Computational methods to explore chromatin state dynamics.

The human genome is marked by several singular and combinatorial histone modifications that shape the different states of chromatin and its three-dimensional organization. Genome-wide mapping of these marks as well as histone variants and open chromatin regions is commonly carried out via profiling DNA-protein binding or via chromatin accessibility methods. After the generation of epigenomic datasets in a cell type, statistical models can be used to annotate the noncoding regions of DNA and infer the combinatorial histone marks or chromatin states (CS). These methods involve partitioning the genome and labeling individual segments based on their CS patterns. Chromatin labels enable the systematic discovery of genomic function and activity and can label the gene body, promoters or enhancers without using other genomic maps. CSs are dynamic and change under different cell conditions, such as in normal, preneoplastic or tumor cells. This review aims to explore the available computational tools that have been developed to capture CS alterations under two or more cellular conditions.

Chromatin state dynamics confers specific therapeutic strategies in enhancer subtypes of colorectal cancer.

OBJECTIVE: Enhancer aberrations are beginning to emerge as a key epigenetic feature of colorectal cancers (CRC), however, a comprehensive knowledge of chromatin state patterns in tumour progression, heterogeneity of these patterns and imparted therapeutic opportunities remain poorly described. DESIGN: We performed comprehensive epigenomic characterisation by mapping 222 chromatin profiles from 69 samples (33 colorectal adenocarcinomas, 4 adenomas, 21 matched normal tissues and 11 colon cancer cell lines) for six histone modification marks: H3K4me3 for Pol II-bound and CpG-rich promoters, H3K4me1 for poised enhancers, H3K27ac for enhancers and transcriptionally active promoters, H3K79me2 for transcribed regions, H3K27me3 for polycomb repressed regions and H3K9me3 for heterochromatin. RESULTS: We demonstrate that H3K27ac-marked active enhancer state could distinguish between different stages of CRC progression. By epigenomic editing, we present evidence that gains of tumour-specific enhancers for crucial oncogenes, such as ASCL2 and FZD10, was required for excessive proliferation. Consistently, combination of MEK plus bromodomain inhibition was found to have synergistic effects in CRC patient-derived xenograft models. Probing intertumour heterogeneity, we identified four distinct enhancer subtypes (EPIgenome-based Classification, EpiC), three of which correlate well with previously defined transcriptomic subtypes (consensus molecular subtypes, CMSs). Importantly, CMS2 can be divided into two EpiC subgroups with significant survival differences. Leveraging such correlation, we devised a combinatorial therapeutic strategy of enhancer-blocking bromodomain inhibitors with pathway-specific inhibitors (PARPi, EGFRi, TGFßi, mTORi and SRCi) for EpiC groups. CONCLUSION: Our data suggest that the dynamics of active enhancer underlies CRC progression and the patient-specific enhancer patterns can be leveraged for precision combination therapy.

Methylation-eQTL analysis in cancer research.

MOTIVATION: DNA methylation is a key epigenetic factor regulating gene expression. While promoter methylation has been well studied, recent publications have revealed that functionally important methylation also occurs in intergenic and distal regions, and varies across genes and tissue types. Given the growing importance of inter-platform integrative genomic analyses, there is an urgent need to develop methods to discover and characterize gene-level relationships between methylation and expression. RESULTS: We introduce a novel sequential penalized regression approach to identify methylation-expression quantitative trait loci (methyl-eQTLs), a term that we have coined to represent, for each gene and tissue type, a sparse set of CpG loci best explaining gene expression and accompanying weights indicating direction and strength of association. Using TCGA and MD Anderson colorectal cohorts to build and validate our models, we demonstrate our strategy better explains expression variability than current commonly used gene-level methylation summaries. The methyl-eQTLs identified by our approach can be used to construct gene-level methylation summaries that are maximally correlated with gene expression for use in integrative models, and produce a tissue-specific summary of which genes appear to be strongly regulated by methylation. Our results introduce an important resource to the biomedical community for integrative genomics analyses involving DNA methylation. AVAILABILITY AND IMPLEMENTATION: We produce an R Shiny app (https://rstudio-prd-c1.pmacs.upenn.edu/methyl-eQTL/) that interactively presents methyl-eQTL results for colorectal, breast and pancreatic cancer. The source R code for this work is provided in the Supplementary Material. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Histone methyltransferase SETDB1 contributes to melanoma tumorigenesis and serves as a new potential therapeutic target.

