Sara A. Grimm

An APOBEC cytidine deaminase mutagenesis pattern is widespread in human cancers

Recent studies indicate that a subclass of APOBEC cytidine deaminases, which convert cytosine to uracil during RNA editing and retrovirus or retrotransposon restriction, may induce mutation clusters in human tumors. We show here that throughout cancer genomes APOBEC-mediated mutagenesis is pervasive and correlates with APOBEC mRNA levels. Mutation clusters in whole-genome and exome data sets conformed to the stringent criteria indicative of an APOBEC mutation pattern. Applying these criteria to 954,247 mutations in 2,680 exomes from 14 cancer types, mostly from The Cancer Genome Atlas (TCGA), showed a significant presence of the APOBEC mutation pattern in bladder, cervical, breast, head and neck, and lung cancers, reaching 68% of all mutations in some samples. Within breast cancer, the HER2-enriched subtype was clearly enriched for tumors with the APOBEC mutation pattern, suggesting that this type of mutagenesis is functionally linked with cancer development. The APOBEC mutation pattern also extended to cancer-associated genes, implying that ubiquitous APOBEC-mediated mutagenesis is carcinogenic.

Research Resource: Whole-Genome Estrogen Receptor α Binding in Mouse Uterine Tissue Revealed by ChIP-Seq

To advance understanding of mechanisms leading to biological and transcriptional endpoints related to estrogen action in the mouse uterus, we have mapped ERα and RNA polymerase II (PolII) binding sites using chromatin immunoprecipitation followed by sequencing of enriched chromatin fragments. In the absence of hormone, 5184 ERα-binding sites were apparent in the vehicle-treated ovariectomized uterine chromatin, whereas 17,240 were seen 1 h after estradiol (E₂) treatment, indicating that some sites are occupied by unliganded ERα, and that ERα binding is increased by E₂. Approximately 15% of the uterine ERα-binding sites were adjacent to (<10 kb) annotated transcription start sites, and many sites are found within genes or are found more than 100 kb distal from mapped genes; however, the density (sites per base pair) of ERα-binding sites is significantly greater adjacent to promoters. An increase in quantity of sites but no significant positional differences were seen between vehicle and E₂-treated samples in the overall locations of ERα-binding sites either distal from, adjacent to, or within genes. Analysis of the PolII data revealed the presence of poised promoter-proximal PolII on some highly up-regulated genes. Additionally, corecruitment of PolII and ERα to some distal enhancer regions was observed. A de novo motif analysis of sequences in the ERα-bound chromatin confirmed that estrogen response elements were significantly enriched. Interestingly, in areas of ERα binding without predicted estrogen response element motifs, homeodomain transcription factor-binding motifs were significantly enriched. The integration of the ERα- and PolII-binding sites from our uterine sequencing of enriched chromatin fragments data with transcriptional responses revealed in our uterine microarrays has the potential to greatly enhance our understanding of mechanisms governing estrogen response in uterine and other estrogen target tissues.

MBD3 Localizes at Promoters, Gene Bodies and Enhancers of Active Genes

The Mi-2/nucleosome remodeling and histone deacetylase (NuRD) complex is a multiprotein machine proposed to regulate chromatin structure by nucleosome remodeling and histone deacetylation activities. Recent reports describing localization of NuRD provide new insights that question previous models on NuRD action, but are not in complete agreement. Here, we provide location analysis of endogenous MBD3, a component of NuRD complex, in two human breast cancer cell lines (MCF-7 and MDA-MB-231) using two independent genomic techniques: DNA adenine methyltransferase identification (DamID) and ChIP-seq. We observed concordance of the resulting genomic localization, suggesting that these studies are converging on a robust map for NuRD in the cancer cell genome. MBD3 preferentially associated with CpG rich promoters marked by H3K4me3 and showed cell-type specific localization across gene bodies, peaking around the transcription start site. A subset of sites bound by MBD3 was enriched in H3K27ac and was in physical proximity to promoters in three-dimensional space, suggesting function as enhancers. MBD3 enrichment was also noted at promoters modified by H3K27me3. Functional analysis of chromatin indicated that MBD3 regulates nucleosome occupancy near promoters and in gene bodies. These data suggest that MBD3, and by extension the NuRD complex, may have multiple roles in fine tuning expression for both active and silent genes, representing an important step in defining regulatory mechanisms by which NuRD complex controls chromatin structure and modification status.

