Swneke D. Bailey

Breast cancer risk-associated SNPs modulate the affinity of chromatin for FOXA1 and alter gene expression

Genome-wide association studies (GWAS) have identified thousands of SNPs that are associated with human traits and diseases. But, because the vast majority of these SNPs are located in non-coding regions of the genome, the mechanisms by which they promote disease risk have remained elusive. Employing a new methodology that combines cistromics, epigenomics and genotype imputation, we annotate the non-coding regions of the genome in breast cancer cells and systematically identify the functional nature of SNPs associated with breast cancer risk. Our results show that breast cancer risk–associated SNPs are enriched in the cistromes of FOXA1 and ESR1 and the epigenome of histone H3 lysine 4 monomethylation (H3K4me1) in a cancer- and cell type–specific manner. Furthermore, the majority of the risk-associated SNPs modulate the affinity of chromatin for FOXA1 at distal regulatory elements, thereby resulting in allele-specific gene expression, which is exemplified by the effect of the rs4784227 SNP on the TOX3 gene within the 16q12.1 risk locus.

Integrative functional genomics identifies an enhancer looping to the SOX9 gene disrupted by the 17q24.3 prostate cancer risk locus

Genome-wide association studies (GWAS) are identifying genetic predisposition to various diseases. The 17q24.3 locus harbors the single nucleotide polymorphism (SNP) rs1859962 that is statistically associated with prostate cancer (PCa). It defines a 130-kb linkage disequilibrium (LD) block that lies in an ∼2-Mb gene desert area. The functional biology driving the risk associated with this LD block is unknown. Here, we integrate genome-wide chromatin landscape data sets, namely, epigenomes and chromatin openness from diverse cell types. This identifies a PCa-specific enhancer within the rs1859962 risk LD block that establishes a 1-Mb chromatin loop with the SOX9 gene. The rs8072254 and rs1859961 SNPs mapping to this enhancer impose allele-specific gene expression. The variant allele of rs8072254 facilitates androgen receptor (AR) binding driving increased enhancer activity. The variant allele of rs1859961 decreases FOXA1 binding while increasing AP-1 binding. The latter is key to imposing allele-specific gene expression. The rs8072254 variant in strong LD with the rs1859962 risk SNP can account for the risk associated with this locus, while rs1859961 is a rare variant less likely to contribute to the risk associated with this LD block. Together, our results demonstrate that multiple genetic variants mapping to a unique enhancer looping to the SOX9 oncogene can account for the risk associated with the PCa 17q24.3 locus. Allele-specific recruitment of the transcription factors androgen receptor (AR) and activating protein-1 (AP-1) account for the increased enhancer activity ascribed to this PCa-risk LD block. This further supports the notion that an integrative genomics approach can identify the functional biology disrupted by genetic risk variants.

ZNF143 provides sequence specificity to secure chromatin interactions at gene promoters

Chromatin interactions connect distal regulatory elements to target gene promoters guiding stimulus- and lineage-specific transcription. Few factors securing chromatin interactions have so far been identified. Here, by integrating chromatin interaction maps with the large collection of transcription factor-binding profiles provided by the ENCODE project, we demonstrate that the zinc-finger protein ZNF143 preferentially occupies anchors of chromatin interactions connecting promoters with distal regulatory elements. It binds directly to promoters and associates with lineage-specific chromatin interactions and gene expression. Silencing ZNF143 or modulating its DNA-binding affinity using single-nucleotide polymorphisms (SNPs) as a surrogate of site-directed mutagenesis reveals the sequence dependency of chromatin interactions at gene promoters. We also find that chromatin interactions alone do not regulate gene expression. Together, our results identify ZNF143 as a novel chromatin-looping factor that contributes to the architectural foundation of the genome by providing sequence specificity at promoters connected with distal regulatory elements. Chromatin interactions can connect distal regulatory elements to promoters via protein factors, but few such factors have been identified. Here, the authors show that zinc-finger protein ZNF143 is a sequence-specific chromatin-looping factor that connects promoters with distal regulatory elements.

