Genome folding principles uncovered in condensin-depleted mitotic chromosomes.

During mitosis, condensin activity is thought to interfere with interphase chromatin structures. To investigate genome folding principles in the absence of chromatin loop extrusion, we codepleted condensin I and condensin II, which triggered mitotic chromosome compartmentalization in ways similar to that in interphase. However, two distinct euchromatic compartments, indistinguishable in interphase, emerged upon condensin loss with different interaction preferences and dependencies on H3K27ac. Constitutive heterochromatin gradually self-aggregated and cocompartmentalized with facultative heterochromatin, contrasting with their separation during interphase. Notably, some cis-regulatory element contacts became apparent even in the absence of CTCF/cohesin-mediated structures. Heterochromatin protein 1 (HP1) proteins, which are thought to partition constitutive heterochromatin, were absent from mitotic chromosomes, suggesting, surprisingly, that constitutive heterochromatin can self-aggregate without HP1. Indeed, in cells traversing from M to G1 phase in the combined absence of HP1α, HP1ß and HP1γ, constitutive heterochromatin compartments are normally re-established. In sum, condensin-deficient mitotic chromosomes illuminate forces of genome compartmentalization not identified in interphase cells.

CLIMB: High-dimensional association detection in large scale genomic data.

Joint analyses of genomic datasets obtained in multiple different conditions are essential for understanding the biological mechanism that drives tissue-specificity and cell differentiation, but they still remain computationally challenging. To address this we introduce CLIMB (Composite LIkelihood eMpirical Bayes), a statistical methodology that learns patterns of condition-specificity present in genomic data. CLIMB provides a generic framework facilitating a host of analyses, such as clustering genomic features sharing similar condition-specific patterns and identifying which of these features are involved in cell fate commitment. We apply CLIMB to three sets of hematopoietic data, which examine CTCF ChIP-seq measured in 17 different cell populations, RNA-seq measured across constituent cell populations in three committed lineages, and DNase-seq in 38 cell populations. Our results show that CLIMB improves upon existing alternatives in statistical precision, while capturing interpretable and biologically relevant clusters in the data.

Subtype-specific 3D genome alteration in acute myeloid leukaemia.

Acute myeloid leukaemia (AML) represents a set of heterogeneous myeloid malignancies, and hallmarks include mutations in epigenetic modifiers, transcription factors and kinases1-5. The extent to which mutations in AML drive alterations in chromatin 3D structure and contribute to myeloid transformation is unclear. Here we use Hi-C and whole-genome sequencing to analyse 25 samples from patients with AML and 7 samples from healthy donors. Recurrent and subtype-specific alterations in A/B compartments, topologically associating domains and chromatin loops were identified. RNA sequencing, ATAC with sequencing and CUT&Tag for CTCF, H3K27ac and H3K27me3 in the same AML samples also revealed extensive and recurrent AML-specific promoter-enhancer and promoter-silencer loops. We validated the role of repressive loops on their target genes by CRISPR deletion and interference. Structural variation-induced enhancer-hijacking and silencer-hijacking events were further identified in AML samples. Hijacked enhancers play a part in AML cell growth, as demonstrated by CRISPR screening, whereas hijacked silencers have a downregulating role, as evidenced by CRISPR-interference-mediated de-repression. Finally, whole-genome bisulfite sequencing of 20 AML and normal samples revealed the delicate relationship between DNA methylation, CTCF binding and 3D genome structure. Treatment of AML cells with a DNA hypomethylating agent and triple knockdown of DNMT1, DNMT3A and DNMT3B enabled the manipulation of DNA methylation to revert 3D genome organization and gene expression. Overall, this study provides a resource for leukaemia studies and highlights the role of repressive loops and hijacked cis elements in human diseases.

HIC2 controls developmental hemoglobin switching by repressing BCL11A transcription.