Alterations in histone modifications play a crucial role in the progression of various types of cancer. The histone methyltransferase SETDB1 catalyzes the addition of methyl groups to histone H3 at lysine 9. Here, we describe how overexpression of SETDB1 contributes to melanoma tumorigenesis. SETDB1 is highly amplified in melanoma cells and in the patient tumors. Increased expression of SETDB1, which correlates with SETDB1 amplification, is associated with a more aggressive phenotype in in vitro and in vivo studies. Mechanistically, SETDB1 implements its effects via regulation of thrombospondin 1, and the SET-domain of SETDB1 is essential for the maintenance of its tumorigenic activity. Inhibition of SETDB1 reduces cell growth in melanomas resistant to targeted treatments. Our results indicate that SETDB1 is a major driver of melanoma development and may serve as a potential future target for the treatment of this disease.

MAP kinase pathway gene copy alterations in NRAS/BRAF wild-type advanced melanoma.

Recent therapeutic advances have improved melanoma patients clinical outcome. Novel therapeutics targeting BRAF, NRAS and cKit mutant melanomas are widely used in clinical practice. However therapeutic options in NRAS(wild-type) /BRAF(wild-type) /cKit(wild-type) melanoma patients are limited. Our study shows that gene copy numbers of members of the MAPK signaling pathway vary in different melanoma subgroups. NRAS(wild-type) /BRAF(wild-type) melanoma metastases are characterized by significant gains of MAP2K1 (MEK1) and MAPK3 (ERK1) gene loci. These additional gene copies could lead to an activation of the MAPK signaling pathway via a gene-dosage effect. Our results suggest that downstream analyses of the pMEK and pERK expression status in NRAS(wild-type) /BRAF(wild-type) melanoma patients identify patients that could benefit from targeted therapies with MEK and ERK inhibitors.

PREVALENCE AND ASSOCIATIONS OF MYELINATED RETINAL NERVE FIBERS: Results From the Population-Based Gutenberg Health Study.

PURPOSE: To determine the prevalence, ocular, and systemic associations of myelinated retinal nerve fibers (MRNF) in a Caucasian cohort. METHODS: The Gutenberg Health Study (GHS) is a population-based, prospective cohort study encompassing 15,010 subjects in Germany. Gutenberg Health Study participants, aged 35 to 74 years, stratified for gender, decades of age, and residence were examined for ophthalmologic and systemic conditions. Optic disc centered and macular photographs were reviewed for the presence of MRNF. RESULTS: In 25,728 eyes of 12,906 participants (86.0% of the cohort), the prevalence of MRNF was 0.4%. In a binary logistic regression analysis, MRNF was positively associated with history of stroke (OR, 6.8; 95% CI, 2.9-16.1; P < 0.001). Myelinated retinal nerve fibers was not associated with age, sex, cardiovascular conditions other than stroke or ocular parameters, such as refraction, visual acuity, intraocular pressure, or central corneal thickness. CONCLUSION: This population-based study provides novel data on the prevalence of MRNF in Western Europe. We report a positive association between history of stroke and MRNF. It adds an additional retinal sign for stroke and calls for further studying of the behavior of oligodendrocytes within cerebrovascular diseases.

Therapeutic effect of Avonex, Rebif and Betaferon on quality of life in multiple sclerosis.

AIMS: The aim of this study was to evaluate the effect of various disease-modifying therapies (DMT) on quality of life in multiple sclerosis (MS). METHODS: This was a three-arm parallel study with balanced randomization in which 90 newly diagnosed, definite MS subjects referred to Ghaem Medical Center, Mashhad, Iran were enrolled between 2006 and 2009. Patients were randomly allocated into three DMT groups: Avonex, Rebif and Betaferon. Health-related quality of life was assessed in MS patients at baseline and 12 months after treatment with DMT using the MS Quality of Life-54 questionnaire. RESULTS: Both mental and physical health scores improved within all three treatment groups after 12 months of treatment; however, this increase was only significant in the mental health composite in the Betaferon group (P = 0.024). Betaferon had the highest mental health score change (14.04) while this change was 7.26 for Avonex (P = 0.031) and 5.08 for Rebif (P = 0.017). A physical health composite score comparison among the three treatment groups revealed no significant results. CONCLUSIONS: With a positive impact of DMT on mental and physical dimensions of QOL in MS patients, initiation of treatment soon after diagnosis is recommended. In MS patients with more mental issues and fewer physical disabilities, Betaferon might be considered as a better choice of treatment.

MYC reshapes CTCF-mediated chromatin architecture in prostate cancer.