DNA methylation profiling in human B cells reveals immune regulatory elements and epigenetic plasticity at Alu elements during B-cell activation

Memory is a hallmark of adaptive immunity, wherein lymphocytes mount a superior response to a previously encountered antigen. It has been speculated that epigenetic alterations in memory lymphocytes contribute to their functional distinction from their naive counterparts. However, the nature and extent of epigenetic alterations in memory compartments remain poorly characterized. Here we profile the DNA methylome and the transcriptome of B-lymphocyte subsets representing stages of the humoral immune response before and after antigen exposure in vivo from multiple humans. A significant percentage of activation-induced losses of DNA methylation mapped to transcription factor binding sites. An additional class of demethylated loci mapped to Alu elements across the genome and accompanied repression of DNA methyltransferase 3A. The activation-dependent DNA methylation changes were largely retained in the progeny of activated B cells, generating a similar epigenetic signature in downstream memory B cells and plasma cells with distinct transcriptional programs. These findings provide insights into the methylation dynamics of the genome during cellular differentiation in an immune response.

GATA3-dependent cellular reprogramming requires activation-domain dependent recruitment of a chromatin remodeler

BackgroundTranscription factor-dependent cellular reprogramming is integral to normal development and is central to production of induced pluripotent stem cells. This process typically requires pioneer transcription factors (TFs) to induce de novo formation of enhancers at previously closed chromatin. Mechanistic information on this process is currently sparse.ResultsHere we explore the mechanistic basis by which GATA3 functions as a pioneer TF in a cellular reprogramming event relevant to breast cancer, the mesenchymal to epithelial transition (MET). In some instances, GATA3 binds previously inaccessible chromatin, characterized by stable, positioned nucleosomes where it induces nucleosome eviction, alters local histone modifications, and remodels local chromatin architecture. At other loci, GATA3 binding induces nucleosome sliding without concomitant generation of accessible chromatin. Deletion of the transactivation domain retains the chromatin binding ability of GATA3 but cripples chromatin reprogramming ability, resulting in failure to induce MET.ConclusionsThese data provide mechanistic insights into GATA3-mediated chromatin reprogramming during MET, and suggest unexpected complexity to TF pioneering. Successful reprogramming requires stable binding to a nucleosomal site; activation domain-dependent recruitment of co-factors including BRG1, the ATPase subunit of the SWI/SNF chromatin remodeling complex; and appropriate genomic context. The resulting model provides a new conceptual framework for de novo enhancer establishment by a pioneer TF.

Obesity, rather than diet, drives epigenomic alterations in colonic epithelium resembling cancer progression.

While obesity represents one of several risk factors for colorectal cancer in humans, the mechanistic underpinnings of this association remain unresolved. Environmental stimuli, including diet, can alter the epigenetic landscape of DNA cis-regulatory elements affecting gene expression and phenotype. Here, we explored the impact of diet and obesity on gene expression and the enhancer landscape in murine colonic epithelium. Obesity led to the accumulation of histone modifications associated with active enhancers at genomic loci downstream of signaling pathways integral to the initiation and progression of colon cancer. Meanwhile, colon-specific enhancers lost the same histone mark, poising cells for loss of differentiation. These alterations reflect a transcriptional program with many features shared with the program driving colon cancer progression. The interrogation of enhancer alterations by diet in colonic epithelium provides insights into the biology underlying high-fat diet and obesity as risk factors for colon cancer.

The Impact of Environmental and Endogenous Damage on Somatic Mutation Load in Human Skin Fibroblasts

Accumulation of somatic changes, due to environmental and endogenous lesions, in the human genome is associated with aging and cancer. Understanding the impacts of these processes on mutagenesis is fundamental to understanding the etiology, and improving the prognosis and prevention of cancers and other genetic diseases. Previous methods relying on either the generation of induced pluripotent stem cells, or sequencing of single-cell genomes were inherently error-prone and did not allow independent validation of the mutations. In the current study we eliminated these potential sources of error by high coverage genome sequencing of single-cell derived clonal fibroblast lineages, obtained after minimal propagation in culture, prepared from skin biopsies of two healthy adult humans. We report here accurate measurement of genome-wide magnitude and spectra of mutations accrued in skin fibroblasts of healthy adult humans. We found that every cell contains at least one chromosomal rearrangement and 600–13,000 base substitutions. The spectra and correlation of base substitutions with epigenomic features resemble many cancers. Moreover, because biopsies were taken from body parts differing by sun exposure, we can delineate the precise contributions of environmental and endogenous factors to the accrual of genetic changes within the same individual. We show here that UV-induced and endogenous DNA damage can have a comparable impact on the somatic mutation loads in skin fibroblasts. Trial Registration ClinicalTrials.gov NCT01087307