The fetal-to-adult switch in hemoglobin production is a model of developmental gene control with relevance to the treatment of hemoglobinopathies. The expression of transcription factor BCL11A, which represses fetal ß-type globin (HBG) genes in adult erythroid cells, is predominantly controlled at the transcriptional level but the underlying mechanism is unclear. We identify HIC2 as a repressor of BCL11A transcription. HIC2 and BCL11A are reciprocally expressed during development. Forced expression of HIC2 in adult erythroid cells inhibits BCL11A transcription and induces HBG expression. HIC2 binds to erythroid BCL11A enhancers to reduce chromatin accessibility and binding of transcription factor GATA1, diminishing enhancer activity and enhancer-promoter contacts. DNA-binding and crystallography studies reveal direct steric hindrance as one mechanism by which HIC2 inhibits GATA1 binding at a critical BCL11A enhancer. Conversely, loss of HIC2 in fetal erythroblasts increases enhancer accessibility, GATA1 binding and BCL11A transcription. HIC2 emerges as an evolutionarily conserved regulator of hemoglobin switching via developmental control of BCL11A.

Identification and characterization of RBM12 as a novel regulator of fetal hemoglobin expression.

The fetal-to-adult hemoglobin transition is clinically relevant because reactivation of fetal hemoglobin (HbF) significantly reduces morbidity and mortality associated with sickle cell disease (SCD) and ß-thalassemia. Most studies on the developmental regulation of the globin genes, including genome-wide genetics screens, have focused on DNA binding proteins, including BCL11A and ZBTB7A/LRF and their cofactors. Our understanding of RNA binding proteins (RBPs) in this process is much more limited. Two RBPs, LIN28B and IGF2BP1, are known posttranscriptional regulators of HbF production, but a global view of RBPs is still lacking. Here, we carried out a CRISPR/Cas9-based screen targeting RBPs harboring RNA methyltransferase and/or RNA recognition motif (RRM) domains and identified RNA binding motif 12 (RBM12) as a novel HbF suppressor. Depletion of RBM12 induced HbF expression and attenuated cell sickling in erythroid cells derived from patients with SCD with minimal detrimental effects on cell maturation. Transcriptome and proteome profiling revealed that RBM12 functions independently of major known HbF regulators. Enhanced cross-linking and immunoprecipitation followed by high-throughput sequencing revealed strong preferential binding of RBM12 to 5' untranslated regions of transcripts, narrowing down the mechanism of RBM12 action. Notably, we pinpointed the first of 5 RRM domains as essential, and, in conjunction with a linker domain, sufficient for RBM12-mediated HbF regulation. Our characterization of RBM12 as a negative regulator of HbF points to an additional regulatory layer of the fetal-to-adult hemoglobin switch and broadens the pool of potential therapeutic targets for SCD and ß-thalassemia.

Genomic landscape of TCRαß and TCRγδ T-large granular lymphocyte leukemia.

Large granular lymphocyte (LGL) leukemia comprises a group of rare lymphoproliferative disorders whose molecular landscape is incompletely defined. We leveraged paired whole-exome and transcriptome sequencing in the largest LGL leukemia cohort to date, which included 105 patients (93 T-cell receptor αß [TCRαß] T-LGL and 12 TCRγδ T-LGL). Seventy-six mutations were observed in 3 or more patients in the cohort, and out of those, STAT3, KMT2D, PIK3R1, TTN, EYS, and SULF1 mutations were shared between both subtypes. We identified ARHGAP25, ABCC9, PCDHA11, SULF1, SLC6A15, DDX59, DNMT3A, FAS, KDM6A, KMT2D, PIK3R1, STAT3, STAT5B, TET2, and TNFAIP3 as recurrently mutated putative drivers using an unbiased driver analysis approach leveraging our whole-exome cohort. Hotspot mutations in STAT3, PIK3R1, and FAS were detected, whereas truncating mutations in epigenetic modifying enzymes such as KMT2D and TET2 were observed. Moreover, STAT3 mutations co-occurred with mutations in chromatin and epigenetic modifying genes, especially KMT2D and SETD1B (P < .01 and P < .05, respectively). STAT3 was mutated in 50.5% of the patients. Most common Y640F STAT3 mutation was associated with lower absolute neutrophil count values, and N647I mutation was associated with lower hemoglobin values. Somatic activating mutations (Q160P, D170Y, L287F) in the STAT3 coiled-coil domain were characterized. STAT3-mutant patients exhibited increased mutational burden and enrichment of a mutational signature associated with increased spontaneous deamination of 5-methylcytosine. Finally, gene expression analysis revealed enrichment of interferon-γ signaling and decreased phosphatidylinositol 3-kinase-Akt signaling for STAT3-mutant patients. These findings highlight the clinical and molecular heterogeneity of this rare disorder.