MYC is a well characterized oncogenic transcription factor in prostate cancer, and CTCF is the main architectural protein of three-dimensional genome organization. However, the functional link between the two master regulators has not been reported. In this study, we find that MYC rewires prostate cancer chromatin architecture by interacting with CTCF protein. Through combining the H3K27ac, AR and CTCF HiChIP profiles with CRISPR deletion of a CTCF site upstream of MYC gene, we show that MYC activation leads to profound changes of CTCF-mediated chromatin looping. Mechanistically, MYC colocalizes with CTCF at a subset of genomic sites, and enhances CTCF occupancy at these loci. Consequently, the CTCF-mediated chromatin looping is potentiated by MYC activation, resulting in the disruption of enhancer-promoter looping at neuroendocrine lineage plasticity genes. Collectively, our findings define the function of MYC as a CTCF co-factor in three-dimensional genome organization.

Single-nucleus multiomic atlas of frontal cortex in amyotrophic lateral sclerosis with a deep learning-based decoding of alternative polyadenylation mechanisms.

The understanding of how different cell types contribute to amyotrophic lateral sclerosis (ALS) pathogenesis is limited. Here we generated a single-nucleus transcriptomic and epigenomic atlas of the frontal cortex of ALS cases with C9orf72 (C9) hexanucleotide repeat expansions and sporadic ALS (sALS). Our findings reveal shared pathways in C9-ALS and sALS, characterized by synaptic dysfunction in excitatory neurons and a disease-associated state in microglia. The disease subtypes diverge with loss of astrocyte homeostasis in C9-ALS, and a more substantial disturbance of inhibitory neurons in sALS. Leveraging high depth 3'-end sequencing, we found a widespread switch towards distal polyadenylation (PA) site usage across ALS subtypes relative to controls. To explore this differential alternative PA (APA), we developed APA-Net, a deep neural network model that uses transcript sequence and expression levels of RNA-binding proteins (RBPs) to predict cell-type specific APA usage and RBP interactions likely to regulate APA across disease subtypes.

A primary hierarchically organized patient-derived model enables in depth interrogation of stemness driven by the coding and non-coding genome.

Many cancers are organized as cellular hierarchies sustained by cancer stem cells (CSC), whose eradication is crucial for achieving long-term remission. Difficulties to isolate and undertake in vitro and in vivo experimental studies of rare CSC under conditions that preserve their original properties currently constitute a bottleneck for identifying molecular mechanisms involving coding and non-coding genomic regions that govern stemness. We focussed on acute myeloid leukemia (AML) as a paradigm of the CSC model and developed a patient-derived system termed OCI-AML22 that recapitulates the cellular hierarchy driven by leukemia stem cells (LSC). Through classical flow sorting and functional analyses, we established that a single phenotypic population is highly enriched for LSC. The LSC fraction can be easily isolated and serially expanded in culture or in xenografts while faithfully recapitulating functional, transcriptional and epigenetic features of primary LSCs. A novel non-coding regulatory element was identified with a new computational approach using functionally validated primary AML LSC fractions and its role in LSC stemness validated through efficient CRISPR editing using methods optimized for OCI-AML22 LSC. Collectively, OCI-AML22 constitutes a valuable resource to uncover mechanisms governing CSC driven malignancies.

IPO11 regulates the nuclear import of BZW1/2 and is necessary for AML cells and stem cells.

AML cells are arranged in a hierarchy with stem/progenitor cells giving rise to more differentiated bulk cells. Despite the importance of stem/progenitors in the pathogenesis of AML, the determinants of the AML stem/progenitor state are not fully understood. Through a comparison of genes that are significant for growth and viability of AML cells by way of a CRISPR screen, with genes that are differentially expressed in leukemia stem cells (LSC), we identified importin 11 (IPO11) as a novel target in AML. Importin 11 (IPO11) is a member of the importin ß family of proteins that mediate transport of proteins across the nuclear membrane. In AML, knockdown of IPO11 decreased growth, reduced engraftment potential of LSC, and induced differentiation. Mechanistically, we identified the transcription factors BZW1 and BZW2 as novel cargo of IPO11. We further show that BZW1/2 mediate a transcriptional signature that promotes stemness and survival of LSC. Thus, we demonstrate for the first time how specific cytoplasmic-nuclear regulation supports stem-like transcriptional signature in relapsed AML.

Reprogramming of bivalent chromatin states in NRAS mutant melanoma suggests PRC2 inhibition as a therapeutic strategy.

