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

Gene expression and H3K4me3 signal at promoters.

<p><b>(A</b>) Correlation of H3K4me3 signal at promoters and gene expression in control and HD. X-axis shows the logarithmic base mean value of normalized expression values CPKM (RNA-seq reads count per kilobase of exon length per millions of total mapped reads), and Y-axis shows the logarithmic signal density of ChIP-seq reads at proximal promoters (i.e. [-1k, 1k] of TSS). It shows that gene expression levels are positively correlated with the density of H3K4me3 signal at promoters in both controls and HD, with Spearman correlation rho = 0.57. (<b>B</b>) Histogram of normalized H3K4me3 signal in all samples. Bars are colored according to the frequency of genes with high (red) and low (blue) expression. (<b>C</b>) Scatter plot of log₂(fold change between HD and control) of H3K4me3 signal at promoter versus gene expression. Genes with differential expression but not differential H3K4me3 are in green; genes with differential H3K4me3 but not differential expression are in red, and genes with both differential expression and H3K4me3 are in blue. In total, 58 genes show both differential expression and H3K4me3, where only 20 of them show the same direction of differentiation between control and HD cases. The remainder (38 of 58) shows a lack of concordance between differential expression and differential H3K4me3 at promoter. <b>(D)</b> Fold changes of expression (in green) and H3K4me3 (in red) between HD and control for the 38 genes with lack of concordance.</p

H3K4me3 proximal and distal peaks in HD and control.

<p><b>(A)</b> The number of H3K4me3 peaks in HD and control. 487 proximal peaks were unique to HD samples while 381 were unique to controls. 474 distal peaks were unique to HD samples and 508 were unique to controls. (<b>B</b>) Histograms of the position of proximal peaks relative to the TSS in HD and control show a slight enrichment upstream the TSS. Small right panels show zoomed-in histograms for unique proximal peaks (highlighted in black). (<b>C</b>) Histograms of the distribution of peak widths for proximal and distal peaks were similar in control and HD samples. Proximal peaks show a bimodal distribution in peak width not seen in distal peaks. The width distributions of unique peaks are in black. (<b>D</b>) Peak intensities of proximal (blue) and distal (red) peaks are plotted for HD versus control.</p

Sub-networks of DEGs and genes with differential H3K4me3.

<p>Each node represents a gene; the fill color of each node indicates the fold-change of gene expression between HD and control, and the outline color of each node indicates the fold-change of H3K4me3 between HD and control. Immune system and ECM-receptor pathway modules were composed of specific gene families. On the other hand, signaling pathway modules were composed of various signaling genes forming hierarchical structure. The directions of all the arrows were drawn toward the bottom of figure.</p

Overlapped regulatory signals and differentially enriched TF binding on distal peaks.

<p><b>(A)</b> UCSC genome browser of a genomic locus (coordinate in hg19 shown on the top) where a distal H3K4me3 peak, called in both control and HD cases, overlaps with enhancer regulatory features including enriched H3K4me1 (“Roadmap H3K4me1”), H3K27ac (“Roadmap H3K27ac”), DNase I (“Roadmap DNase I”), TFs binding hotspot (“ENCODE TFs ChIP-seq”), and bidirectional CAGE signals (“FANTOM CAGE”). <b>(B)</b> Aggregation plots of several types of regulatory signals, including CAGE (1^st row), histone marks (2^nd row), TF binding (3^rd row), and DNase I hypersensitivity (4^th row), centered on the middle of H3K4me3 peaks. <b>(C)</b> Directionality of H3K4me3 peaks represented by CAGE tags shows that, unlike the strong bias towards sense strand for proximal peaks, distal peaks show similar level of CAGE tags in both directions. <b>(D)</b> Fraction of distal peaks overlapping with different sources of known enhancers occurred at rates greater than expected (All = blue, control = red, HD = green, Expected = grey). <b>(E)</b> Transcription factors binding in differentially enriched distal peaks.</p

Genes that correspond to the top 22 differential proximal H3K4me3 peaks.

<p>Genes that correspond to the top 22 differential proximal H3K4me3 peaks.</p

GO terms enriched with proximal peaks in control and HD.

