Joseph R. Boyd

Histone H3 lysine 4 acetylation and methylation dynamics define breast cancer subtypes

The onset and progression of breast cancer are linked to genetic and epigenetic changes that alter the normal programming of cells. Epigenetic modifications of DNA and histones contribute to chromatin structure that result in the activation or repression of gene expression. Several epigenetic pathways have been shown to be highly deregulated in cancer cells. Targeting specific histone modifications represents a viable strategy to prevent oncogenic transformation, tumor growth or metastasis. Methylation of histone H3 lysine 4 has been extensively studied and shown to mark genes for expression; however this residue can also be acetylated and the specific function of this alteration is less well known. To define the relative roles of histone H3 methylation (H3K4me3) and acetylation (H3K4ac) in breast cancer, we determined genomic regions enriched for both marks in normal-like (MCF10A), transformed (MCF7) and metastatic (MDA-MB-231) cells using a genome-wide ChIP-Seq approach. Our data revealed a genome-wide gain of H3K4ac associated with both early and late breast cancer cell phenotypes, while gain of H3K4me3 was predominantly associated with late stage cancer cells. Enrichment of H3K4ac was over-represented at promoters of genes associated with cancer-related phenotypic traits, such as estrogen response and epithelial-to-mesenchymal transition pathways. Our findings highlight an important role for H3K4ac in predicting epigenetic changes associated with early stages of transformation. In addition, our data provide a valuable resource for understanding epigenetic signatures that correlate with known breast cancer-associated oncogenic pathways.

MicroRNA-378-mediated suppression of Runx1 alleviates the aggressive phenotype of triple-negative MDA-MB-231 human breast cancer cells.

The Runx1 transcription factor, known for its essential role in normal hematopoiesis, was reported in limited studies to be mutated or associated with human breast tumor tissues. Runx1 increases concomitantly with disease progression in the MMTV-PyMT transgenic mouse model of breast cancer. Compelling questions relate to mechanisms that regulate Runx1 expression in breast cancer. Here, we tested the hypothesis that dysregulation of Runx1-targeting microRNAs (miRNAs) allows for pathologic increase of Runx1 during breast cancer progression. Microarray profiling of the MMTV-PyMT model revealed significant downregulation of numerous miRNAs predicted to target Runx1. One of these, miR-378, was inversely correlated with Runx1 expression during breast cancer progression in mice and in human breast cancer cell lines MCF7 and triple-negative MDA-MB-231 that represent early- and late-stage diseases, respectively. MiR-378 is nearly absent in MDA-MB-231 cells. Luciferase reporter assays revealed that miR-378 binds the Runx1 3′ untranslated region (3′UTR) and inhibits Runx1 expression. Functionally, we demonstrated that ectopic expression of miR-378 in MDA-MB-231 cells inhibited Runx1 and suppressed migration and invasion, while inhibition of miR-378 in MCF7 cells increased Runx1 levels and cell migration. Depletion of Runx1 in late-stage breast cancer cells resulted in increased expression of both the miR-378 host gene PPARGC1B and pre-miR-378, suggesting a feedback loop. Taken together, our study identifies a novel and clinically relevant mechanism for regulation of Runx1 in breast cancer that is mediated by a PPARGC1B-miR-378-Runx1 regulatory pathway. Our results highlight the translational potential of miRNA replacement therapy for inhibiting Runx1 in breast cancer.

Oncofetal Epigenetic Bivalency in Breast Cancer Cells: H3K4 and H3K27 Tri-Methylation as a Biomarker for Phenotypic Plasticity.

Alterations in the epigenetic landscape are fundamental drivers of aberrant gene expression that contribute to cancer progression and pathology. Understanding specific modes of epigenetic regulation can be used to identify novel biomarkers or targets for therapeutic intervention to clinically treat solid tumors and leukemias. The bivalent marking of gene promoters by H3K4me3 and H3K27me3 is a primary mechanism to poise genes for expression in pluripotent embryonic stem cells (ESC). In this study we interrogated three well‐established mammary cell lines to model epigenetic programming observed among breast cancer subtypes. Evidence is provided for a distinct bivalent signature, activating and repressive histone marks co‐residing at the same gene promoter, in the MCF7 (ESR/PGR+) luminal breast cancer cell line. We identified a subset of genes, enriched for developmental pathways that regulate cellular phenotype and signaling, and partially recapitulate the bivalent character observed in ESC. We validated the biological relevance of this “oncofetal epigenetic” signature using data from ESR/PGR+ tumor samples from breast cancer patients. This signature of oncofetal epigenetic control is an informative biomarker and may provide novel therapeutic targets, selective for both recurring and treatment‐resistant cancers. J. Cell. Physiol. 231: 2474–2481, 2016.