The dynamic evolution of chromatin state patterns during metastasis, their relationship with bona fide genetic drivers, and their therapeutic vulnerabilities are not completely understood. Combinatorial chromatin state profiling of 46 melanoma samples reveals an association of NRAS mutants with bivalent histone H3 lysine 27 trimethylation (H3K27me3) and Polycomb repressive complex 2. Reprogramming of bivalent domains during metastasis occurs on master transcription factors of a mesenchymal phenotype, including ZEB1, TWIST1, and CDH1. Resolution of bivalency using pharmacological inhibition of EZH2 decreases invasive capacity of melanoma cells and markedly reduces tumor burden in vivo, specifically in NRAS mutants. Coincident with bivalent reprogramming, the increased expression of pro-metastatic and melanocyte-specific cell-identity genes is associated with exceptionally wide H3K4me3 domains, suggesting a role for this epigenetic element. Overall, we demonstrate that reprogramming of bivalent and broad domains represents key epigenetic alterations in metastatic melanoma and that EZH2 plus MEK inhibition may provide a promising therapeutic strategy for NRAS mutant melanoma patients.

Microembolic signals in patients with systemic lupus erythematosus.

INTRODUCTION: Central nervous system (CNS) involvement is a common and less understood aspect of systemic lupus erythematosus (SLE). Microembolic signals (MES) have been reported in SLE. We conducted a prospective study to evaluate the frequency of MES among patients with CNS involvement and those without. The main aim of the study is to clarify the pathophysiology of the CNS involvement in SLE. METHODS AND MATERIALS: Sixty eight patients with a diagnosis of SLE (60 females, 8 males) participated in the study. Both middle cerebral arteries were monitored using transcranial Doppler for 60 min to detect MES. All cases underwent neurology and psychiatry assessments. RESULTS: MES were detected in 7/68 patients (10.3%) with the mean number of 3.5 per hour. MES were significantly higher in patients with CNS involvement (6/24, 25%) than those without (1/44, 2.2%) (P=0.006). SLE disease activity index, duration of disease, plaque formation, intima-media thickness, and antiphospholipid antibodies were not associated with MES. MES were more frequent in patients receiving Aspirin and/or Warfarin (p=0.02). CONCLUSIONS: MES may be a predictor for CNS involvement in SLE patients at risk for neuropsychiatric syndromes. Cerebral embolism may be implicated in the pathophysiology of neuropsychiatric SLE.

Oncogenic Role of an Epigenetic Reader of m6A RNA Modification: YTHDF1 in Merkel Cell Carcinoma.

Merkel cell carcinoma is a deadly skin cancer, which in the majority of cases is caused by the Merkel cell polyomavirus (MCPyV). The viral small T antigen is regarded as the dominant oncoprotein expressed in the tumor cells. We used genomic screening of copy number aberrations along with transcriptomic analysis to investigate regions with amplification that harbor differentially expressed genes. We identified YTHDF1, a protein that is a reader of N6-methyladenosine (m6A) RNA modifications, to have high copy gains and to be highly expressed in Merkel cell carcinoma. Importantly, we identified the presence of m6A on small T antigen mRNA suggesting a relation between YTHDF1 amplification and MCPyV gene expression. Interestingly, knockdown of YTHDF1 in Merkel cell carcinoma (MCC) cell lines negatively affected the translation initiation factor eIF3 and reduced proliferation and clonogenic capacity in vitro. Furthermore, analysis of survival data revealed worse overall survival in YTHDF1high MCC patients compared to YTHDF1low patients. Our findings indicate a novel oncogenic role of YTHDF1 through m6A machinery in the tumorigenesis of MCC.

Unique Role of Histone Methyltransferase PRDM8 in the Tumorigenesis of Virus-Negative Merkel Cell Carcinoma.

Merkel cell carcinoma (MCC) is a deadly skin cancer, and about 80% of its cases have been shown to harbor integrated Merkel polyomavirus in the tumor cell genome. Viral oncoproteins expressed in the tumor cells are considered as the oncogenic factors of these virus-positive Merkel cell carcinoma (VP-MCC). In contrast, the molecular pathogenesis of virus-negative MCC (VN-MCC) is less well understood. Using gene expression analysis of MCC cell lines, we found histone methyltransferase PRDM8 to be elevated in VN-MCC. This finding was confirmed by immunohistochemical analysis of MCC tumors, revealing that increased PRDM8 expression in VN-MCC is also associated with increased H3K9 methylation. CRISPR-mediated silencing of PRDM8 in MCC cells further supported the histone methylating role of this protein in VN-MCC. We also identified miR-20a-5p as a negative regulator of PRDM8. Taken together, our findings provide insights into the role of PRDM8 as a histone methyltransferase in VN-MCC tumorigenesis.

Enhancer Reprogramming Confers Dependence on Glycolysis and IGF Signaling in KMT2D Mutant Melanoma.

