generation-native-chromatin-immunoprecipitation-sequencing-libraries

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

University of British Columbia

The normal adult human mammary gland is a continuous bilayered epithelial system. Bipotent and myoepithelial progenitors are prominent and unique components of the outer (basal) layer. The inner (luminal) layer includes both luminal-restricted progenitors and a phenotypically separable fraction that lacks progenitor activity. We now report an epigenomic comparison of these three subsets with one another, with their associated stromal cells, and with three immortalized, non-tumorigenic human mammary cell lines. Each genome-wide analysis contains profiles for six histone marks, methylated DNA, and RNA transcripts. Analysis of these datasets shows that each cell type has unique features, primarily within genomic regulatory regions, and that the cell lines group together. Analyses of the promoter and enhancer profiles place the luminal progenitors in between the basal cells and the non-progenitor luminal subset. Integrative analysis reveals networks of subset-specific transcription factors.

Analysis of Normal Human Mammary Epigenomes Reveals Cell-Specific Active Enhancer States and Associated Transcription Factor Networks.

Genome sequences of chicken (low pathogenic avian influenza [LPAI] and highly pathogenic avian influenza [HPAI]) and human isolates from a 2004 outbreak of H7N3 avian influenza in Canada showed a novel insertion in the HA0 cleavage site of the human and HPAI isolate. This insertion likely occurred by recombination between the hemagglutination and matrix genes in the LPAI virus.

Novel avian influenza H7N3 strain outbreak, British Columbia.

Follicular lymphoma (FL) and the GCB subtype of diffuse large B-cell lymphoma (DLBCL) derive from germinal center B cells. Targeted resequencing studies have revealed mutations in various genes encoding proteins in the NF-kappaB pathway that contribute to the activated B-cell (ABC) DLBCL subtype, but thus far few GCB-specific mutations have been identified. Here we report recurrent somatic mutations affecting the polycomb-group oncogene EZH2, which encodes a histone methyltransferase responsible for trimethylating Lys27 of histone H3 (H3K27). After the recent discovery of mutations in KDM6A (UTX), which encodes the histone H3K27me3 demethylase UTX, in several cancer types, EZH2 is the second histone methyltransferase gene found to be mutated in cancer. These mutations, which result in the replacement of a single tyrosine in the SET domain of the EZH2 protein (Tyr641), occur in 21.7% of GCB DLBCLs and 7.2% of FLs and are absent from ABC DLBCLs. Our data are consistent with the notion that EZH2 proteins with mutant Tyr641 have reduced enzymatic activity in vitro.

Somatic mutations altering EZH2 (Tyr641) in follicular and diffuse large B-cell lymphomas of germinal-center origin.

Follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) are the two most common non-Hodgkin lymphomas (NHLs). Here we sequenced tumour and matched normal DNA from 13 DLBCL cases and one FL case to identify genes with mutations in B-cell NHL. We analysed RNA-seq data from these and another 113 NHLs to identify genes with candidate mutations, and then re-sequenced tumour and matched normal DNA from these cases to confirm 109 genes with multiple somatic mutations. Genes with roles in histone modification were frequent targets of somatic mutation. For example, 32% of DLBCL and 89% of FL cases had somatic mutations in MLL2, which encodes a histone methyltransferase, and 11.4% and 13.4% of DLBCL and FL cases, respectively, had mutations in MEF2B, a calcium-regulated gene that cooperates with CREBBP and EP300 in acetylating histones. Our analysis suggests a previously unappreciated disruption of chromatin biology in lymphomagenesis.

Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma.

Lymphome folliculaire (FL) et lymphome diffus de B-cellule grand (LMNH) sont les deux les lymphomes non hodgkiniens plus courantes (NHLs). Ici nous avons s&eacute;quenc&eacute; tumeur et mis en correspondance l&#39;ADN normal de 13 cas de LMNH et FL un cas pour identifier les g&egrave;nes pr&eacute;sentant des mutations dans les cellules B NHL. Nous avons analys&eacute; RNA-seq donn&eacute;es provenant de ces et un autre 113 NHLs d&#39;identifier les g&egrave;nes pr&eacute;sentant des mutations de candidat et puis divis&eacute; la tumeur et appari&eacute;s ADN normal de ces arr&ecirc;ts pour confirmer 109 g&egrave;nes avec plusieurs mutations somatiques. G&egrave;nes avec des r&ocirc;les dans la modification des histones sont fr&eacute;quemment la cible d&#39;une mutation somatique. Par exemple, 32 % des LMNH et 89 % des cas de FL eu mutations somatiques dans MLL2, qui code une histone-m&eacute;thyltransf&eacute;rase et 11,4 % et 13,4 % des cas de LMNH et FL, respectivement, ont des mutations dans MEF2B, un g&egrave;ne de calcium r&eacute;glement&eacute;s qui coop&egrave;re avec CREBBP et EP300 dans l&#39;ac&eacute;tylation des histones. Notre analyse sugg&egrave;re une perturbation pr&eacute;c&eacute;demment incomprises de la biologie de la chromatine en lymphomagen&egrave;se.