Genetic analysis of Ikaros target genes and tumor suppressor function in BCR-ABL1+ pre–B ALL

Inactivation of the tumor suppressor gene encoding the transcriptional regulator Ikaros (IKZF1) is a hallmark of BCR-ABL1+ precursor B cell acute lymphoblastic leukemia (pre–B ALL). However, the mechanisms by which Ikaros functions as a tumor suppressor in pre–B ALL remain poorly understood. Here, we analyzed a mouse model of BCR-ABL1+ pre–B ALL together with a new model of inducible expression of wild-type Ikaros in IKZF1 mutant human BCR-ABL1+ pre–B ALL. We performed integrated genome-wide chromatin and expression analyses and identified Ikaros target genes in mouse and human BCR-ABL1+ pre–B ALL, revealing novel conserved gene pathways associated with Ikaros tumor suppressor function. Notably, genetic depletion of different Ikaros targets, including CTNND1 and the early hematopoietic cell surface marker CD34, resulted in reduced leukemic growth. Our results suggest that Ikaros mediates tumor suppressor function by enforcing proper developmental stage–specific expression of multiple genes through chromatin compaction at its target genes.

Intranuclear and higher-order chromatin organization of the major histone gene cluster in breast cancer†

Alterations in nuclear morphology are common in cancer progression. However, the degree to which gross morphological abnormalities translate into compromised higher‐order chromatin organization is poorly understood. To explore the functional links between gene expression and chromatin structure in breast cancer, we performed RNA‐seq gene expression analysis on the basal breast cancer progression model based on human MCF10A cells. Positional gene enrichment identified the major histone gene cluster at chromosome 6p22 as one of the most significantly upregulated (and not amplified) clusters of genes from the normal‐like MCF10A to premalignant MCF10AT1 and metastatic MCF10CA1a cells. This cluster is subdivided into three sub‐clusters of histone genes that are organized into hierarchical topologically associating domains (TADs). Interestingly, the sub‐clusters of histone genes are located at TAD boundaries and interact more frequently with each other than the regions in‐between them, suggesting that the histone sub‐clusters form an active chromatin hub. The anchor sites of loops within this hub are occupied by CTCF, a known chromatin organizer. These histone genes are transcribed and processed at a specific sub‐nuclear microenvironment termed the major histone locus body (HLB). While the overall chromatin structure of the major HLB is maintained across breast cancer progression, we detected alterations in its structure that may relate to gene expression. Importantly, breast tumor specimens also exhibit a coordinate pattern of upregulation across the major histone gene cluster. Our results provide a novel insight into the connection between the higher‐order chromatin organization of the major HLB and its regulation during breast cancer progression.

Bivalent Epigenetic Control of Oncofetal Gene Expression in Cancer

ABSTRACT Multiple mechanisms of epigenetic control that include DNA methylation, histone modification, noncoding RNAs, and mitotic gene bookmarking play pivotal roles in stringent gene regulation during lineage commitment and maintenance. Experimental evidence indicates that bivalent chromatin domains, i.e., genome regions that are marked by both H3K4me3 (activating) and H3K27me3 (repressive) histone modifications, are a key property of pluripotent stem cells. Bivalency of developmental genes during the G1 phase of the pluripotent stem cell cycle contributes to cell fate decisions. Recently, some cancer types have been shown to exhibit partial recapitulation of bivalent chromatin modifications that are lost along with pluripotency, suggesting a mechanism by which cancer cells reacquire properties that are characteristic of undifferentiated, multipotent cells. This bivalent epigenetic control of oncofetal gene expression in cancer cells may offer novel insights into the onset and progression of cancer and may provide specific and selective options for diagnosis as well as for therapeutic intervention.

