Alina Saiakhova

Epigenomic Enhancer Profiling Defines a Signature of Colon Cancer

Colorectal Cancer Signature The mutations and genome aberrations that characterize cancer result in often dramatically altered gene and protein expression patterns. It is these altered expression patterns that directly and indirectly drive progression of the disease. In human primary colorectal cancer cells, Akhtar-Zaidi et al. (p. 736, published online 12 April) analyzed the pattern of epigenetically modified chromatin at “enhancer” sequences that are known to be critical in the control of gene expression. An epigenetic enhancer signature was defined that was specifically associated with colorectal cancer cells. Methylation tags at long-distance gene regulatory elements provide a signature specific to cancer cells. Cancer is characterized by gene expression aberrations. Studies have largely focused on coding sequences and promoters, even though distal regulatory elements play a central role in controlling transcription patterns. We used the histone mark H3K4me1 to analyze gain and loss of enhancer activity genome-wide in primary colon cancer lines relative to normal colon crypts. We identified thousands of variant enhancer loci (VELs) that comprise a signature that is robustly predictive of the in vivo colon cancer transcriptome. Furthermore, VELs are enriched in haplotype blocks containing colon cancer genetic risk variants, implicating these genomic regions in colon cancer pathogenesis. We propose that reproducible changes in the epigenome at enhancer elements drive a specific transcriptional program to promote colon carcinogenesis.

Epigenomic Comparison Reveals Activation of “Seed” Enhancers during Transition from Naive to Primed Pluripotency

Naive mouse embryonic stem cells (mESCs) and primed epiblast stem cells (mEpiSCs) represent successive snapshots of pluripotency during embryogenesis. Using transcriptomic and epigenomic mapping we show that a small fraction of transcripts are differentially expressed between mESCs and mEpiSCs and that these genes show expected changes in chromatin at their promoters and enhancers. Unexpectedly, the cis-regulatory circuitry of genes that are expressed at identical levels between these cell states also differs dramatically. In mESCs, these genes are associated with dominant proximal enhancers and dormant distal enhancers, which we term seed enhancers. In mEpiSCs, the naive-dominant enhancers are lost, and the seed enhancers take up primary transcriptional control. Seed enhancers have increased sequence conservation and show preferential usage in downstream somatic tissues, often expanding into super enhancers. We propose that seed enhancers ensure proper enhancer utilization and transcriptional fidelity as mammalian cells transition from naive pluripotency to a somatic regulatory program.

Integrative genomic analysis of human ribosomal DNA

The transcription of ribosomal RNA (rRNA) is critical to life. Despite its importance, ribosomal DNA (rDNA) is not included in current genome assemblies and, consequently, genomic analyses to date have excluded rDNA. Here, we show that short sequence reads can be aligned to a genome assembly containing a single rDNA repeat. Integrated analysis of ChIP-seq, DNase-seq, MNase-seq and RNA-seq data reveals several novel findings. First, the coding region of active rDNA is contained within nucleosome-depleted open chromatin that is highly transcriptionally active. Second, histone modifications are located not only at the rDNA promoter but also at novel sites within the intergenic spacer. Third, the distributions of active modifications are more similar within and between different cell types than repressive modifications. Fourth, UBF, a positive regulator of rRNA transcription, binds to sites throughout the genome. Lastly, the insulator binding protein CTCF associates with the spacer promoter of rDNA, suggesting that transcriptional insulation plays a role in regulating the transcription of rRNA. Taken together, these analyses confirm and expand the results of previous ChIP studies of rDNA and provide novel avenues for exploration of chromatin-mediated regulation of rDNA.

Trithorax monomethylates histone H3K4 and interacts directly with CBP to promote H3K27 acetylation and antagonize Polycomb silencing

Trithorax (TRX) antagonizes epigenetic silencing by Polycomb group (PcG) proteins, stimulates enhancer-dependent transcription, and establishes a ‘cellular memory’ of active transcription of PcG-regulated genes. The mechanisms underlying these TRX functions remain largely unknown, but are presumed to involve its histone H3K4 methyltransferase activity. We report that the SET domains of TRX and TRX-related (TRR) have robust histone H3K4 monomethyltransferase activity in vitro and that Tyr3701 of TRX and Tyr2404 of TRR prevent them from being trimethyltransferases. The trxZ11 missense mutation (G3601S), which abolishes H3K4 methyltransferase activity in vitro, reduces the H3K4me1 but not the H3K4me3 level in vivo. trxZ11 also suppresses the impaired silencing phenotypes of the Pc3 mutant, suggesting that H3K4me1 is involved in antagonizing Polycomb silencing. Polycomb silencing is also antagonized by TRX-dependent H3K27 acetylation by CREB-binding protein (CBP). We show that perturbation of Polycomb silencing by TRX overexpression requires CBP. We also show that TRX and TRR are each physically associated with CBP in vivo, that TRX binds directly to the CBP KIX domain, and that the chromatin binding patterns of TRX and TRR are highly correlated with CBP and H3K4me1 genome-wide. In vitro acetylation of H3K27 by CBP is enhanced on K4me1-containing H3 substrates, and independently altering the H3K4me1 level in vivo, via the H3K4 demethylase LSD1, produces concordant changes in H3K27ac. These data indicate that the catalytic activities of TRX and CBP are physically coupled and suggest that both activities play roles in antagonizing Polycomb silencing, stimulating enhancer activity and cellular memory.

