Motif Vations OCTOBER 2016 | VOLUME 17 NUMBER 2
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THE NEWSLETTER OF ACTIVE MOTIF NEUROEPIGENETICS EDITION motif vations OCTOBER 2016 | VOLUME 17 NUMBER 2 Enabling Epigenetics Research Antibodies, Kits and Services for Epigenetics & Gene Regulation Research ACTIVE MOTIF – Advancing Neuroepigenetics Research Epigenetic modifications including histone modifications, DNA methylation, and non-coding RNAs influence chromatin structure and, ultimately, gene regulation. Epigenetic modifications are heritable but, unlike genetic modifications, are dynamic and responsive to external stimuli such as stress, toxins and learning. Neuroepi- genetics is a growing area of research focused on the study of epigenetics in various neural processes, including development and plasticity, behavior, stress, aging, and brain disorders. Below are two publication summaries illustrating how Active Motif, the leading provider of innovative epigenetic research tools, is aiding IN THIS ISSUE scientists every day in advancing their neuroepigenetics research. 2 The Role of Epigenetic Modulation in THE ROLE OF EPIGENETIC Dr. Emma Whitelaw performed a Brain Disorders MODULATION IN BRAIN DISORDERS mutagenesis mouse screen in which they There is increasing evidence that identified the gene Widely interspaced 6 NEW: High Quality ChIP-Seq Data from several neurodevelopmental, zinc finger motifs,or Wiz, as a modulator as Few as 5,000 Cells neurodegenerative and psychiatric of gene expression. Although little is 7 NEW: NGS DNA Library Preparation disorders are, in part, caused by aberrant known about Wiz, it is highly expressed with Molecular Identifiers Eliminates epigenetic modifications. As a result, in both the embryonic and adult brain Bias from PCR Duplicates researchers in neurogenetics are now and is known to mediate protein-protein beginning to investigate the mecha- and protein-DNA interactions of the 8 NEW: Reveal Histone Modification nisms underlying epigenetic regulation G9a/GLP histone methyltransferase Patterns in Clinical Samples with in neuronal gene expression and how complex which catalyzes methylation Active Motif’s FFPE ChIP-Seq Service this impacts normal brain function. of H3K9me1 and H3K9me2, marks that 9 NEW: Easily Isolate Intact Histones Recently, Active Motif collaborated in are associated with transcriptional for PTM Analysis two separate published studies where repression. G9a is also associated with loss-of-function mutations of chromatin transcriptional activation independent 10 NEW: RRBS Service for Genome-scale modifiers Wiz and Kdm5C were used as of its histone methyltransferase activity, DNA Methylation Profiling mouse models to study how disrupting and it has been suggested that Wiz 11 NEW: Is DNA Methylation a Biomarker the function of epigenetic modifiers may also be involved in G9a-mediated in Your FFPE Sample? contributes to brain disorders. These transcriptional activation. recent publications in eLife by the 12 NEW: Simple High-throughput Screen laboratory of Dr. Emma Whitelaw at Reduced levels of Wiz are associated to Evaluate Changes in Global DNA La Trobe University in Melbourne, with gene silencing Methylation Australia and in Cell Reports by the In the mutagenesis screen, haploin- MD30/+ 13 NEW: Sortase 2.0 – A Versatile laboratory of Dr. Yang Shi at Harvard sufficiency for Wiz (Wiz ) resulted Platform for Rapid, Controlled, Medical School, respectively, are in silencing of both the targeted GFP Site-specific Protein Modification summarized below. transgene reporter under the control of erythroid promoter/enhancer 14 A Genome-wide 3’UTR Collection for Identification of a role for the epigenetic sequences as well as an independent vy High-throughput Functional Screening modifier Wiz in neural function locus, the Agouti viable yellow (A ) of miRNA Targets This following article is a summary of the research published in allele, known to be sensitive to levels eLIfe.: Isbel et al., 2016;5:e15082. DOI: 10.7554/eLife.15082. of epigenetic modifiers, lending further To identify novel modifiers of epigenetic evidence that Wiz is involved in regulation reprogramming, the laboratory of of G9a-mediated transcriptional 2 www.activemotif.com ADVANCES IN NEUROEPIGENETICS RESEARCH activation. Furthermore, a reduction in causes reduced expression of a levels of G9a/GLP are associated with number of genes genome-wide. Kleefstra syndrome, a neurological However, differential expression is disorder that manifests in a range of observed mainly within gene clusters, symptoms including anxiety. Because in particular within the protocadherin of the high levels of Wiz in the brain, b (mainly expressed in neurons) and the researchers postulated that cadherin 11-like clusters, and adjacent reduced levels of Wiz might also have to