<p><b>(A)</b> GO terms enriched with genes marked with down-regulated H3K4me3 signals in HD. The GO terms overlapped with those derived from differentially expressed genes are highlighted in red text, with iron ion binding (in black) representing the only term not seen in the differentially expressed genes. X-axis shows the negative logarithm FDR values. <b>(B)</b> GO terms enriched with genes marked with unique proximal peaks in HD (right; orange) and in control (left; green). These terms also overlap with those seen in HD differentially down-regulated genes.</p

Image_1_Chronic Caffeine Treatment Protects Against α-Synucleinopathy by Reestablishing Autophagy Activity in the Mouse Striatum.PDF

<p>Despite converging epidemiological evidence for the inverse relationship of regular caffeine consumption and risk of developing Parkinson's disease (PD) with animal studies demonstrating protective effect of caffeine in various neurotoxin models of PD, whether caffeine can protect against mutant α-synuclein (α-Syn) A53T-induced neurotoxicity in intact animals has not been examined. Here, we determined the effect of chronic caffeine treatment using the α-Syn fibril model of PD by intra-striatal injection of preformed A53T α-Syn fibrils. We demonstrated that chronic caffeine treatment blunted a cascade of pathological events leading to α-synucleinopathy, including pSer129α-Syn-rich aggregates, apoptotic neuronal cell death, microglia, and astroglia reactivation. Importantly, chronic caffeine treatment did not affect autophagy processes in the normal striatum, but selectively reversed α-Syn-induced defects in macroautophagy (by enhancing microtubule-associated protein 1 light chain 3, and reducing the receptor protein sequestosome 1, SQSTM1/p62) and chaperone-mediated autophagy (CMA, by enhancing LAMP2A). These findings support that caffeine—a strongly protective environment factor as suggested by epidemiological evidence—may represent a novel pharmacological therapy for PD by targeting autophagy pathway.</p

Image_2_Chronic Caffeine Treatment Protects Against α-Synucleinopathy by Reestablishing Autophagy Activity in the Mouse Striatum.pdf

<p>Despite converging epidemiological evidence for the inverse relationship of regular caffeine consumption and risk of developing Parkinson's disease (PD) with animal studies demonstrating protective effect of caffeine in various neurotoxin models of PD, whether caffeine can protect against mutant α-synuclein (α-Syn) A53T-induced neurotoxicity in intact animals has not been examined. Here, we determined the effect of chronic caffeine treatment using the α-Syn fibril model of PD by intra-striatal injection of preformed A53T α-Syn fibrils. We demonstrated that chronic caffeine treatment blunted a cascade of pathological events leading to α-synucleinopathy, including pSer129α-Syn-rich aggregates, apoptotic neuronal cell death, microglia, and astroglia reactivation. Importantly, chronic caffeine treatment did not affect autophagy processes in the normal striatum, but selectively reversed α-Syn-induced defects in macroautophagy (by enhancing microtubule-associated protein 1 light chain 3, and reducing the receptor protein sequestosome 1, SQSTM1/p62) and chaperone-mediated autophagy (CMA, by enhancing LAMP2A). These findings support that caffeine—a strongly protective environment factor as suggested by epidemiological evidence—may represent a novel pharmacological therapy for PD by targeting autophagy pathway.</p

Immunofluorescence double staining of c-Fos and enkephalin in forebrain-WT and forebrain-A_2AR KO mice after saline or cocaine treatment.

<p>(<b>A</b>) Representative merged images of immunofluorescence double staining of c-Fos (red) with enkephalin (Enk, green) in cocaine- <i>vs.</i> saline-treated fb-A_2AR KO and fb-WT mice. (<b>B</b>) Quantitative analysis demonstrating the percentage of total c-Fos positive [c-Fos⁽⁺⁾]cells out of the total cells. (<b>C</b>) Quantitative analysis showing the percentage of c-Fos and Enk double positive [c-Fos⁽⁺⁾Enk⁽⁺⁾] stained cells out of the total cells. (<b>D</b>) Quantitative analysis demonstrating the percentage of c-Fos positive but Enk negative [c-Fos⁽⁺⁾Enk⁽⁻⁾] cells out of the total cells. Data in the bar graphs are mean ± SEM, n = 6-10 per group. * p< 0.05, <i>vs</i>. groups of same genotype with saline treatment; # p< 0.05 <i>vs</i>. cocaine-treated WT groups. ^△ p< 0.05, <i>vs</i>. saline-treated fb-A_2AR group. Scale bar  =  50 µm. Yellow arrows indicate neurons with c-Fos positive but Enk negative [c-Fos⁽⁺⁾Enk⁽⁻⁾] staining; Red arrows indicate neurons with c-Fos and Enk double positive [c-Fos⁽⁺⁾Enk⁽⁺⁾] staining.</p