The reference human genome sequence set the stage for studies of genetic variation and its association with human disease, but epigenomic studies lack a similar reference. To address this need, the NIH Roadmap Epigenomics Consortium generated the largest collection so far of human epigenomes for primary cells and tissues. Here we describe the integrative analysis of 111 reference human epigenomes generated as part of the programme, profiled for histone modification patterns, DNA accessibility, DNA methylation and RNA expression. We establish global maps of regulatory elements, define regulatory modules of coordinated activity, and their likely activators and repressors. We show that disease- and trait-associated genetic variants are enriched in tissue-specific epigenomic marks, revealing biologically relevant cell types for diverse human traits, and providing a resource for interpreting the molecular basis of human disease. Our results demonstrate the central role of epigenomic information for understanding gene regulation, cellular differentiation and human disease.

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Integrative analysis of 111 reference human epigenomes

DNA methylation is frequently described as a 'silencing' epigenetic mark, and indeed this function of 5-methylcytosine was originally proposed in the 1970s. Now, thanks to improved genome-scale mapping of methylation, we can evaluate DNA methylation in different genomic contexts: transcriptional start sites with or without CpG islands, in gene bodies, at regulatory elements and at repeat sequences. The emerging picture is that the function of DNA methylation seems to vary with context, and the relationship between DNA methylation and transcription is more nuanced than we realized at first. Improving our understanding of the functions of DNA methylation is necessary for interpreting changes in this mark that are observed in diseases such as cancer.

Functions of DNA methylation: islands, start sites, gene bodies and beyond

The transcription factor NF-kappaB has been the focus of intense investigation for nearly two decades. Over this period, considerable progress has been made in determining the function and regulation of NF-kappaB, although there are nuances in this important signaling pathway that still remain to be understood. The challenge now is to reconcile the regulatory complexity in this pathway with the complexity of responses in which NF-kappaB family members play important roles. In this review, we provide an overview of established NF-kappaB signaling pathways with focus on the current state of research into the mechanisms that regulate IKK activation and NF-kappaB transcriptional activity.

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Signaling to NF-kappaB.

Background The incidence of hematologic cancers increases with age. These cancers are associated with recurrent somatic mutations in specific genes. We hypothesized that such mutations would be detectable in the blood of some persons who are not known to have hematologic disorders. Methods We analyzed whole-exome sequencing data from DNA in the peripheral-blood cells of 17,182 persons who were unselected for hematologic phenotypes. We looked for somatic mutations by identifying previously characterized single-nucleotide variants and small insertions or deletions in 160 genes that are recurrently mutated in hematologic cancers. The presence of mutations was analyzed for an association with hematologic phenotypes, survival, and cardiovascular events. Results Detectable somatic mutations were rare in persons younger than 40 years of age but rose appreciably in frequency with age. Among persons 70 to 79 years of age, 80 to 89 years of age, and 90 to 108 years of age, these clonal mutations were observed in 9.5% (219 of 2300 persons), 11.7% (37 of 317), and 18.4% (19 of 103), respectively. The majority of the variants occurred in three genes: DNMT3A, TET2, and ASXL1. The presence of a somatic mutation was associated with an increase in the risk of hematologic cancer (hazard ratio, 11.1; 95% confidence interval [CI], 3.9 to 32.6), an increase in all-cause mortality (hazard ratio, 1.4; 95% CI, 1.1 to 1.8), and increases in the risks of incident coronary heart disease (hazard ratio, 2.0; 95% CI, 1.2 to 3.4) and ischemic stroke (hazard ratio, 2.6; 95% CI, 1.4 to 4.8). Conclusions Age-related clonal hematopoiesis is a common condition that is associated with increases in the risk of hematologic cancer and in all-cause mortality, with the latter possibly due to an increased risk of cardiovascular disease. (Funded by the National Institutes of Health and others.)

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Age-Related Clonal Hematopoiesis Associated with Adverse Outcomes

A wide range of chromosomal rearrangements have been identified in human cancers especially in leukemia. Murine models that recapitulate chromosomal translocations are essential for understanding the determinants of malignancies and as a basis for preclinical development of rational therapeutic methods. Genetically engineered murine models involving chromosomal translocation genes have been generated using transgenic approaches or gene knockin. However, the closest model is the creation of bona fide chromosomal translocations, which recapitulate the physiologic levels of the fusion genes, the reduced dosage of the wildtype alleles and the contribution of the reciprocal products of the translocations. This has been achieved in vivo using the Cre-loxP system in embryonal stem cells, which is labor intensive and time consuming. The CRISPR/Cas9 system is a versatile tool for genomic engineering. Transduction of target cells with vectors encoding Cas9 and sgRNAs targeting two genomic loci causes two DNA double strand breaks (DSBs), which could induce chromosomal rearrangements as a result from non-homologous end joining-based DNA repair. This strategy has been used to model oncogenic chromosomal rearrangements in murine or human cells in vivo. However, given the challenges of delivering CRISPR/Cas9 vectors into hematopoietic cells, the penetrance of leukemogenesis has been shown with low frequency in vivo. Here we establish an efficient methodology to generate chromosomal translocations in mice hematopoietic cells based on a vector design enabling convenient lentiviral delivery of multiple sgRNAs to hematopoietic cells from a Cas9 expressing mouse strain. TheKMT2A(MLL)gene constitutes ahotspot for chromosomal translocations in human leukemia, resulting in over 135 different leukemia-associated KMT2A gene fusions.KMT2A-MLLT1 defines a leukemia subtype with poor outcome. This translocation frequently constitutes the sole genomic lesion to drive the

https://www.zora.uzh.ch/id/eprint/193335/1/Huang_hs9-4-e456.pdf

Rapid Generation of Leukemogenic Chromosomal Translocations in Vivo Using CRISPR/Cas9