Single-nucleotide-level mapping of DNA regulatory elements that control fetal hemoglobin expression.

Pinpointing functional noncoding DNA sequences and defining their contributions to health-related traits is a major challenge for modern genetics. We developed a high-throughput framework to map noncoding DNA functions with single-nucleotide resolution in four loci that control erythroid fetal hemoglobin (HbF) expression, a genetically determined trait that modifies sickle cell disease (SCD) phenotypes. Specifically, we used the adenine base editor ABEmax to introduce 10,156 separate A•T to G•C conversions in 307 predicted regulatory elements and quantified the effects on erythroid HbF expression. We identified numerous regulatory elements, defined their epigenomic structures and linked them to low-frequency variants associated with HbF expression in an SCD cohort. Targeting a newly discovered γ-globin gene repressor element in SCD donor CD34+ hematopoietic progenitors raised HbF levels in the erythroid progeny, inhibiting hypoxia-induced sickling. Our findings reveal previously unappreciated genetic complexities of HbF regulation and provide potentially therapeutic insights into SCD.

Frequent somatic TET2 mutations in chronic NK-LGL leukemia with distinct patterns of cytopenias.

Chronic natural killer large granular lymphocyte (NK-LGL) leukemia, also referred to as chronic lymphoproliferative disorder of NK cells, is a rare disorder defined by prolonged expansion of clonal NK cells. Similar prevalence of STAT3 mutations in chronic T-LGL and NK-LGL leukemia is suggestive of common pathogenesis. We undertook whole-genome sequencing to identify mutations unique to NK-LGL leukemia. The results were analyzed to develop a resequencing panel that was applied to 58 patients. Phosphatidylinositol 3-kinase pathway gene mutations (PIK3CD/PIK3AP1) and TNFAIP3 mutations were seen in 5% and 10% of patients, respectively. TET2 was exceptional in that mutations were present in 16 (28%) of 58 patient samples, with evidence that TET2 mutations can be dominant and exclusive to the NK compartment. Reduced-representation bisulfite sequencing revealed that methylation patterns were significantly altered in TET2 mutant samples. The promoter of TET2 and that of PTPRD, a negative regulator of STAT3, were found to be methylated in additional cohort samples, largely confined to the TET2 mutant group. Mutations in STAT3 were observed in 19 (33%) of 58 patient samples, 7 of which had concurrent TET2 mutations. Thrombocytopenia and resistance to immunosuppressive agents were uniquely observed in those patients with only TET2 mutation (Games-Howell post hoc test, P = .0074; Fisher's exact test, P = .00466). Patients with STAT3 mutation, inclusive of those with TET2 comutation, had lower hematocrit, hemoglobin, and absolute neutrophil count compared with STAT3 wild-type patients (Welch's t test, P ≤ .015). We present the discovery of TET2 mutations in chronic NK-LGL leukemia and evidence that it identifies a unique molecular subtype.

Effects of sheared chromatin length on ChIP-seq quality and sensitivity.

