Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner.

BackgroundRewriting of the epigenome has risen as a promising alternative to gene editing for precision medicine. In nature, epigenetic silencing can result in complete attenuation of target gene expression over multiple mitotic divisions. However, persistent repression has been difficult to achieve in a predictable manner using targeted systems.ResultsHere, we report that persistent epigenetic memory required both a DNA methyltransferase (DNMT3A-dCas9) and a histone methyltransferase (Ezh2-dCas9 or KRAB-dCas9). We demonstrate that the histone methyltransferase requirement can be locus specific. Co-targeting Ezh2-dCas9, but not KRAB-dCas9, with DNMT3A-dCas9 and DNMT3L induced long-term HER2 repression over at least 50 days (approximately 57 cell divisions) and triggered an epigenetic switch to a heterochromatic environment. An increase in H3K27 trimethylation and DNA methylation was stably maintained and accompanied by a sustained loss of H3K27 acetylation. Interestingly, substitution of Ezh2-dCas9 with KRAB-dCas9 enabled long-term repression at some target genes (e.g., SNURF) but not at HER2, at which H3K9me3 and DNA methylation were transiently acquired and subsequently lost. Off-target DNA hypermethylation occurred at many individual CpG sites but rarely at multiple CpGs in a single promoter, consistent with no detectable effect on transcription at the off-target loci tested. Conversely, robust hypermethylation was observed at HER2. We further demonstrated that Ezh2-dCas9 required full-length DNMT3L for maximal activity and that co-targeting DNMT3L was sufficient for persistent repression by Ezh2-dCas9 or KRAB-dCas9.ConclusionsThese data demonstrate that targeting different combinations of histone and DNA methyltransferases is required to achieve maximal repression at different loci. Fine-tuning of targeting tools is a necessity to engineer epigenetic memory at any given locus in any given cell type

Nucleosome positioning and histone modifications define relationships between regulatory elements and nearby gene expression in breast epithelial cells

Abstract\ud \ud Background\ud The precise nature of how cell type specific chromatin structures at enhancer sites affect gene expression is largely unknown. Here we identified cell type specific enhancers coupled with gene expression in two different types of breast epithelial cells, HMEC (normal breast epithelial cells) and MDAMB231 (triple negative breast cancer cell line).\ud \ud \ud Results\ud Enhancers were defined by modified neighboring histones [using chromatin immunoprecipitation followed by sequencing (ChIP-seq)] and nucleosome depletion [using formaldehyde-assisted isolation of regulatory elements followed by sequencing (FAIRE-seq)]. Histone modifications at enhancers were related to the expression levels of nearby genes up to 750 kb away. These expression levels were correlated with enhancer status (poised or active), defined by surrounding histone marks. Furthermore, about fifty percent of poised and active enhancers contained nucleosome-depleted regions. We also identified response element motifs enriched at these enhancer sites that revealed key transcription factors (e.g. TP63) likely involved in regulating breast epithelial enhancer-mediated gene expression. By utilizing expression data, potential target genes of more than 600 active enhancers were identified. These genes were involved in proteolysis, epidermis development, cell adhesion, mitosis, cell cycle, and DNA replication.\ud \ud \ud Conclusions\ud These findings facilitate the understanding of epigenetic regulation specifically, such as the relationships between regulatory elements and gene expression and generally, how breast epithelial cellular phenotypes are determined by cell type specific enhancers.National Institutes of Health [R01 CA136924 to GAC, T32CA009320 to HN]Genetic Associations and Mechanisms in Oncology (GAME-ON

Local labor markets and the persistence of population shocks

The research in this paper was funded by Deutsche Forschungsgemeinschaft (grant no. BR 4979/1-1, “Die volkswirtschaftlichen Effekte der Vertriebenen und ihre Integration in Westdeutschland, 1945-70”).This paper studies the persistence of a large, unexpected, and regionally very unevenly distributed population shock, the inflow of eight million ethnic Germans from Eastern Europe to West Germany after World War II. Using detailed census data from 1939 to 1970, we show that the shock had a persistent effect on the distribution of population within local labor markets, but only a temporary effect on the distribution between labor markets. These results suggest that locational fundamentals determine population patterns across but not within local labor markets, and they can help to explain why previous studies on the persistence of population shocks reached such different conclusions.Publisher PD

Using 3D epigenomic maps of primary olfactory neuronal cells from living individuals to understand gene regulation

As part of PsychENCODE, we developed a three-dimensional (3D) epigenomic map of primary cultured neuronal cells derived from olfactory neuroepithelium (CNON). We mapped topologically associating domains and high-resolution chromatin interactions using Hi-C and identified regulatory elements using chromatin immunoprecipitation and nucleosome positioning assays. Using epigenomic datasets from biopsies of 63 living individuals, we found that epigenetic marks at distal regulatory elements are more variable than marks at proximal regulatory elements. By integrating genotype and metadata, we identified enhancers that have different levels corresponding to differences in genetic variation, gender, smoking, and schizophrenia. Motif searches revealed that many CNON enhancers are bound by neuronal-related transcription factors. Last, we combined 3D epigenomic maps and gene expression profiles to predict enhancer-target gene interactions on a genome-wide scale. This study not only provides a framework for understanding individual epigenetic variation using a primary cell model system but also contributes valuable data resources for epigenomic studies of neuronal epithelium

Comprehensive functional annotation of 77 prostate cancer risk loci.