Fr&eacute;quentes mutations de modification des histones g&egrave;nes dans les lymphomes non hodgkiniens.

Linfoma follicolare (FL) e diffusa grande linfoma a cellule B (DLBCL) sono i due pi&ugrave; comuni linfomi non-Hodgkin (NHLs). Qui abbiamo sequenziato il tumore e abbinate normale DNA da 13 casi DLBCL e FL uno caso per identificare geni con mutazioni in cellule B NHL. Abbiamo analizzato RNA-seq dati da questi e un altro 113 NHLs per identificare geni con mutazioni del candidato e poi re-sequenced tumore e abbinate normale DNA da questi casi per confermare 109 geni con mutazioni somatiche multiple. Geni con ruoli in modifiche dell&#39;istone erano frequenti obiettivi di mutazione somatica. Ad esempio, il 32% dei DLBCL e 89% dei casi di FL avevano mutazioni somatiche nel MLL2, che codifica un istone metiltransferasi e 11,4% e 13,4% dei casi DLBCL e FL, rispettivamente, presentavano mutazioni nel MEF2B, un gene calcio-regolato che coopera con CREBBP ed EP300 acetilare istoni. La nostra analisi suggerisce una perturbazione precedentemente incompreso della biologia della cromatina in lymphomagenesis.

Frequente mutazione di modifica dell&#39;istone geni nel linfoma non-Hodgkin.

Linfoma folicular (FL) y linfoma difuso de c&eacute;lulas B grandes (DLBCL) son los dos m&aacute;s comunes no-Hodgkin (NHLs). Aqu&iacute; ordenamos tumor y ADN normal de 13 casos DLBCL y FL un caso para identificar genes con mutaciones en c&eacute;lulas B NHL. Analizaron datos de RNA-seq de estos y otros 113 NHLs identificar genes con mutaciones del candidato y luego re-sequenced tumor y emparejado el ADN normal de estos casos para confirmar 109 genes con m&uacute;ltiples mutaciones som&aacute;ticas. Genes con funciones en la modificaci&oacute;n de las histonas son blancos frecuentes de mutaci&oacute;n som&aacute;tica. Por ejemplo, 32% de DLBCL y 89% de los casos de FL ten&iacute;a mutaciones som&aacute;ticas en MLL2, que codifica una Histona metiltransferasa y 11,4% y 13,4% de los casos DLBCL y FL, respectivamente, ten&iacute;an mutaciones en MEF2B, un gen regulado por calcio que coopera con CREBBP y EP300 en acetylating histonas. Nuestro an&aacute;lisis sugiere una interrupci&oacute;n previamente apreciada de la biolog&iacute;a de la cromatina en lymphomagenesis.

Frecuente mutaci&oacute;n de histona-modificar genes en linfoma non-Hodgkin.

Endotoxin is recognized as one of the virulence factors of the Bordetella avium bird pathogen, and characterization of its structure and corresponding genomic features are important for an understanding of its role in pathogenicity and for an improved general knowledge of Bordetella spp virulence factors. The structure of the biologically active part of B. avium LPS, lipid A, is described and compared to those of another bird pathogen, opportunistic in humans, Bordetella hinzii, and to that of Bordetella trematum, a human pathogen. Sequence analyses showed that the three strains have homologues of acyl-chain modifying enzymes PagL, PagP and LpxO, of the 1-phosphatase LpxE, in addition to LgmA, LgmB and LgmC, which are required for the glucosamine modification. MALDI mass spectrometry identified a high amount of glucosamine substituting the phosphate groups of B. avium lipid A; this modification was absent from B. hinzii and B. trematum. The acylation patterns of the three lipid As were similar, but they differed from those of Bordetella pertussis and Bordetella parapertussis. They were also found to be close to the lipid A structure of Bordetella bronchiseptica, a mammalian pathogen, only differing from the latter by the degree of hydroxylation of the branched fatty acid.

Complete Bordetella avium, Bordetella hinzii and Bordetella trematum lipid A structures and genomic sequence analyses of the loci involved in their modifications.