Chromatin immunoprecipitation followed by massively parallel, high throughput sequencing (ChIP-seq) is the method of choice for genome-wide identification of DNA segments bound by specific transcription factors or in chromatin with particular histone modifications. However, the quality of ChIP-seq datasets varies widely, with a substantial fraction being of intermediate to poor quality. Thus, it is important to discern and control the factors that contribute to variation in ChIP-seq. In this study, we focused on sonication, a user-controlled variable, to produce sheared chromatin. We systematically varied the amount of shearing of fixed chromatin from a mouse erythroid cell line, carefully measuring the distribution of resultant fragment lengths prior to ChIP-seq. This systematic study was complemented with a retrospective analysis of additional experiments. We found that the level of sonication had a pronounced impact on the quality of ChIP-seq signals. Over-sonication consistently reduced quality, while the impact of under-sonication differed among transcription factors, with no impact on sites bound by CTCF but frequently leading to the loss of sites occupied by TAL1 or bound by POL2. The bound sites not observed in low-quality datasets were inferred to be a mix of both direct and indirect binding. We leveraged these findings to produce a set of CTCF ChIP-seq datasets in rare, primary hematopoietic progenitor cells. Our observation that the amount of chromatin sonication is a key variable in success of ChIP-seq experiments indicates that monitoring the level of sonication can improve ChIP-seq quality and reproducibility and facilitate ChIP-seq in rare cell types.

An integrative view of the regulatory and transcriptional landscapes in mouse hematopoiesis.

Thousands of epigenomic data sets have been generated in the past decade, but it is difficult for researchers to effectively use all the data relevant to their projects. Systematic integrative analysis can help meet this need, and the VISION project was established for validated systematic integration of epigenomic data in hematopoiesis. Here, we systematically integrated extensive data recording epigenetic features and transcriptomes from many sources, including individual laboratories and consortia, to produce a comprehensive view of the regulatory landscape of differentiating hematopoietic cell types in mouse. By using IDEAS as our integrative and discriminative epigenome annotation system, we identified and assigned epigenetic states simultaneously along chromosomes and across cell types, precisely and comprehensively. Combining nuclease accessibility and epigenetic states produced a set of more than 200,000 candidate cis-regulatory elements (cCREs) that efficiently capture enhancers and promoters. The transitions in epigenetic states of these cCREs across cell types provided insights into mechanisms of regulation, including decreases in numbers of active cCREs during differentiation of most lineages, transitions from poised to active or inactive states, and shifts in nuclease accessibility of CTCF-bound elements. Regression modeling of epigenetic states at cCREs and gene expression produced a versatile resource to improve selection of cCREs potentially regulating target genes. These resources are available from our VISION website to aid research in genomics and hematopoiesis.

Systematic integration of GATA transcription factors and epigenomes via IDEAS paints the regulatory landscape of hematopoietic cells.

Members of the GATA family of transcription factors play key roles in the differentiation of specific cell lineages by regulating the expression of target genes. Three GATA factors play distinct roles in hematopoietic differentiation. In order to better understand how these GATA factors function to regulate genes throughout the genome, we are studying the epigenomic and transcriptional landscapes of hematopoietic cells in a model-driven, integrative fashion. We have formed the collaborative multi-lab VISION project to conduct ValIdated Systematic IntegratiON of epigenomic data in mouse and human hematopoiesis. The epigenomic data included nuclease accessibility in chromatin, CTCF occupancy, and histone H3 modifications for 20 cell types covering hematopoietic stem cells, multilineage progenitor cells, and mature cells across the blood cell lineages of mouse. The analysis used the Integrative and Discriminative Epigenome Annotation System (IDEAS), which learns all common combinations of features (epigenetic states) simultaneously in two dimensions-along chromosomes and across cell types. The result is a segmentation that effectively paints the regulatory landscape in readily interpretable views, revealing constitutively active or silent loci as well as the loci specifically induced or repressed in each stage and lineage. Nuclease accessible DNA segments in active chromatin states were designated candidate cis-regulatory elements in each cell type, providing one of the most comprehensive registries of candidate hematopoietic regulatory elements to date. Applications of VISION resources are illustrated for the regulation of genes encoding GATA1, GATA2, GATA3, and Ikaros. VISION resources are freely available from our website http://usevision.org.