Nuclear organization mediates cancer-compromised genetic and epigenetic control

Nuclear organization is functionally linked to genetic and epigenetic regulation of gene expression for biological control and is modified in cancer. Nuclear organization supports cell growth and phenotypic properties of normal and cancer cells by facilitating physiologically responsive interactions of chromosomes, genes and regulatory complexes at dynamic three-dimensional microenvironments. We will review nuclear structure/function relationships that include: 1. Epigenetic bookmarking of genes by phenotypic transcription factors to control fidelity and plasticity of gene expression as cells enter and exit mitosis; 2. Contributions of chromatin remodeling to breast cancer nuclear morphology, metabolism and effectiveness of chemotherapy; 3. Relationships between fidelity of nuclear organization and metastasis of breast cancer to bone; 4. Dynamic modifications of higher-order inter- and intra-chromosomal interactions in breast cancer cells; 5. Coordinate control of cell growth and phenotype by tissue-specific transcription factors; 6. Oncofetal epigenetic control by bivalent histone modifications that are functionally related to sustaining the stem cell phenotype; and 7. Noncoding RNA-mediated regulation in the onset and progression of breast cancer. The discovery of components to nuclear organization that are functionally related to cancer and compromise gene expression have the potential for translation to innovative cancer diagnosis and targeted therapy.

Expression of Ribosomal RNA and Protein Genes in Human Embryonic Stem Cells Is Associated With the Activating H3K4me3 Histone Mark.

Embryonic stem cells (ESCs) exhibit unrestricted and indefinite, but stringently controlled, proliferation, and can differentiate into any lineage in the body. In the current study, we test the hypothesis that expression of ribosomal RNA (rRNA) and ribosomal protein genes (RPGs) contribute to the ability of hESCs to proliferate indefinitely. Consistent with the accelerated growth rate of hESCs, we find that hESC lines H1 and H9 both exhibit significantly higher levels of rRNA when compared to a panel of normal and cancer human cell lines. Although many RPGs are expressed at levels that comparable to other human cell lines, a few RPGs also exhibit higher expression levels. In situ nuclear run‐on assays reveal that both nucleoli in hESCs actively transcribe nascent rRNA. Employing genome‐wide chromatin immunoprecipitation‐deep sequencing and bioinformatics approaches, we discovered that, RPGs are dominantly marked by the activating H3K4me3 histone mark in the G1, M, and G2 phases of the cell cycle. Interestingly, the rDNA repeats are marked by the activating H3K4me3 only in the M phase, and repressive H3K27me3 histone mark in all three cell cycle phases. Bioinformatics analyses also reveal that Myc, a known regulator of cell growth and proliferation, occupies both the rRNA genes and RPGs. Functionally, down‐regulation of Myc expression by siRNA results in a concomitant decrease in rRNA levels. Together, our results show that expression of rRNA, which is regulated by the Myc pluripotency transcription factor, and of RPGs in hESCs is associated with the activating H3K4me3 modification. J. Cell. Physiol. 231: 2007–2013, 2016.