Genome-wide association studies (GWAS) have revolutionized the field of cancer genetics, but the causal links between increased genetic risk and onset/progression of disease processes remain to be identified. Here we report the first step in such an endeavor for prostate cancer. We provide a comprehensive annotation of the 77 known risk loci, based upon highly correlated variants in biologically relevant chromatin annotations--we identified 727 such potentially functional SNPs. We also provide a detailed account of possible protein disruption, microRNA target sequence disruption and regulatory response element disruption of all correlated SNPs at r(2) ≥ 0.88%. 88% of the 727 SNPs fall within putative enhancers, and many alter critical residues in the response elements of transcription factors known to be involved in prostate biology. We define as risk enhancers those regions with enhancer chromatin biofeatures in prostate-derived cell lines with prostate-cancer correlated SNPs. To aid the identification of these enhancers, we performed genomewide ChIP-seq for H3K27-acetylation, a mark of actively engaged enhancers, as well as the transcription factor TCF7L2. We analyzed in depth three variants in risk enhancers, two of which show significantly altered androgen sensitivity in LNCaP cells. This includes rs4907792, that is in linkage disequilibrium (r(2) = 0.91) with an eQTL for NUDT11 (on the X chromosome) in prostate tissue, and rs10486567, the index SNP in intron 3 of the JAZF1 gene on chromosome 7. Rs4907792 is within a critical residue of a strong consensus androgen response element that is interrupted in the protective allele, resulting in a 56% decrease in its androgen sensitivity, whereas rs10486567 affects both NKX3-1 and FOXA-AR motifs where the risk allele results in a 39% increase in basal activity and a 28% fold-increase in androgen stimulated enhancer activity. Identification of such enhancer variants and their potential target genes represents a preliminary step in connecting risk to disease process

Characterizing Genetic Susceptibility to Breast Cancer in Women of African Ancestry

Background: Genome-wide association studies have identified approximately 100 common genetic variants associated with breast cancer risk, the majority of which were discovered in women of European ancestry. Because of different patterns of linkage disequilibrium, many of these genetic markers may not represent signals in populations of African ancestry. Methods: We tested 74 breast cancer risk variants and conducted fine-mapping of these susceptibility regions in 6,522 breast cancer cases and 7,643 controls of African ancestry from three genetic consortia (AABC, AMBER, and ROOT). Results: Fifty-four of the 74 variants (73%) were found to have ORs that were directionally consistent with those previously reported, of which 12 were nominally statistically significant ( P < 0.05). Through fine-mapping, in six regions ( 3p24, 12p11, 14q13, 16q12/FTO, 16q23, 19p13 ), we observed seven markers that better represent the underlying risk variant for overall breast cancer or breast cancer subtypes, whereas in another two regions ( 11q13, 16q12/TOX3 ), we identified suggestive evidence of signals that are independent of the reported index variant. Overlapping chromatin features and regulatory elements suggest that many of the risk alleles lie in regions with biological functionality. Conclusions: Through fine-mapping of known susceptibility regions, we have revealed alleles that better characterize breast cancer risk in women of African ancestry. Impact: The risk alleles identified represent genetic markers for modeling and stratifying breast cancer risk in women of African ancestry. Cancer Epidemiol Biomarkers Prev; 26(7); 1-11. ©2017 AACR

Genome-wide association studies identify four ER negative–specific breast cancer risk loci

Estrogen receptor (ER)-negative tumors represent 20–30% of all breast cancers, with a higher proportion occurring in younger women and women of African ancestry1. The etiology2 and clinical behavior3 of ER-negative tumors are different from those of tumors expressing ER (ER positive), including differences in genetic predisposition4. To identify susceptibility loci specific to ER-negative disease, we combined in a meta-analysis 3 genome-wide association studies of 4,193 ER-negative breast cancer cases and 35,194 controls with a series of 40 follow-up studies (6,514 cases and 41,455 controls), genotyped using a custom Illumina array, iCOGS, developed by the Collaborative Oncological Gene-environment Study (COGS). SNPs at four loci, 1q32.1 (MDM4, P = 2.1 × 10−12 and LGR6, P = 1.4 × 10−8), 2p24.1 (P = 4.6 × 10−8) and 16q12.2 (FTO, P = 4.0 × 10−8), were associated with ER-negative but not ER-positive breast cancer (P > 0.05). These findings provide further evidence for distinct etiological pathways associated with invasive ER-positive and ER-negative breast cancers

A meta-analysis identifies new loci associated with body mass index in individuals of African ancestry

Genome-wide association studies (GWAS) have identified 36 loci associated with body mass index (BMI), predominantly in populations of European ancestry. We conducted a meta-analysis to examine the association of >3.2 million SNPs with BMI in 39,144 men and women of African ancestry, and followed up the most significant associations in an additional 32,268 individuals of African ancestry. We identified one novel locus at 5q33 (GALNT10, rs7708584, p=3.4×10−11) and another at 7p15 when combined with data from the Giant consortium (MIR148A/NFE2L3, rs10261878, p=1.2×10−10). We also found suggestive evidence of an association at a third locus at 6q16 in the African ancestry sample (KLHL32, rs974417, p=6.9×10−8). Thirty-two of the 36 previously established BMI variants displayed directionally consistent effect estimates in our GWAS (binomial p=9.7×10−7), of which five reached genome-wide significance. These findings provide strong support for shared BMI loci across populations as well as for the utility of studying ancestrally diverse populations