Transcriptional Burst Initiation and Polymerase Pause Release Are Key Control Points of Transcriptional Regulation.

Transcriptional regulation occurs via changes to rates of different biochemical steps of transcription, but it remains unclear which rates are subject to change upon biological perturbation. Biochemical studies have suggested that stimuli predominantly affect the rates of RNA polymerase II (Pol II) recruitment and polymerase release from promoter-proximal pausing. Single-cell studies revealed that transcription occurs in discontinuous bursts, suggesting that features of such bursts like frequency and intensity could also be regulated. We combined Pol II chromatin immunoprecipitation sequencing (ChIP-seq) and single-cell transcriptional measurements to show that an independently regulated burst initiation step is required before polymerase recruitment can occur. Using a number of global and targeted transcriptional regulatory perturbations, we showed that biological perturbations regulated both burst initiation and polymerase pause release rates but seemed not to regulate polymerase recruitment rate. Our results suggest that transcriptional regulation primarily acts by changing the rates of burst initiation and polymerase pause release.

Integrative detection and analysis of structural variation in cancer genomes.

Structural variants (SVs) can contribute to oncogenesis through a variety of mechanisms. Despite their importance, the identification of SVs in cancer genomes remains challenging. Here, we present a framework that integrates optical mapping, high-throughput chromosome conformation capture (Hi-C), and whole-genome sequencing to systematically detect SVs in a variety of normal or cancer samples and cell lines. We identify the unique strengths of each method and demonstrate that only integrative approaches can comprehensively identify SVs in the genome. By combining Hi-C and optical mapping, we resolve complex SVs and phase multiple SV events to a single haplotype. Furthermore, we observe widespread structural variation events affecting the functions of noncoding sequences, including the deletion of distal regulatory sequences, alteration of DNA replication timing, and the creation of novel three-dimensional chromatin structural domains. Our results indicate that noncoding SVs may be underappreciated mutational drivers in cancer genomes.

Establishment of regulatory elements during erythro-megakaryopoiesis identifies hematopoietic lineage-commitment points.

BACKGROUND: Enhancers and promoters are cis-acting regulatory elements associated with lineage-specific gene expression. Previous studies showed that different categories of active regulatory elements are in regions of open chromatin, and each category is associated with a specific subset of post-translationally marked histones. These regulatory elements are systematically activated and repressed to promote commitment of hematopoietic stem cells along separate differentiation paths, including the closely related erythrocyte (ERY) and megakaryocyte (MK) lineages. However, the order in which these decisions are made remains unclear. RESULTS: To characterize the order of cell fate decisions during hematopoiesis, we collected primary cells from mouse bone marrow and isolated 10 hematopoietic populations to generate transcriptomes and genome-wide maps of chromatin accessibility and histone H3 acetylated at lysine 27 binding (H3K27ac). Principle component analysis of transcriptional and open chromatin profiles demonstrated that cells of the megakaryocyte lineage group closely with multipotent progenitor populations, whereas erythroid cells form a separate group distinct from other populations. Using H3K27ac and open chromatin profiles, we showed that 89% of immature MK (iMK)-specific active regulatory regions are present in the most primitive hematopoietic cells, 46% of which contain active enhancer marks. These candidate active enhancers are enriched for transcription factor binding site motifs for megakaryopoiesis-essential proteins, including ERG and ETS1. In comparison, only 64% of ERY-specific active regulatory regions are present in the most primitive hematopoietic cells, 20% of which containing active enhancer marks. These regions were not enriched for any transcription factor consensus sequences. Incorporation of genome-wide DNA methylation identified significant levels of de novo methylation in iMK, but not ERY. CONCLUSIONS: Our results demonstrate that megakaryopoietic profiles are established early in hematopoiesis and are present in the majority of the hematopoietic progenitor population. However, megakaryopoiesis does not constitute a "default" differentiation pathway, as extensive de novo DNA methylation accompanies megakaryopoietic commitment. In contrast, erythropoietic profiles are not established until a later stage of hematopoiesis, and require more dramatic changes to the transcriptional and epigenetic programs. These data provide important insights into lineage commitment and can contribute to ongoing studies related to diseases associated with differentiation defects.