Hotspots of aberrant enhancer activity punctuate the colorectal cancer epigenome

In addition to mutations in genes, aberrant enhancer element activity at non-coding regions of the genome is a key driver of tumorigenesis. Here, we perform epigenomic enhancer profiling of a cohort of more than forty genetically diverse human colorectal cancer (CRC) specimens. Using normal colonic crypt epithelium as a comparator, we identify enhancers with recurrently gained or lost activity across CRC specimens. Of the enhancers highly recurrently activated in CRC, most are constituents of super enhancers, are occupied by AP-1 and cohesin complex members, and originate from primed chromatin. Many activate known oncogenes, and CRC growth can be mitigated through pharmacologic inhibition or genome editing of these loci. Nearly half of all GWAS CRC risk loci co-localize to recurrently activated enhancers. These findings indicate that the CRC epigenome is defined by highly recurrent epigenetic alterations at enhancers which activate a common, aberrant transcriptional programme critical for CRC growth and survival.

Transcriptome-wide identification of mRNAs and lincRNAs associated with trastuzumab-resistance in HER2-positive breast cancer

Approximately, 25–30% of early-stage breast tumors are classified at the molecular level as HER2-positive, which is an aggressive subtype of breast cancer. Amplification of the HER2 gene in these tumors results in a substantial increase in HER2 mRNA levels, and consequently, HER2 protein levels. HER2, a transmembrane receptor tyrosine kinase (RTK), is targeted therapeutically by a monoclonal antibody, trastuzumab (Tz), which has dramatically improved the prognosis of HER2-driven breast cancers. However, ~30% of patients develop resistance to trastuzumab and recur; and nearly all patients with advanced disease develop resistance over time and succumb to the disease. Mechanisms of trastuzumab resistance (TzR) are not well understood, although some studies suggest that growth factor signaling through other receptors may be responsible. However, these studies were based on cell culture models of the disease, and thus, it is not known which pathways are driving the resistance in vivo. Using an integrative transcriptomic approach of RNA isolated from trastuzumab-sensitive and trastuzumab-resistant HER2+ tumors, and isogenic cell culture models, we identified a small set of mRNAs and lincRNAs that are associated with trastuzumab-resistance (TzR). Functional analysis of a top candidate gene, S100P, demonstrated that inhibition of S100P results in reversing TzR. Mechanistically, S100P activates the RAS/MEK/MAPK pathway to compensate for HER2 inhibition by trastuzumab. Finally, we demonstrated that the upregulation of S100P appears to be driven by epigenomic changes at the enhancer level. Our current findings should pave the path toward new therapies for breast cancer patients.

Therapeutic targeting of ependymoma as informed by oncogenic enhancer profiling

Genomic sequencing has driven precision-based oncology therapy; however, the genetic drivers of many malignancies remain unknown or non-targetable, so alternative approaches to the identification of therapeutic leads are necessary. Ependymomas are chemotherapy-resistant brain tumours, which, despite genomic sequencing, lack effective molecular targets. Intracranial ependymomas are segregated on the basis of anatomical location (supratentorial region or posterior fossa) and further divided into distinct molecular subgroups that reflect differences in the age of onset, gender predominance and response to therapy. The most common and aggressive subgroup, posterior fossa ependymoma group A (PF-EPN-A), occurs in young children and appears to lack recurrent somatic mutations. Conversely, posterior fossa ependymoma group B (PF-EPN-B) tumours display frequent large-scale copy number gains and losses but have favourable clinical outcomes. More than 70% of supratentorial ependymomas are defined by highly recurrent gene fusions in the NF-κB subunit gene RELA (ST-EPN-RELA), and a smaller number involve fusion of the gene encoding the transcriptional activator YAP1 (ST-EPN-YAP1). Subependymomas, a distinct histologic variant, can also be found within the supratetorial and posterior fossa compartments, and account for the majority of tumours in the molecular subgroups ST-EPN-SE and PF-EPN-SE. Here we describe mapping of active chromatin landscapes in 42 primary ependymomas in two non-overlapping primary ependymoma cohorts, with the goal of identifying essential super-enhancer-associated genes on which tumour cells depend. Enhancer regions revealed putative oncogenes, molecular targets and pathways; inhibition of these targets with small molecule inhibitors or short hairpin RNA diminished the proliferation of patient-derived neurospheres and increased survival in mouse models of ependymomas. Through profiling of transcriptional enhancers, our study provides a framework for target and drug discovery in other cancers that lack known genetic drivers and are therefore difficult to treat.