Cellular barcoding offers a powerful approach to characterize the growth and differentiation activity of large numbers of cotransplanted stem cells. Here, we describe a lentiviral genomic-barcoding and analysis strategy and its use to compare the clonal outputs of transplants of purified mouse and human basal mammary epithelial cells. We found that both sources of transplanted cells produced many bilineage mammary epithelial clones in primary recipients, although primary clones containing only one detectable mammary lineage were also common. Interestingly, regardless of the species of origin, many clones evident in secondary recipients were not detected in the primary hosts, and others that were changed from appearing luminal-restricted to appearing bilineage. This barcoding methodology has thus revealed conservation between mice and humans of a previously unknown diversity in the growth and differentiation activities of their basal mammary epithelial cells stimulated to grow in transplanted hosts.

Clonal analysis via barcoding reveals diverse growth and differentiation of transplanted mouse and human mammary stem cells.

Rhodobacter capsulatus is a model organism for studying a novel type of horizontal gene transfer mediated by a phage-like gene transfer agent (RcGTA). Here we report the draft genome sequences of six R. capsulatus strains that exhibit different RcGTA properties, including RcGTA overproducers, RcGTA nonproducers, and/or RcGTA nonreceivers.

Draft Genome Sequences of Six Rhodobacter capsulatus Strains, YW1, YW2, B6, Y262, R121, and DE442.

Genomic and phenotypic analyses indicate extensive intra- as well as intertumoral heterogeneity in primary human malignant cell populations despite their clonal origin. Cellular DNA barcoding offers a powerful and unbiased alternative to track the number and size of multiple subclones within a single human tumour xenograft and their response to continued in vivo passaging. Using this approach we find clone-initiating cell frequencies that vary from ~1/10 to ~1/10,000 cells transplanted for two human breast cancer cell lines and breast cancer xenografts derived from three different patients. For the cell lines, these frequencies are negatively affected in transplants of more than 20,000 cells. Serial transplants reveal five clonal growth patterns (unchanging, expanding, diminishing, fluctuating or of delayed onset), whose predominance is highly variable both between and within original samples. This study thus demonstrates the high growth potential and diverse growth properties of xenografted human breast cancer cells.

DNA barcoding reveals diverse growth kinetics of human breast tumour subclones in serially passaged xenografts.

While significant effort has been dedicated to the characterization of epigenetic changes associated with prenatal differentiation, relatively little is known about the epigenetic changes that accompany post-natal differentiation where fully functional differentiated cell types with limited lifespans arise. Here we sought to address this gap by generating epigenomic and transcriptional profiles from primary human breast cell types isolated from disease-free human subjects. From these data we define a comprehensive human breast transcriptional network, including a set of myoepithelial- and luminal epithelial-specific intronic retention events. Intersection of epigenetic states with RNA expression from distinct breast epithelium lineages demonstrates that mCpG provides a stable record of exonic and intronic usage, whereas H3K36me3 is dynamic. We find a striking asymmetry in epigenomic reprogramming between luminal and myoepithelial cell types, with the genomes of luminal cells harbouring more than twice the number of hypomethylated enhancer elements compared with myoepithelial cells.

Epigenetic and transcriptional determinants of the human breast.

Myocyte enhancer factor 2B (MEF2B) is a transcription factor with mutation hotspots at K4, Y69 and D83 in diffuse large B-cell lymphoma (DLBCL). To provide insight into the regulatory network of MEF2B, in this study, we analyse global gene expression and DNA-binding patterns. We find that candidate MEF2B direct target genes include RHOB, RHOD, CDH13, ITGA5 and CAV1, and that indirect target genes of MEF2B include MYC, TGFB1, CARD11, MEF2C, NDRG1 and FN1. MEF2B overexpression increases HEK293A cell migration and epithelial-mesenchymal transition, and decreases DLBCL cell chemotaxis. K4E, Y69H and D83V MEF2B mutations decrease the capacity of MEF2B to activate transcription and decrease its' effects on cell migration. The K4E and D83V mutations decrease MEF2B DNA binding. In conclusion, our map of the MEF2B regulome connects MEF2B to drivers of oncogenesis.

MEF2B mutations in non-Hodgkin lymphoma dysregulate cell migration by decreasing MEF2B target gene activation.