The BET Protein BRD2 Cooperates with CTCF to Enforce Transcriptional and Architectural Boundaries.

Bromodomain and extraterminal motif (BET) proteins are pharmacologic targets for the treatment of diverse diseases, yet the roles of individual BET family members remain unclear. We find that BRD2, but not BRD4, co-localizes with the architectural/insulator protein CCCTC-binding factor (CTCF) genome-wide. CTCF recruits BRD2 to co-bound sites whereas BRD2 is dispensable for CTCF occupancy. Disruption of a CTCF/BRD2-occupied element positioned between two unrelated genes enables regulatory influence to spread from one gene to another, suggesting that CTCF and BRD2 form a transcriptional boundary. Accordingly, single-molecule mRNA fluorescence in situ hybridization (FISH) reveals that, upon site-specific CTCF disruption or BRD2 depletion, expression of the two genes becomes increasingly correlated. HiC shows that BRD2 depletion weakens boundaries co-occupied by CTCF and BRD2, but not those that lack BRD2. These findings indicate that BRD2 supports boundary activity, and they raise the possibility that pharmacologic BET inhibitors can influence gene expression in part by perturbing domain boundary function.

Finding partners to play the music of regulation.

In this issue of Blood, Wilson et al generate and analyze a treasure trove of epigenetic data, such as transcription factor occupancy, histone modifications, and chromatin interaction frequencies, genome-wide (ie, epigenomic data), in a cell line model of hematopoietic stem/progenitor cells (HSPCs). To appreciate the importance of these data, consider an analogy of gene expression being a song or symphony (transcripts) played by musicians (transcription factors and transcriptional machinery) reading the score encoded in the genome sequence. Previous studies revealed the positions of a few transcription factors across the genome, so we only knew about, for example, the violinists and oboists. No wonder we did not understand how the music was being generated (how expression was regulated). By mapping the sites of occupancy of many more transcription factors (now a total of 29), as well as positions of 4 histone modifications and DNase hypersensitive sites, Wilson et al reveal many more of the players and their partners. Furthermore, their data on 3-dimensional interaction frequencies of chromatin show how groups of musicians (protein complexes) come together in an orchestra to read the score and perform a symphony.

Genome-Wide Organization of GATA1 and TAL1 Determined at High Resolution.

Erythroid development and differentiation from multiprogenitor cells into red blood cells requires precise transcriptional regulation. Key erythroid transcription factors, GATA1 and TAL1, cooperate, along with other proteins, to regulate many aspects of this process. How GATA1 and TAL1 are juxtaposed along the DNA and their cognate DNA binding site across the mouse genome remains unclear. We applied high-resolution ChIP-exo (chromatin immunoprecipitation followed by 5'-to-3' exonuclease treatment and then massively parallel DNA sequencing) to GATA1 and TAL1 to study their positional organization across the mouse genome during GATA1-dependent maturation. Two complementary methods, MultiGPS and peak pairing, were used to determine high-confidence binding locations by ChIP-exo. We identified ∼10,000 GATA1 and ∼15,000 TAL1 locations, which were essentially confirmed by ChIP-seq (chromatin immunoprecipitation followed by massively parallel DNA sequencing). Of these, ∼4,000 locations were bound by both GATA1 and TAL1. About three-quarters of them were tightly linked to a partial E-box located 7 or 8 bp upstream of a WGATAA motif. Both TAL1 and GATA1 generated distinct characteristic ChIP-exo peaks around WGATAA motifs that reflect their positional arrangement within a complex. We show that TAL1 and GATA1 form a precisely organized complex at a compound motif consisting of a TG 7 or 8 bp upstream of a WGATAA motif across thousands of genomic locations.