Simple methods with straightforward readouts that enable real-time interrogation of protein quaternary structure are much needed to facilitate the physicochemical characterization of proteins at the single-cell level. After screening over a series of microtubule (MT) binders, we report herein the development of two genetically encoded tags (designated as “MoTags” for the monomer/oligomer detection tag) that can be conveniently fused to a given protein to probe its oligomeric state in cellulo when combined with routine fluorescence microscopy. In their monomeric form, MoTags are evenly distributed in the cytosol; whereas oligomerization enables MoTags to label MT or track MT tips in an oligomeric state-dependent manner. We demonstrate here the broad utility of engineered MoTags to aid the determination of protein oligomeric states, dissection of protein structure and function, and monitoring of protein–target interactions under physiological conditions in living cells.

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Genetically encoded tags for real time dissection of protein assembly in living cells

The oncogenic fusion transcription factor TCF3-HLF identifies an aggressive subtype of acute lymphoblastic leukemia. TCF3-HLF imposes a malignant program by activation of lineage-specific oncogenic enhancers. Among critical cofactors of the TCF3-HLF complex we identified EP300, which functional inhibition results in potent anti-leukemic activity by interference with the specific gene expression.

Targeting the oncogenic activity of TCF3-HLF in leukemia

Follicle arrest is one of the main characteristics of polycystic ovary syndrome (PCOS), the most common endocrinological disorder in reproductive-aged women. Increasing evidence proves that high anti-Mullerian hormone (AMH) levels may play an important role in follicular development. Long noncoding RNA (lncRNA) with a length of more than 200 nt is widely involved in the directional differentiation, growth, and development of cells, whereas whether lncRNA is involved in AMH’s role in follicular development is unknown. In this study, we analyzed lncRNA expression in ovarian granulosa cells (GCs) collected from women with and without PCOS via high-throughput sequencing. The results showed that a total of 79 noncoding transcripts were differently expressed in GCs of PCOS patients, including upregulated lncRNA MALAT1. The upregulation of MALAT1 was further confirmed by RT-qPCR in GCs from a larger cohort of PCOS patients. Furthermore, knockdown MALAT1 can promote the proliferation of KGN cell in vitro. These data suggested a role for MALAT1 in the development of PCOS. Meanwhile, MALAT1 and phosphorylated SMAD 1/5 (Ser463/465) protein were upregulated in KGN cells after exogenous AMH stimulation, which identified AMH perhaps as a regulator for the expression of MALAT1. We also found that MALAT1 can predict clinical pregnancy outcome to a certain extent by ROC curve analysis (area: 0.771, p = 0.007, 95% CI: 0.617–0.925, sensitivity: 57.1%, specificity: 91.7%). Thus, our findings revealed a role of lncRNA MALAT1 in inhibiting granulosa cell proliferation and may be correlated with pregnancy outcome in PCOS.

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Effect of lncRNA MALAT1 on the Granulosa Cell Proliferation and Pregnancy Outcome in Patients With PCOS

DNA methylation is a stable and heritable epigenetic modification in the mammalian genome and is involved in regulating gene expression to control cellular functions. The reversal of DNA methylation, or DNA demethylation, is mediated by the ten-eleven translocation (TET) protein family of dioxygenases. Although it has been widely reported that aberrant DNA methylation and demethylation are associated with developmental defects and cancer, how these epigenetic changes directly contribute to the subsequent alteration in gene expression or disease progression remains unclear, largely owing to the lack of reliable tools to accurately add or remove DNA modifications in the genome at defined temporal and spatial resolution. To overcome this hurdle, we designed a split-TET2 enzyme to enable temporal control of 5-methylcytosine (5mC) oxidation and subsequent remodeling of epigenetic states in mammalian cells by simply adding chemicals. Here, we describe methods for introducing a chemical-inducible epigenome remodeling tool (CiDER), based on an engineered split-TET2 enzyme, into mammalian cells and quantifying the chemical inducible production of 5-hydroxymethylcytosine (5hmC) with immunostaining, flow cytometry or a dot-blot assay. This chemical-inducible epigenome remodeling tool will find broad use in interrogating cellular systems without altering the genetic code, as well as in probing the epigenotype−phenotype relations in various biological systems.

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Yun Huang

Papers

Rapid Generation of Leukemogenic Chromosomal Translocations in Vivo Using CRISPR/Cas9

Genetically encoded tags for real time dissection of protein assembly in living cells

Targeting the oncogenic activity of TCF3-HLF in leukemia

Effect of lncRNA MALAT1 on the Granulosa Cell Proliferation and Pregnancy Outcome in Patients With PCOS

An Engineered Split-TET2 Enzyme for Chemical-inducible DNA Hydroxymethylation and Epigenetic Remodeling.