Epigenetic deregulation in myeloid malignancies.

Epigenetic deregulation is now a well-recognized although not yet fully understood mechanism that contributes to the development and progression of myeloid malignancies. In the past 15 years, next-generation sequencing studies have revealed patterns of aberrant DNA methylation, altered chromatin states, and mutations in chromatin modifiers across the spectrum of myeloid malignancies. Studies into the mechanisms that drive these diseases through mouse modeling have helped identify new avenues for therapeutic interventions, from initial treatment to resistant or relapsed disease. This is particularly significant when chemotherapy with cytotoxic agents remains the general standard of care. In this review, we will discuss some of the recent findings of epigenetic mechanisms and how these are informing the development of more targeted strategies for therapeutic intervention in myeloid malignancies.

CHD7 and Runx1 interaction provides a braking mechanism for hematopoietic differentiation.

Hematopoietic stem and progenitor cell (HSPC) formation and lineage differentiation involve gene expression programs orchestrated by transcription factors and epigenetic regulators. Genetic disruption of the chromatin remodeler chromodomain-helicase-DNA-binding protein 7 (CHD7) expanded phenotypic HSPCs, erythroid, and myeloid lineages in zebrafish and mouse embryos. CHD7 acts to suppress hematopoietic differentiation. Binding motifs for RUNX and other hematopoietic transcription factors are enriched at sites occupied by CHD7, and decreased RUNX1 occupancy correlated with loss of CHD7 localization. CHD7 physically interacts with RUNX1 and suppresses RUNX1-induced expansion of HSPCs during development through modulation of RUNX1 activity. Consequently, the RUNX1:CHD7 axis provides proper timing and function of HSPCs as they emerge during hematopoietic development or mature in adults, representing a distinct and evolutionarily conserved control mechanism to ensure accurate hematopoietic lineage differentiation.

Direct recruitment of polycomb repressive complex 1 to chromatin by core binding transcription factors.

Polycomb repressive complexes (PRCs) play key roles in developmental epigenetic regulation. Yet the mechanisms that target PRCs to specific loci in mammalian cells remain incompletely understood. In this study we show that Bmi1, a core component of Polycomb Repressive Complex 1 (PRC1), binds directly to the Runx1/CBFß transcription factor complex. Genome-wide studies in megakaryocytic cells demonstrate significant chromatin occupancy overlap between the PRC1 core component Ring1b and Runx1/CBFß and functional regulation of a considerable fraction of commonly bound genes. Bmi1/Ring1b and Runx1/CBFß deficiencies generate partial phenocopies of one another in vivo. We also show that Ring1b occupies key Runx1 binding sites in primary murine thymocytes and that this occurs via PRC2-independent mechanisms. Genetic depletion of Runx1 results in reduced Ring1b binding at these sites in vivo. These findings provide evidence for site-specific PRC1 chromatin recruitment by core binding transcription factors in mammalian cells.

lncRNA-screen: an interactive platform for computationally screening long non-coding RNAs in large genomics datasets.

BACKGROUND: Long non-coding RNAs (lncRNAs) have emerged as a class of factors that are important for regulating development and cancer. Computational prediction of lncRNAs from ultra-deep RNA sequencing has been successful in identifying candidate lncRNAs. However, the complexity of handling and integrating different types of genomics data poses significant challenges to experimental laboratories that lack extensive genomics expertise. RESULT: To address this issue, we have developed lncRNA-screen, a comprehensive pipeline for computationally screening putative lncRNA transcripts over large multimodal datasets. The main objective of this work is to facilitate the computational discovery of lncRNA candidates to be further examined by functional experiments. lncRNA-screen provides a fully automated easy-to-run pipeline which performs data download, RNA-seq alignment, assembly, quality assessment, transcript filtration, novel lncRNA identification, coding potential estimation, expression level quantification, histone mark enrichment profile integration, differential expression analysis, annotation with other type of segmented data (CNVs, SNPs, Hi-C, etc.) and visualization. Importantly, lncRNA-screen generates an interactive report summarizing all interesting lncRNA features including genome browser snapshots and lncRNA-mRNA interactions based on Hi-C data. CONCLUSION: lncRNA-screen provides a comprehensive solution for lncRNA discovery and an intuitive interactive report for identifying promising lncRNA candidates. lncRNA-screen is available as open-source software on GitHub.