Laying a solid foundation for Manhattan – ‘setting the functional basis for the post-GWAS era’

Genome-wide association studies (GWAS) have identified more than 8900 genetic variants, mainly single-nucleotide polymorphisms (SNPs), associated with hundreds of human traits and diseases, which define risk-associated loci. Variants that map to coding regions can affect protein sequence, translation rate, and alternative splicing, all of which influence protein function. However, the vast majority of sequence variants map to non-coding intergenic and intronic regions, and it has been much more challenging to assess the functional nature of these variants. Recent work annotating the non-coding regions of the genome has contributed to post-GWAS studies by facilitating the identification of the functional targets of risk-associated loci. Many non-coding genetic variants within risk-associated loci alter gene expression by modulating the activity of cis-regulatory elements. We review here these recent findings, discuss their implication for the post-GWAS era, and relate their importance to the interpretation of disease-associated mutations identified through whole-genome sequencing.

BioID identifies novel c-MYC interacting partners in cultured cells and xenograft tumors

UNLABELLEDnThe BioID proximity-based biotin labeling technique was recently developed for the characterization of protein-protein interaction networks [1]. To date, this method has been applied to a number of different polypeptides expressed in cultured cells. Here we report the adaptation of BioID to the identification of protein-protein interactions surrounding the c-MYC oncoprotein in human cells grown both under standard culture conditions and in mice as tumor xenografts. Notably, in vivo BioID yielded >100 high confidence MYC interacting proteins, including >30 known binding partners. Putative novel MYC interactors include components of the STAGA/KAT5 and SWI/SNF chromatin remodeling complexes, DNA repair and replication factors, general transcription and elongation factors, and transcriptional co-regulators such as the DNA helicase protein chromodomain 8 (CHD8). Providing additional confidence in these findings, ENCODE ChIP-seq datasets highlight significant coincident binding throughout the genome for the MYC interactors identified here, and we validate the previously unreported MYC-CHD8 interaction using both a yeast two hybrid analysis and the proximity-based ligation assay. In sum, we demonstrate that BioID can be utilized to identify bona fide interacting partners for a chromatin-associated protein in vivo. This technique will allow for a much improved understanding of protein-protein interactions in a previously inaccessible biological setting.nnnBIOLOGICAL SIGNIFICANCEnThe c-MYC (MYC) oncogene is a transcription factor that plays important roles in cancer initiation and progression. MYC expression is deregulated in more than 50% of human cancers, but the role of this protein in normal cell biology and tumor progression is still not well understood, in part because identifying MYC-interacting proteins has been technically challenging: MYC-containing chromatin-associated complexes are difficult to isolate using traditional affinity purification methods, and the MYC protein is exceptionally labile, with a half-life of only ~30 min. Developing a new strategy to gain insight into MYC-containing protein complexes would thus mark a key advance in cancer research. The recently described BioID proximity-based labeling technique represents a promising new complementary approach for the characterization of protein-protein interactions (PPIs) in cultured cells. Here we report that BioID can also be used to characterize protein-protein interactions for a chromatin-associated protein in tumor xenografts, and present a comprehensive, high confidence in vivo MYC interactome. This article is part of a Special Issue entitled: Protein dynamics in health and disease. Guest Editors: Pierre Thibault and Anne-Claude Gingras.

Human-Chromatin-Related Protein Interactions Identify a Demethylase Complex Required for Chromosome Segregation

Chromatin regulation is driven by multicomponent protein complexes, which form functional modules. Deciphering the components of these modules and their interactions is central to understanding the molecular pathways these proteins are regulating, their functions, and their relation to both normal development and disease. We describe the use of affinity purifications of tagged human proteins coupled with mass spectrometry to generate a protein-protein interaction map encompassing known and predicted chromatin-related proteins. On the basis of 1,394 successful purifications of 293 proteins, we report a high-confidence (85% precision) network involving 11,464 protein-protein interactions among 1,738 different human proteins, grouped into 164 often overlapping protein complexes with a particular focus on the family of JmjC-containing lysine demethylases, their partners, and their roles in chromatin remodeling. We show that RCCD1 is a partner of histone H3K36 demethylase KDM8 and demonstrate that both are important for cell-cycle-regulated transcriptional repression in centromeric regions and accurate mitotic division.