Most human breast cancers have diversified genomically and biologically by the time they become clinically evident. Early events involved in their genesis and the cellular context in which these events occur have thus been difficult to characterize. Here we present the first formal evidence of the shared and independent ability of basal cells and luminal progenitors, isolated from normal human mammary tissue and transduced with a single oncogene (KRAS(G12D)), to produce serially transplantable, polyclonal, invasive ductal carcinomas within 8 weeks of being introduced either subrenally or subcutaneously into immunodeficient mice. DNA barcoding of the initial cells revealed a dramatic change in the numbers and sizes of clones generated from them within 2 weeks, and the first appearance of many 'new' clones in tumours passaged into secondary recipients. Both primary and secondary tumours were phenotypically heterogeneous and primary tumours were categorized transcriptionally as 'normal-like'. This system challenges previous concepts that carcinogenesis in normal human epithelia is necessarily a slow process requiring the acquisition of multiple driver mutations. It also presents the first description of initial events that accompany the genesis and evolution of malignant human mammary cell populations, thereby contributing new understanding of the rapidity with which heterogeneity in their properties can develop.

Barcoding reveals complex clonal dynamics of de novo transformed human mammary cells.

The genomes of large numbers of single cells must be sequenced to further understanding of the biological significance of genomic heterogeneity in complex systems. Whole genome amplification (WGA) of single cells is generally the first step in such studies, but is prone to nonuniformity that can compromise genomic measurement accuracy. Despite recent advances, robust performance in high-throughput single-cell WGA remains elusive. Here, we introduce droplet multiple displacement amplification (MDA), a method that uses commercially available liquid dispensing to perform high-throughput single-cell MDA in nanoliter volumes. The performance of droplet MDA is characterized using a large dataset of 129 normal diploid cells, and is shown to exceed previously reported single-cell WGA methods in amplification uniformity, genome coverage, and/or robustness. We achieve up to 80% coverage of a single-cell genome at 5× sequencing depth, and demonstrate excellent single-nucleotide variant (SNV) detection using targeted sequencing of droplet MDA product to achieve a median allelic dropout of 15%, and using whole genome sequencing to achieve false and true positive rates of 9.66 × 10(-6) and 68.8%, respectively, in a G1-phase cell. We further show that droplet MDA allows for the detection of copy number variants (CNVs) as small as 30 kb in single cells of an ovarian cancer cell line and as small as 9 Mb in two high-grade serous ovarian cancer samples using only 0.02× depth. Droplet MDA provides an accessible and scalable method for performing robust and accurate CNV and SNV measurements on large numbers of single cells.

Robust high-performance nanoliter-volume single-cell multiple displacement amplification on planar substrates.

Nucleosome position, density, and post-translational modification are widely accepted components of mechanisms regulating DNA transcription but still incompletely understood. We present a modified native ChIP-seq method combined with an analytical framework that allows MNase accessibility to be integrated with histone modification profiles. Application of this methodology to the primitive (CD34+) subset of normal human cord blood cells enabled genomic regions enriched in one versus two nucleosomes marked by histone 3 lysine 4 trimethylation (H3K4me3) and/or histone 3 lysine 27 trimethylation (H3K27me3) to be associated with their transcriptional and DNA methylation states. From this analysis, we defined four classes of promoter-specific profiles and demonstrated that a majority of bivalent marked promoters are heterogeneously marked at a single-cell level in this primitive cell type. Interestingly, extension of this approach to human embryonic stem cells revealed an altered relationship between chromatin modification state and nucleosome content at promoters, suggesting developmental stage-specific organization of histone methylation states.

Nucleosome Density ChIP-Seq Identifies Distinct Chromatin Modification Signatures Associated with MNase Accessibility.

Mutations in human Atrophin1, a transcriptional corepressor, cause dentatorubral-pallidoluysian atrophy, a neurodegenerative disease. Drosophila Atrophin (Atro) mutants display many phenotypes, including neurodegeneration, segmentation, patterning and planar polarity defects. Despite Atro's critical role in development and disease, relatively little is known about Atro's binding partners and downstream targets. We present the first genomic analysis of Atro using ChIP-seq against endogenous Atro. ChIP-seq identified 1300 potential direct targets of Atro including engrailed, and components of the Dpp and Notch signaling pathways. We show that Atro regulates Dpp and Notch signaling in larval imaginal discs, at least partially via regulation of thickveins and fringe. In addition, bioinformatics analyses, sequential ChIP and coimmunoprecipitation experiments reveal that Atro interacts with the Drosophila GAGA Factor, Trithorax-like (Trl), and they bind to the same loci simultaneously. Phenotypic analyses of Trl and Atro clones suggest that Atro is required to modulate the transcription activation by Trl in larval imaginal discs. Taken together, these data indicate that Atro is a major Trl cofactor that functions to moderate developmental gene transcription.

Atrophin controls developmental signaling pathways via interactions with Trithorax-like.