MIR retrotransposons link the epigenome and the transcriptome of coding genes in acute myeloid leukemia.

DNMT3A and IDH1/2 mutations combinatorically regulate the transcriptome and the epigenome in acute myeloid leukemia; yet the mechanisms of this interplay are unknown. Using a systems approach within topologically associating domains, we find that genes with significant expression-methylation correlations are enriched in signaling and metabolic pathways. The common denominator across these methylation-regulated genes is the density in MIR retrotransposons of their introns. Moreover, a discrete number of CpGs overlapping enhancers are responsible for regulating most of these genes. Established mouse models recapitulate the dependency of MIR-rich genes on the balanced expression of epigenetic modifiers, while projection of leukemic profiles onto normal hematopoiesis ones further consolidates the dependencies of methylation-regulated genes on MIRs. Collectively, MIR elements on genes and enhancers are susceptible to changes in DNA methylation activity and explain the cooperativity of proteins in this pathway in normal and malignant hematopoiesis.

LncTx: A network-based method to repurpose drugs acting on the survival-related lncRNAs in lung cancer.

Despite the fact that an increased amount of survival-related lncRNAs have been found in cancer, few drugs that target lncRNAs are approved for treatment. Here, we developed a network-based algorithm, LncTx, to repurpose the medications that potentially act on survival-related lncRNAs in lung cancer. We used eight survival-related lncRNAs derived from our previous study to test the efficacy of this method. LncTx calculates the shortest path length (proximity) between the drug targets and the lncRNA-correlated proteins in the protein-protein interaction network (interactome). LncTx contains seven different proximity measures, which are calculated in the unweighted or weighted interactome. First, to test the performance of LncTx in predicting correct indication of drugs, we benchmarked the proximity measures based on the accuracy of differentiating anticancer drugs from non-anticancer drugs. The closest proximity weighted by clustering coefficient (closestCC) has the best performance (AUC around 0.8) compared to other proximity measures across all survival-related lncRNAs. The majority of the other six proximity measures have decent performance as well, with AUC greater than 0.7. Second, to evaluate whether LncTx can repurpose the drugs effectively acting on the lncRNAs, we clustered the drugs according to their proximities by hierarchical clustering. The drugs with smaller proximity (proximal drugs) were proved to be more effective than the drugs with larger proximity (distal drugs). In conclusion, LncTx enables us to accurately identify anticancer drugs and can potentially be an index to repurpose effective agents acting on survival-related lncRNAs in lung cancer.

Aging Human Hematopoietic Stem Cells Manifest Profound Epigenetic Reprogramming of Enhancers That May Predispose to Leukemia.

Aging is associated with functional decline of hematopoietic stem cells (HSC) as well as an increased risk of myeloid malignancies. We performed an integrative characterization of epigenomic and transcriptomic changes, including single-cell RNA sequencing, during normal human aging. Lineage-CD34+CD38- cells [HSC-enriched (HSCe)] undergo age-associated epigenetic reprogramming consisting of redistribution of DNA methylation and reductions in H3K27ac, H3K4me1, and H3K4me3. This reprogramming of aged HSCe globally targets developmental and cancer pathways that are comparably altered in acute myeloid leukemia (AML) of all ages, encompassing loss of 4,646 active enhancers, 3,091 bivalent promoters, and deregulation of several epigenetic modifiers and key hematopoietic transcription factors, such as KLF6, BCL6, and RUNX3. Notably, in vitro downregulation of KLF6 results in impaired differentiation, increased colony-forming potential, and changes in expression that recapitulate aging and leukemia signatures. Thus, age-associated epigenetic reprogramming may form a predisposing condition for the development of age-related AML. SIGNIFICANCE: AML, which is more frequent in the elderly, is characterized by epigenetic deregulation. We demonstrate that epigenetic reprogramming of human HSCs occurs with age, affecting cancer and developmental pathways. Downregulation of genes epigenetically altered with age leads to impairment in differentiation and partially recapitulates aging phenotypes.This article is highlighted in the In This Issue feature, p. 983.

A network of epigenetic regulators guides developmental haematopoiesis in vivo.