SBR-Blood: systems biology repository for hematopoietic cells.

Extensive research into hematopoiesis (the development of blood cells) over several decades has generated large sets of expression and epigenetic profiles in multiple human and mouse blood cell types. However, there is no single location to analyze how gene regulatory processes lead to different mature blood cells. We have developed a new database framework called hematopoietic Systems Biology Repository (SBR-Blood), available online at http://sbrblood.nhgri.nih.gov, which allows user-initiated analyses for cell type correlations or gene-specific behavior during differentiation using publicly available datasets for array- and sequencing-based platforms from mouse hematopoietic cells. SBR-Blood organizes information by both cell identity and by hematopoietic lineage. The validity and usability of SBR-Blood has been established through the reproduction of workflows relevant to expression data, DNA methylation, histone modifications and transcription factor occupancy profiles.

Occupancy by key transcription factors is a more accurate predictor of enhancer activity than histone modifications or chromatin accessibility.

BACKGROUND: Regulated gene expression controls organismal development, and variation in regulatory patterns has been implicated in complex traits. Thus accurate prediction of enhancers is important for further understanding of these processes. Genome-wide measurement of epigenetic features, such as histone modifications and occupancy by transcription factors, is improving enhancer predictions, but the contribution of these features to prediction accuracy is not known. Given the importance of the hematopoietic transcription factor TAL1 for erythroid gene activation, we predicted candidate enhancers based on genomic occupancy by TAL1 and measured their activity. Contributions of multiple features to enhancer prediction were evaluated based on the results of these and other studies. RESULTS: TAL1-bound DNA segments were active enhancers at a high rate both in transient transfections of cultured cells (39 of 79, or 56%) and transgenic mice (43 of 66, or 65%). The level of binding signal for TAL1 or GATA1 did not help distinguish TAL1-bound DNA segments as active versus inactive enhancers, nor did the density of regulation-related histone modifications. A meta-analysis of results from this and other studies (273 tested predicted enhancers) showed that the presence of TAL1, GATA1, EP300, SMAD1, H3K4 methylation, H3K27ac, and CAGE tags at DNase hypersensitive sites gave the most accurate predictors of enhancer activity, with a success rate over 80% and a median threefold increase in activity. Chromatin accessibility assays and the histone modifications H3K4me1 and H3K27ac were sensitive for finding enhancers, but they have high false positive rates unless transcription factor occupancy is also included. CONCLUSIONS: Occupancy by key transcription factors such as TAL1, GATA1, SMAD1, and EP300, along with evidence of transcription, improves the accuracy of enhancer predictions based on epigenetic features.

The effects of chromatin organization on variation in mutation rates in the genome.

The variation in local rates of mutations can affect both the evolution of genes and their function in normal and cancer cells. Deciphering the molecular determinants of this variation will be aided by the elucidation of distinct types of mutations, as they differ in regional preferences and in associations with genomic features. Chromatin organization contributes to regional variation in mutation rates, but its contribution differs among mutation types. In both germline and somatic mutations, base substitutions are more abundant in regions of closed chromatin, perhaps reflecting error accumulation late in replication. By contrast, a distinctive mutational state with very high levels of insertions and deletions (indels) and substitutions is enriched in regions of open chromatin. These associations indicate an intricate interplay between the nucleotide sequence of DNA and its dynamic packaging into chromatin, and have important implications for current biomedical research. This Review focuses on recent studies showing associations between chromatin state and mutation rates, including pairwise and multivariate investigations of germline and somatic (particularly cancer) mutations.