 250 kb of the PD associated risk loci.

<p><i>Cis</i>-Acting SNP effects on gene expression for probes within 250 kb of the PD associated risk loci.</p

Brain Sample Characteristics.

*<p> <i>significantly different between cases and controls (p = 0.02).</i></p

HLA region probes and SNPs involved in significant <i>cis</i> eSNP relationships.

<p>Two probes in the HLA region on chromosme 6 showing significant association with PD risk SNPs are shown on the alternative sequence haplotype chr6_ssto_hap7. Six PD risk SNPs (shown in green) showed significant association to A_24_P326084 located in <i>HLA-DQA1</i>, while four SNPs (shown in red) had significant association to A_24P852756, located in <i>HLA-DQA2</i>.</p

<i>MAPT</i> region probes and SNPs involved in significant <i>cis</i> eSNP relationships.

<p>Three probes in the <i>MAPT</i> region on chromosme 17 showing significant association with PD risk SNPs are displayed. Significant SNP associations with A_24_P110521 located in the duplicated genes <i>LRRC37A</i> and <i>LRRC37A2</i> are shown in blue, while SNPs associated with A_24_P221327 located in <i>LOC644246</i> are shown in green and SNPs associated with A_24_P58331 (in <i>DCAKD</i>) are shown in red.</p

The Role of H3K4me3 in Transcriptional Regulation Is Altered in Huntington’s Disease

<div><p>Huntington’s disease (HD) is an autosomal-dominant neurodegenerative disorder resulting from expansion of CAG repeats in the Huntingtin (<i>HTT</i>) gene. Previous studies have shown mutant <i>HTT</i> can alter expression of genes associated with dysregulated epigenetic modifications. One of the most widely studied chromatin modifications is trimethylated lysine 4 of histone 3 (H3K4me3). Here, we conducted the first comprehensive study of H3K4me3 ChIP-sequencing in neuronal chromatin from the prefrontal cortex of six HD cases and six non-neurologic controls, and its association with gene expression measured by RNA-sequencing. We detected 2,830 differentially enriched H3K4me3 peaks between HD and controls, with 55% of them down-regulated in HD. Although H3K4me3 signals are expected to be associated with mRNA levels, we found an unexpected discordance between altered H3K4me3 peaks and mRNA levels. Gene ontology (GO) term enrichment analysis of the genes with differential H3K4me3 peaks, revealed statistically significantly enriched GO terms only in the genes with down-regulated signals in HD. The most frequently implicated biological process terms are organ morphogenesis and positive regulation of gene expression. More than 9,000 H3K4me3 peaks were located not near any recognized transcription start sites and approximately 36% of these “distal” peaks co-localized to known enhancer sites. Six transcription factors and chromatin remodelers are differentially enriched in HD H3K4me3 distal peaks, including EZH2 and SUZ12, two core subunits of the polycomb repressive complex 2 (PRC2). Moreover, PRC2 repressive state was significantly depleted in HD-enriched peaks, suggesting the epigenetic role of PRC2 inhibition associated with up-regulated H3K4me3 in Huntington’s disease. In summary, our study provides new insights into transcriptional dysregulation of Huntington’s disease by analyzing the differentiation of H3K4me3 enrichment.</p></div