telomeres, showing significant neurological effects. Utilizing a suite of overlap with embryonic and adult molecular and bioinformatics tools, the expression patterns (Figure 3A). researchers demonstrate that Wiz Telomeres are well-known hetero- binds active promoters and they chromatic regions, and protocadherin provide evidence that suggests Wiz clusters exist as large tandem arrays functions as a transcriptional activator important for gene silencing during in neural tissue. Furthermore, they heterochromatization, suggesting a role show that Wiz mutant mice present with for Wiz in chromosome looping. Analysis behavioral abnormalities, revealing an of ChIP-Seq read density at the TSS anxiety-like phenotype similar to what of the differentially regulated genes in is observed with reduced G9a/GLP levels. the WizMD30/+ mice showed enrichment of Wiz at the TSS of genes with Wiz binds promoters and shows high Figure 2: Wiz binding consensus shows a high degree decreased expression, but not at the consensus overlap with CTCF of overlap with CTCF. (A) The six most enriched motifs genes with increased expression, In order to determine where Wiz was in Wiz ChIP-Seq peaks (n = 39,781) and their distribution further lending evidence that Wiz bound within the genome with respect inside peaks are shown. (B) The most significant of these acts as a transcriptional activator matches the CTCF-binding site consensus sequence to active and inactive genes, cerebellums (JASPAR CORE database - MA0139.1). (C) Read density rather than repressor (Figure 3B). +/+ of adult Wiz (WT, wild-type) mice were for Wiz and the Encode CTCF and H3K4me3 ChIP-Seq collected and submitted to Active Motif datasets were calculated across a 4 kb region centered Wiz haploinsufficiency in mice results for chromatin immunoprecipitation on the ~40,000 Wiz ChIP-Seq peaks. Input sequencing in phenotypic alterations in behavior followed by sequencing (ChIP-Seq) from the Wiz ChIP-Seq experiment is also shown. (D) Venn To characterize any changes in behavior diagram showing the overlap for Wiz, CTCF and H3K4me3 services. ChIP-Seq analysis confirmed ChIP-Seq peaks. resulting from Wiz haploinsufficiency, that Wiz binds across the genome at A promoters and showed strong enrich- ment at the transcription start site (TSS) (Figure 1). A Motif analysis revealed that one of the common Wiz motifs showed correlation with the CTCF binding motifs (Figure 2A B and 2B). A comparison of Wiz ChIP-Seq data with existing ENCODE ChIP-Seq B data sets for CTCF and H3K4me3, a marker of active transcription, verified strong correlation with Wiz ChIP-Seq peaks, with almost all CTCF peaks overlapping with Wiz peaks (Figure 2C and 2D). Figure 3: Wiz binding at the protocadherin b locus in the adult cerebellum. (A) Genome browser screenshots of the protocadherin b locus and the HS5-1bL enhancer Wiz is associated with transcriptional (*indicated) located ~400 Kb downstream showing Wiz Figure 1: Wiz binds across the genome and at promoter activation in neural tissue binding. (B) Shown is Wiz ChIP-Seq read density in adult elements. (A) Pie graph depicting the percentage of Wiz To identify sites within the genome cerebellum as a heatplot for the 29 genes that decreased peaks that overlap with the promoter (up to 2 kb from a where Wiz haploinsufficiency was and the 53 genes that increased in expression in the cer- MD30/+ TSS) and genic sequence of active or silenced genes, causing differential gene expression, ebellum of Wiz mice. Also shown is CTCF ChIP-Seq according to RNA-Seq data mapped to Ensembl genes read density. ChIP-Seq density was calculated for the 1 annotations. (B) Wiz ChIP-Seq occupancy over all RNA-Seq was performed using brains Kb surrounding the TSS of each gene showing that those MD30/+ Ensembl gene bodies is shown as deep sequencing read from both E13.5 and adult Wiz and with decreased expression showed high enrichment for Wiz density along the transcription unit, including 2 kb up and Wiz+/+ male and female mice. The and some for CTCF, whereas upregulated genes showed downstream of the transcriptional start and stop site. results show that Wiz haploinsufficiency little enrichment for Wiz and no enrichment for CTCF. [email protected] 3 ADVANCES IN NEUROEPIGENETICS RESEARCH the researchers conducted behavioral studies on cohorts of WizMD30/+ and WT male mice. Their findings show various abnormalities in behavioral activity exhibited by the WizMD30/+ mutant vs. WT mice, including increased displays of anxiety-like behavior. Kdm5c-knockout mouse model of X-linked intellectual disability (XLID) This article is a summary of the research published in Cell Reports.: Iwase et al., A Mouse Model of X-linked Intellectual Disability Associated with Impaired Removal of Histone Methyl- ation (2016), http://dx.doi.org/10.1016/j.celrep.2015.12.091.