The yeast Sup35 protein is a subunit of the translation termination factor, and its conversion to the [PSI +] prion state leads to more translational read-through. Although extensive studies have been done on [PSI +], changes at the proteomic level have not been performed exhaustively. We therefore used a SILAC-based quantitative mass spectrometry approach and identified 4187 proteins from both [psi -] and [PSI +] strains. Surprisingly, there was very little difference between the two proteomes under standard growth conditions. We found however that several [PSI +] strains harbored an additional chromosome, such as chromosome I. Albeit, we found no evidence to support that [PSI +] induces chromosomal instability (CIN). Instead we hypothesized that the selective pressure applied during the establishment of [PSI +]-containing strains could lead to a supernumerary chromosome due to the presence of the ade1-14 selective marker for translational read-through. We therefore verified that there was no prevalence of disomy among newly generated [PSI +] strains in absence of strong selection pressure. We also noticed that low amounts of adenine in media could lead to higher levels of mitochondrial DNA in [PSI +] in ade1-14 cells. Our study has important significance for the establishment and manipulation of yeast strains with the Sup35 prion.

The [PSI +] yeast prion does not wildly affect proteome composition whereas selective pressure exerted on [PSI +] cells can promote aneuploidy.

Human glioblastomas harbour a subpopulation of glioblastoma stem cells that drive tumorigenesis. However, the origin of intratumoural functional heterogeneity between glioblastoma cells remains poorly understood. Here we study the clonal evolution of barcoded glioblastoma cells in an unbiased way following serial xenotransplantation to define their individual fate behaviours. Independent of an evolving mutational signature, we show that the growth of glioblastoma clones in vivo is consistent with a remarkably neutral process involving a conserved proliferative hierarchy rooted in glioblastoma stem cells. In this model, slow-cycling stem-like cells give rise to a more rapidly cycling progenitor population with extensive self-maintenance capacity, which in turn generates non-proliferative cells. We also identify rare 'outlier' clones that deviate from these dynamics, and further show that chemotherapy facilitates the expansion of pre-existing drug-resistant glioblastoma stem cells. Finally, we show that functionally distinct glioblastoma stem cells can be separately targeted using epigenetic compounds, suggesting new avenues for glioblastoma-targeted therapy.

Article	Year
Analysis of Normal Human Mammary Epigenomes Reveals Cell-Specific Active Enhancer States and Associated Transcription Factor Networks. Cell reports\| PubMed ID: 27851968	2016
Novel avian influenza H7N3 strain outbreak, British Columbia. Emerging infectious diseases\| PubMed ID: 15663859	2004
Somatic mutations altering EZH2 (Tyr641) in follicular and diffuse large B-cell lymphomas of germinal-center origin. Nature genetics\| PubMed ID: 20081860	2010
Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma. Nature\| PubMed ID: 21796119	2011
Complete Bordetella avium, Bordetella hinzii and Bordetella trematum lipid A structures and genomic sequence analyses of the loci involved in their modifications. Innate immunity\| PubMed ID: 24127384	2014
Clonal analysis via barcoding reveals diverse growth and differentiation of transplanted mouse and human mammary stem cells. Cell stem cell\| PubMed ID: 24440600	2014
Draft Genome Sequences of Six Rhodobacter capsulatus Strains, YW1, YW2, B6, Y262, R121, and DE442. Genome announcements\| PubMed ID: 24526637	2014
DNA barcoding reveals diverse growth kinetics of human breast tumour subclones in serially passaged xenografts. Nature communications\| PubMed ID: 25532760	2014
Epigenetic and transcriptional determinants of the human breast. Nature communications\| PubMed ID: 25690954	2015
MEF2B mutations in non-Hodgkin lymphoma dysregulate cell migration by decreasing MEF2B target gene activation. Nature communications\| PubMed ID: 26245647	2015
Barcoding reveals complex clonal dynamics of de novo transformed human mammary cells. Nature\| PubMed ID: 26633636	2015
Robust high-performance nanoliter-volume single-cell multiple displacement amplification on planar substrates. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 27412862	2016
Nucleosome Density ChIP-Seq Identifies Distinct Chromatin Modification Signatures Associated with MNase Accessibility. Cell reports\| PubMed ID: 27851972	2016
Atrophin controls developmental signaling pathways via interactions with Trithorax-like. eLife\| PubMed ID: 28327288	2017
The [PSI +] yeast prion does not wildly affect proteome composition whereas selective pressure exerted on [PSI +] cells can promote aneuploidy. Scientific reports\| PubMed ID: 28814753	2017
Fate mapping of human glioblastoma reveals an invariant stem cell hierarchy. Nature\| PubMed ID: 28854171	2017

Michelle Moksa

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