Noncoding somatic and inherited single-nucleotide variants converge to promote ESR1 expression in breast cancer.

Sustained expression of the estrogen receptor-α (ESR1) drives two-thirds of breast cancer and defines the ESR1-positive subtype. ESR1 engages enhancers upon estrogen stimulation to establish an oncogenic expression program. Somatic copy number alterations involving the ESR1 gene occur in approximately 1% of ESR1-positive breast cancers, suggesting that other mechanisms underlie the persistent expression of ESR1. We report significant enrichment of somatic mutations within the set of regulatory elements (SRE) regulating ESR1 in 7% of ESR1-positive breast cancers. These mutations regulate ESR1 expression by modulating transcription factor binding to the DNA. The SRE includes a recurrently mutated enhancer whose activity is also affected by rs9383590, a functional inherited single-nucleotide variant (SNV) that accounts for several breast cancer risk–associated loci. Our work highlights the importance of considering the combinatorial activity of regulatory elements as a single unit to delineate the impact of noncoding genetic alterations on single genes in cancer.

Human somatic cell mutagenesis creates genetically tractable sarcomas

Creating spontaneous yet genetically tractable human tumors from normal cells presents a fundamental challenge. Here we combined retroviral and transposon insertional mutagenesis to enable cancer gene discovery starting with human primary cells. We used lentiviruses to seed gain- and loss-of-function gene disruption elements, which were further deployed by Sleeping Beauty transposons throughout the genome of human bone explant mesenchymal cells. De novo tumors generated rapidly in this context were high-grade myxofibrosarcomas. Tumor insertion sites were enriched in recurrent somatic copy-number aberration regions from multiple cancer types and could be used to pinpoint new driver genes that sustain somatic alterations in patients. We identified HDLBP, which encodes the RNA-binding protein vigilin, as a candidate tumor suppressor deleted at 2q37.3 in greater than one out of ten tumors across multiple tissues of origin. Hybrid viral-transposon systems may accelerate the functional annotation of cancer genomes by enabling insertional mutagenesis screens in higher eukaryotes that are not amenable to germline transgenesis.

Enhancer alterations in cancer: a source for a cell identity crisis

Enhancers are selectively utilized to orchestrate gene expression programs that first govern pluripotency and then proceed to highly specialized programs required for the process of cellular differentiation. Whereas gene-proximal promoters are typically active across numerous cell types, distal enhancer activation is cell-type-specific and central to cell fate determination, thereby accounting for cell identity. Recent studies have highlighted the diversity of enhancer usage, cataloguing millions of such elements in the human genome. The disruption of enhancer activity, through genetic or epigenetic alterations, can impact cell-type-specific functions, resulting in a wide range of pathologies. In cancer, these alterations can promote a `cell identity crisis, in which enhancers associated with oncogenes and multipotentiality are activated, while those promoting cell fate commitment are inactivated. Overall, these alterations favor an undifferentiated cellular phenotype. Here, we review the current knowledge regarding the role of enhancers in normal cell function, and discuss how genetic and epigenetic changes in enhancer elements potentiate oncogenesis. In addition, we discuss how understanding the mechanisms regulating enhancer activity can inform therapeutic opportunities in cancer cells and highlight key challenges that remain in understanding enhancer biology as it relates to oncology.

Pre-neoplastic epigenetic disruption of transcriptional enhancers in chronic inflammation

Chronic periodontitis (CP) is a chronic inflammatory disease independently associated with higher incidence of oral cavity squamous cell carcinoma (OSCC). However, the molecular mechanism responsible for this increased incidence is unknown. Here we profiled the DNA methylome of CP patients and healthy controls and compared to a large set of OSCC samples from TCGA. We observed a significant overlap between the altered DNA methylation patterns in CP and in OSCC, suggesting an emergence of a pre-neoplastic epigenome in CP. Remarkably, the hypermethylated CpGs in CP were significantly enriched for enhancer elements. This aberrant enhancer methylation is functional and able to disrupt enhancer activity by preventing the binding of chromatin looping factors. This study provides new insights on the molecular mechanisms linking chronic inflammation and tumor predisposition, highlighting the role of epigenetic disruption of transcriptional enhancers.