Histone methyltransferase KMT2D harbors frequent loss-of-function somatic point mutations in several tumor types, including melanoma. Here, we identify KMT2D as a potent tumor suppressor in melanoma through an in vivo epigenome-focused pooled RNAi screen and confirm the finding by using a genetically engineered mouse model (GEMM) based on conditional and melanocyte-specific deletion of KMT2D. KMT2D-deficient tumors show substantial reprogramming of key metabolic pathways, including glycolysis. KMT2D deficiency aberrantly upregulates glycolysis enzymes, intermediate metabolites, and glucose consumption rates. Mechanistically, KMT2D loss causes genome-wide reduction of H3K4me1-marked active enhancer chromatin states. Enhancer loss and subsequent repression of IGFBP5 activates IGF1R-AKT to increase glycolysis in KMT2D-deficient cells. Pharmacological inhibition of glycolysis and insulin growth factor (IGF) signaling reduce proliferation and tumorigenesis preferentially in KMT2D-deficient cells. We conclude that KMT2D loss promotes tumorigenesis by facilitating an increased use of the glycolysis pathway for enhanced biomass needs via enhancer reprogramming, thus presenting an opportunity for therapeutic intervention through glycolysis or IGF pathway inhibitors.

Disrupting Mitochondrial Copper Distribution Inhibits Leukemic Stem Cell Self-Renewal.

Leukemic stem cells (LSCs) rely on oxidative metabolism and are differentially sensitive to targeting mitochondrial pathways, which spares normal hematopoietic cells. A subset of mitochondrial proteins is folded in the intermembrane space via the mitochondrial intermembrane assembly (MIA) pathway. We found increased mRNA expression of MIA pathway substrates in acute myeloid leukemia (AML) stem cells. Therefore, we evaluated the effects of inhibiting this pathway in AML. Genetic and chemical inhibition of ALR reduces AML growth and viability, disrupts LSC self-renewal, and induces their differentiation. ALR inhibition preferentially decreases its substrate COX17, a mitochondrial copper chaperone, and knockdown of COX17 phenocopies ALR loss. Inhibiting ALR and COX17 increases mitochondrial copper levels which in turn inhibit S-adenosylhomocysteine hydrolase (SAHH) and lower levels of S-adenosylmethionine (SAM), DNA methylation, and chromatin accessibility to lower LSC viability. These results provide insight into mechanisms through which mitochondrial copper controls epigenetic status and viability of LSCs.

Tackling malignant melanoma epigenetically: histone lysine methylation.

Post-translational histone modifications such as acetylation and methylation can affect gene expression. Histone acetylation is commonly associated with activation of gene expression whereas histone methylation is linked to either activation or repression of gene expression. Depending on the site of histone modification, several histone marks can be present throughout the genome. A combination of these histone marks can shape global chromatin architecture, and changes in patterns of marks can affect the transcriptomic landscape. Alterations in several histone marks are associated with different types of cancers, and these alterations are distinct from marks found in original normal tissues. Therefore, it is hypothesized that patterns of histone marks can change during the process of tumorigenesis.This review focuses on histone methylation changes (both removal and addition of methyl groups) in malignant melanoma, a deadly skin cancer, and the implications of specific inhibitors of these modifications as a combinatorial therapeutic approach.

An Integrated Platform for Genome-wide Mapping of Chromatin States Using High-throughput ChIP-sequencing in Tumor Tissues.

Histone modifications constitute a major component of the epigenome and play important regulatory roles in determining the transcriptional status of associated loci. In addition, the presence of specific modifications has been used to determine the position and identity non-coding functional elements such as enhancers. In recent years, chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) has become a powerful tool in determining the genome-wide profiles of individual histone modifications. However, it has become increasingly clear that the combinatorial patterns of chromatin modifications, referred to as Chromatin States, determine the identity and nature of the associated genomic locus. Therefore, workflows consisting of robust high-throughput (HT) methodologies for profiling a number of histone modification marks, as well as computational analyses pipelines capable of handling myriads of ChIP-Seq profiling datasets, are needed for comprehensive determination of epigenomic states in large number of samples. The HT-ChIP-Seq workflow presented here consists of two modules: 1) an experimental protocol for profiling several histone modifications from small amounts of tumor samples and cell lines in a 96-well format; and 2) a computational data analysis pipeline that combines existing tools to compute both individual mark occupancy and combinatorial chromatin state patterns. Together, these two modules facilitate easy processing of hundreds of ChIP-Seq samples in a fast and efficient manner. The workflow presented here is used to derive chromatin state patterns from 6 histone mark profiles in melanoma tumors and cell lines. Overall, we present a comprehensive ChIP-seq workflow that can be applied to dozens of human tumor samples and cancer cell lines to determine epigenomic aberrations in various malignancies.