LIN28A modulates splicing and gene expression programs in breast cancer cells

ABSTRACT LIN28 is an evolutionarily conserved RNA-binding protein with critical functions in developmental timing and cancer. However, the molecular mechanisms underlying LIN28s oncogenic properties are yet to be described. RNA-protein immunoprecipitation coupled with genome-wide sequencing (RIP-Seq) analysis revealed significant LIN28 binding within 843 mRNAs in breast cancer cells. Many of the LIN28-bound mRNAs are implicated in the regulation of RNA and cell metabolism. We identify heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), a protein with multiple roles in mRNA metabolism, as a LIN28-interacting partner. Subsequently, we used a custom computational method to identify differentially spliced gene isoforms in LIN28 and hnRNP A1 small interfering RNA (siRNA)-treated cells. The results reveal that these proteins regulate alternative splicing and steady-state mRNA expression of genes implicated in aspects of breast cancer biology. Notably, cells lacking LIN28 undergo significant isoform switching of the ENAH gene, resulting in a decrease in the expression of the ENAH exon 11a isoform. The expression of ENAH isoform 11a has been shown to be elevated in breast cancers that express HER2. Intriguingly, analysis of publicly available array data from the Cancer Genome Atlas (TCGA) reveals that LIN28 expression in the HER2 subtype is significantly different from that in other breast cancer subtypes. Collectively, our data suggest that LIN28 may regulate splicing and gene expression programs that drive breast cancer subtype phenotypes.

Fine-tuning of epigenetic regulation with respect to promoter CpG content in a cell type-specific manner.

Epigenetic regulation of gene expression is fundamental for cell type-specific gene expression. However, integrated comparative transcriptomic and epigenomic analyses in various adult primary differentiated cells remain underrepresented. We generated promoter landscapes of DNA methylation and three important histone methylation marks (H3K4me3, H3K9me2, and H3K27me3) in two primary cell types (B lymphocytes and liver) from adult mice. In line with previous studies, we also observed distinct H3K4me3 patterns at promoters dictated by CpG content in differentiated primary cells. We further explored the distribution of initiating RNA polymerase II and elongating RNA polymerase II across genes within different promoter classes, suggesting different rate-limiting steps at CpG-rich vs. CpG-poor genes. Examination of differentially expressed genes revealed that regulation of tissue-specific genes is closely related to gene function regardless of promoter type. Although repressive chromatin marks displayed differential preference to promoters based on CpG content, we observed fine-tuning of the pattern of association of these marks with specific promoter types in a cell type-specific manner. The distribution of H3K9me2 and H3K27me3, relative to CpG content, differed substantially between the two cell types. Cell-type specific accumulation of repressive chromatin marks was also observed at silent genes in both cell types, suggesting that differentiated primary cells may exhibit cell-type specificity in the distribution of repressive chromatin marks. Epigenetic regulation of gene expression and the association of specific histone marks with promoter sequence classes are fine-tuned in a cell type-specific manner. This unexpected finding underscores the value of extensive study of epigenetic marks across cell and tissue types.

The novel p53 target TNFAIP8 variant 2 is increased in cancer and offsets p53-dependent tumor suppression

Tumor necrosis factor-α-induced protein 8 (TNFAIP8) is a stress-response gene that has been associated with cancer, but no studies have differentiated among or defined the regulation or function of any of its several recently described expression variants. We found that TNFAIP8 variant 2 (v2) is overexpressed in multiple human cancers, whereas other variants are commonly downregulated in cancer (v1) or minimally expressed in cancer or normal tissue (v3–v6). Silencing v2 in cancer cells induces p53-independent inhibition of DNA synthesis, widespread binding of p53, and induction of target genes and p53-dependent cell cycle arrest and DNA damage sensitization. Cell cycle arrest induced by v2 silencing requires p53-dependent induction of p21. In response to the chemotherapeutic agent doxorubicin, p53 regulates v2 through binding to an intragenic enhancer, together indicating that p53 and v2 engage in complex reciprocal regulation. We propose that TNFAIP8 v2 promotes human cancer by broadly repressing p53 function, in essence offsetting p53-dependent tumor suppression.