The initiation of cellular programs is orchestrated by key transcription factors and chromatin regulators that activate or inhibit target gene expression. To generate a compendium of chromatin factors that establish the epigenetic code during developmental haematopoiesis, a large-scale reverse genetic screen was conducted targeting orthologues of 425 human chromatin factors in zebrafish. A set of chromatin regulators was identified that target different stages of primitive and definitive blood formation, including factors not previously implicated in haematopoiesis. We identified 15 factors that regulate development of primitive erythroid progenitors and 29 factors that regulate development of definitive haematopoietic stem and progenitor cells. These chromatin factors are associated with SWI/SNF and ISWI chromatin remodelling, SET1 methyltransferase, CBP-p300-HBO1-NuA4 acetyltransferase, HDAC-NuRD deacetylase, and Polycomb repressive complexes. Our work provides a comprehensive view of how specific chromatin factors and their associated complexes play a major role in the establishment of haematopoietic cells in vivo.

The transcriptional landscape of hematopoietic stem cell ontogeny.

Transcriptome analysis of adult hematopoietic stem cells (HSCs) and their progeny has revealed mechanisms of blood differentiation and leukemogenesis, but a similar analysis of HSC development is lacking. Here, we acquired the transcriptomes of developing HSCs purified from >2,500 murine embryos and adult mice. We found that embryonic hematopoietic elements clustered into three distinct transcriptional states characteristic of the definitive yolk sac, HSCs undergoing specification, and definitive HSCs. We applied a network-biology-based analysis to reconstruct the gene regulatory networks of sequential stages of HSC development and functionally validated candidate transcriptional regulators of HSC ontogeny by morpholino-mediated knockdown in zebrafish embryos. Moreover, we found that HSCs from in vitro differentiated embryonic stem cells closely resemble definitive HSCs, yet lack a Notch-signaling signature, likely accounting for their defective lymphopoiesis. Our analysis and web resource will enhance efforts to identify regulators of HSC ontogeny and facilitate the engineering of hematopoietic specification.

CHMP1A encodes an essential regulator of BMI1-INK4A in cerebellar development.

Charged multivesicular body protein 1A (CHMP1A; also known as chromatin-modifying protein 1A) is a member of the ESCRT-III (endosomal sorting complex required for transport-III) complex but is also suggested to localize to the nuclear matrix and regulate chromatin structure. Here, we show that loss-of-function mutations in human CHMP1A cause reduced cerebellar size (pontocerebellar hypoplasia) and reduced cerebral cortical size (microcephaly). CHMP1A-mutant cells show impaired proliferation, with increased expression of INK4A, a negative regulator of stem cell proliferation. Chromatin immunoprecipitation suggests loss of the normal INK4A repression by BMI in these cells. Morpholino-based knockdown of zebrafish chmp1a resulted in brain defects resembling those seen after bmi1a and bmi1b knockdown, which were partially rescued by INK4A ortholog knockdown, further supporting links between CHMP1A and BMI1-mediated regulation of INK4A. Our results suggest that CHMP1A serves as a critical link between cytoplasmic signals and BMI1-mediated chromatin modifications that regulate proliferation of central nervous system progenitor cells.

Myomaxin is a novel transcriptional target of MEF2A that encodes a Xin-related alpha-actinin-interacting protein.

The physiological targets regulated by MEF2 in striated muscle are not completely known. Several recent studies have identified novel downstream target genes and shed light on the global transcriptional network regulated by MEF2 in muscle. In our continuing effort to identify novel, downstream pathways controlled by MEF2, we have used mef2a knock-out mice to find those genes dependent on MEF2A transcriptional activity. Here, we describe the characterization of a direct, downstream target gene for the MEF2A transcription factor encoding a large, muscle-specific protein that localizes to the Z-disc/costameric region in striated muscle. This gene, called myomaxin, was identified as a gene markedly down-regulated in MEF2A knock-out hearts. Myomaxin is the mouse ortholog of a partial human cDNA of unknown function named cardiomyopathy associated gene 3 (CMYA3). Myomaxin is expressed as a single, large transcript of approximately 11 kilobases in adult heart and skeletal muscle with an open reading frame of 3,283 amino acids. The protein encoded by the myomaxin gene is related to the actin-binding protein Xin and interacts with the sarcomeric Z-disc protein, alpha-actinin-2. Our findings demonstrate that Myomaxin functions directly downstream of MEF2A at the peripheral Z-disc complex in striated muscle potentially playing a role in regulating cytoarchitectural integrity.