Positively selected enhancer elements endow osteosarcoma cells with metastatic competence

Metastasis results from a complex set of traits acquired by tumor cells, distinct from those necessary for tumorigenesis. Here, we investigate the contribution of enhancer elements to the metastatic phenotype of osteosarcoma. Through epigenomic profiling, we identify substantial differences in enhancer activity between primary and metastatic human tumors and between near isogenic pairs of highly lung metastatic and nonmetastatic osteosarcoma cell lines. We term these regions metastatic variant enhancer loci (Met-VELs). Met-VELs drive coordinated waves of gene expression during metastatic colonization of the lung. Met-VELs cluster nonrandomly in the genome, indicating that activity of these enhancers and expression of their associated gene targets are positively selected. As evidence of this causal association, osteosarcoma lung metastasis is inhibited by global interruptions of Met-VEL-associated gene expression via pharmacologic BET inhibition, by knockdown of AP-1 transcription factors that occupy Met-VELs, and by knockdown or functional inhibition of individual genes activated by Met-VELs, such as that encoding coagulation factor III/tissue factor (F3). We further show that genetic deletion of a single Met-VEL at the F3 locus blocks metastatic cell outgrowth in the lung. These findings indicate that Met-VELs and the genes they regulate play a functional role in metastasis and may be suitable targets for antimetastatic therapies.

Enhancer mapping uncovers phenotypic heterogeneity and evolution in patients with luminal breast cancer

The degree of intrinsic and interpatient phenotypic heterogeneity and its role in tumor evolution is poorly understood. Phenotypic drifts can be transmitted via inheritable transcriptional programs. Cell-type specific transcription is maintained through the activation of epigenetically defined regulatory regions including promoters and enhancers. Here we have annotated the epigenome of 47 primary and metastatic estrogen-receptor (ERα)-positive breast cancer clinical specimens and inferred phenotypic heterogeneity from the regulatory landscape, identifying key regulatory elements commonly shared across patients. Shared regions contain a unique set of regulatory information including the motif for transcription factor YY1. We identify YY1 as a critical determinant of ERα transcriptional activity promoting tumor growth in most luminal patients. YY1 also contributes to the expression of genes mediating resistance to endocrine treatment. Finally, we used H3K27ac levels at active enhancer elements as a surrogate of intra-tumor phenotypic heterogeneity to track the expansion and contraction of phenotypic subpopulations throughout breast cancer progression. By tracking the clonality of SLC9A3R1-positive cells, a bona fide YY1-ERα-regulated gene, we show that endocrine therapies select for phenotypic clones under-represented at diagnosis. Collectively, our data show that epigenetic mechanisms significantly contribute to phenotypic heterogeneity and evolution in systemically treated breast cancer patients.Topographic analysis of the active regulatory landscape in estrogen receptor-positive breast cancer uncovers a role for transcription factor YY1 in modulating phenotypic heterogeneity during tumor evolution and endocrine resistance.

Positively Selected Enhancer Elements Endow Tumor Cells with Metastatic Competence

Metastasis results from a complex set of traits acquired by tumor cells, distinct from those necessary for tumorigenesis. Here, we investigate the contribution of enhancer elements to the metastatic phenotype of osteosarcoma. Through epigenomic profiling, we identify substantial differences in enhancer activity between primary and metastatic tumors in human patients as well as nearisogenic pairs of high and low lung-metastatic osteosarcoma cells. We term these regions Metastatic Variant Enhancer Loci (Met-VELs). We demonstrate that these Met-VELs drive coordinated waves of gene expression during metastatic colonization of the lung. Met-VELs cluster non-randomly, indicating that activity of these enhancers and their associated gene targets are positively selected. As evidence of this causal association, osteosarcoma lung metastasis is inhibited by global interruptions of Met-VEL-associated gene expression via pharmacologic BET inhibition, by knockdown of AP-1 transcription factors that occupy Met-VELs, and by knockdown or functional inhibition of individual genes activated by Met-VELs, such as F3. We further show that genetic deletion of a single Met-VEL at the F3 locus blocks metastatic cell outgrowth in the lung. These findings indicate that Met-VELs and the genes they regulate play a functional role in metastasis and may be suitable targets for anti-metastatic therapies.