Oncogenic epigenetic control

There is growing recognition that epigenetic regulation contributes significantly to the fidelity of biological control. Compromised epigenetic pathways have now been shown to provide indications of transformation and tumor progression; they suggest potential targets for cancer prevention and intervention [1]. A formidable challenge is to deconvolute the complexity and plasticity of histone modifications associated with tumorigenesis. Although challenging, identification of epigenetic signals that are functionally associated with oncogenesis will provide a better understanding of the mechanisms underlying their impact. Post translational modifications of histone3 lysine4 (H3K4) may constitute epigenetically mediated regulatory signatures that can be informative for the molecular characterization of breast cancer pathology. Both methylation and acetylation of H3K4 are associated with gene activation. Tri-methylation of histone3 lysine4 has been extensively studied and is a well-documented marker for active or poised gene transcription. The enzyme complexes that regulate H3K4me3 have been implicated in tumorigenesis, pointing to the relevance of this epigenetic modification in cancer. Histone acetylation as well as histone acetyltransferases and deacetylating enzymes are recognized as important in tumor progression. However, the acetylation of H3K4 in particular has been less extensively investigated to date. Messier, et al., addressed the coordinated contributions of H3K4me3 and H3K4ac to breast cancer tumorigenesis in three well-established human mammary cell lines that represent a normal-like subtype (MCF10A; fibrocystic disease) and two cancer subtypes, luminal (MCF7; ER+/PR+) and basal-like metastatic (MDA-MB-231; ER−/PR−/HER2−) [2]. These cell lines recapitulate the phenotypic and proliferative transitions that accompany tumor progression states. Utilizing ChIP-seq analysis, a genome-wide map of H3K4me3 and H3K4ac patterns was developed, establishing dynamic alterations in the overall histone H3K4 epigenetic landscape in the three cell lines. An observed global increase in H3K4ac in MCF7 and MDA-MB-231 cell lines that is accompanied by a global increase in H3K4me3 in the MDA-MB-231 metastatic cell line is consistent with increased representation of both histone marks during oncogenic progression. Modifications in H3K4 tri-methylation and acetylation were found to occur on specific chromosomes, including the X-chromosome. This striking change in H3K4 tri-methylation and acetylation may account for the loss of X-chromosome inactivation and epigenomic instability of the inactive X-chromosome seen in breast cancer [3]. In contrast to methylation and acetylation of H3K9 and H3K27, which activate or repress transcription respectively, methylation and acetylation of H3K4 are both associated with gene activation. Observed increases in H3K4 acetylation marks at specific gene promoters in both the MCF7 and MDA-MB-231 cell lines, compared to normal-like MCF10A cells, suggests that H3K4 acetylation may be an early step in cancer initiation or progression. The global increase in H3K4me3 at gene promoters in MDA-MB-231 cells suggests alterations in the epigenetic landscape that may be functionally related to late stage, metastatic breast cancer. From a mechanistic perspective, the presence of H3K4ac and H3K4me3 at specific gene promoters provides a blueprint for regulatory pathways that are functionally linked to epigenetic control of breast cancer onset and progression. The dynamic acetylation of H3K4 is associated with estrogen-receptor responsiveness in the MCF7 cells and the initial loss of mammary tissue–specific gene expression in MCF7 cells that represent early stage breast cancer. Enhancing the pivotal role of H3K4ac in initial stages of breast cancer tumorigenesis, this epigenetic mark appears to poise genes for expression that leverages continued acquisition and persistence of metastatic properties. In contrast, H3K4me3, with persistence of H3K4ac reflects the epithelial to mesenchymal transition in MDA-MB-231 cells that represent late stage breast cancer. Important mechanistic linkages between histone H3K4ac and H3K4me3 modifications and breast cancer tumorigenesis remain to be established. While H3K4ac is emerging as an informative predictor of pathways that are deregulated during the onset and progression of breast cancer, the full potential for exploitation of this epigenetic modification as a biomarker and therapeutic target remains to be explored. Future studies must further define the enzymes that support acetylation and deacetylation of H3K4. Furthermore, the dynamic balance between H3K4 acetylation and methylation must be elucidated within the context of the full spectrum of histone modifications that contribute to combinatorial control of the epigenetic landscape. This will provide a drastically improved understanding of how epigenetic modifications and chromatin organization regulate mammary epithelial phenotype and gene expression related to transformation and tumor progression—the complexity of chromatin organization that supports breast cancer initiation and progression is increasingly evident. Consequently, the clinical value of epigenetic biomarkers and related therapeutic targets can be expanded by detailed characterization of the regulatory signals responsible for the deposition of specific “histone marks”. Adding to the biological challenge inherent in these analyses, the plasticity observed in post translational histone modifications must also be considered. It will be important to establish the relationships between histone modifications and other parameters of epigenetic control and chromatin organization in breast cancer that include perturbations in inter- and intra-chromosomal interactions [4] and oncofetal recapitulation of bivalent epigenetic regulatory mechanisms [5-8]. Although challenging, the complexity of epigenetic control in breast cancer provides the potential for increased capabilities in breast cancer risk assessment, early diagnosis and therapy with enhanced specificity and minimal off-target consequences. Figure 1 Dynamics of H3K4ac epigenetic modifications in cell lines representative of breast/breast cancer subtypes

Higher order genomic organization and regulatory compartmentalization for cell cycle control at the G1/S-phase transition

Fidelity of histone gene regulation, and ultimately of histone protein biosynthesis, is obligatory for packaging of newly replicated DNA into chromatin. Control of histone gene expression within the 3‐dimensional context of nuclear organization is reflected by two well documented observations. DNA replication‐dependent histone mRNAs are synthesized at specialized subnuclear domains designated histone locus bodies (HLBs), in response to activation of the growth factor dependent Cyclin E/CDK2/HINFP/NPAT pathway at the G1/S transition in mammalian cells. Complete loss of the histone gene regulatory factors HINFP or NPAT disrupts HLB integrity that is necessary for coordinate control of DNA replication and histone gene transcription. Here we review the molecular histone‐related requirements for G1/S‐phase progression during the cell cycle. Recently developed experimental strategies, now enable us to explore mechanisms involved in dynamic control of histone gene expression in the context of the temporal (cell cycle) and spatial (HLBs) remodeling of the histone gene loci.