Human CCR4-NOT is a global regulator of gene expression and is a novel silencer of retrotransposon activation.

CCR4-NOT regulates multiple steps in gene regulation, including transcription, mRNA decay, protein ubiquitylation, and translation. It has been well studied in budding yeast; however, relatively less is known about its regulation and functions in mammals. To characterize the functions of the human CCR4-NOT complex, we developed a rapid auxin-induced degron system to deplete CNOT1 (the scaffold of the complex) and CNOT4 (E3 ubiquitin ligase) in cell culture. Transcriptome-wide measurements of gene-expression revealed that depleting CNOT1 changed several thousand transcripts, wherein most mRNAs were increased and resulted in a global decrease in mRNA decay rates. In contrast to what was observed in CNOT1-depleted cells, CNOT4 depletion only modestly changed RNA steady-state levels and, surprisingly, led to a global acceleration in mRNA decay. To further investigate the role of CCR4-NOT in transcription, we used transient transcriptome sequencing (TT-seq) to measure ongoing RNA synthesis. Depletion of either subunit resulted in increased RNA synthesis of several thousand genes. In contrast to most of the genome, a rapid reduction in the synthesis of KRAB-Zinc-Finger-proteins (KZNFs) genes, especially those clustered on chromosome 19, was observed. KZNFs are transcriptional repressors of retro-transposable elements (rTEs), and consistent with the decreased KZNFs expression, we observed a significant and rapid activation of rTEs, mainly Long interspersed Nuclear Elements (LINEs). Our data reveal that CCR4-NOT regulates gene expression and silences retrotransposons across the genome by maintaining KZNF expression. These data establish CCR4-NOT as a global regulator of gene expression, and we have identified a novel mammalian-specific function of the complex, the suppression of rTEs.

LDB1 establishes multi-enhancer networks to regulate gene expression.

How specific enhancer-promoter pairing is established is still mostly unclear. Besides the CTCF/cohesin machinery, only a few nuclear factors have been studied for a direct role in physically connecting regulatory elements. Here, we show via acute degradation experiments that LDB1 directly and broadly promotes enhancer-promoter loops. Most LDB1-mediated contacts, even those spanning hundreds of kb, can form in the absence of CTCF, cohesin, or YY1 as determined via the use of multiple degron systems. Moreover, an engineered LDB1-driven chromatin loop is cohesin independent. Cohesin-driven loop extrusion does not stall at LDB1 occupied sites but may aid the formation of a subset of LDB1 anchored loops. Leveraging the dynamic reorganization of nuclear architecture during the transition from mitosis to G1-phase, we establish a relationship between LDB1-dependent interactions in the context of TAD organization and gene activation. Lastly, Tri-C and Region Capture Micro-C reveal that LDB1 organizes multi-enhancer networks to activate transcription. This establishes LDB1 as a direct driver of regulatory network inter-connectivity.

YY1-controlled regulatory connectivity and transcription are influenced by the cell cycle.

Few transcription factors have been examined for their direct roles in physically connecting enhancers and promoters. Here acute degradation of Yin Yang 1 (YY1) in erythroid cells revealed its requirement for the maintenance of numerous enhancer-promoter loops, but not compartments or domains. Despite its reported ability to interact with cohesin, the formation of YY1-dependent enhancer-promoter loops does not involve stalling of cohesin-mediated loop extrusion. Integrating mitosis-to-G1-phase dynamics, we observed partial retention of YY1 on mitotic chromatin, predominantly at gene promoters, followed by rapid rebinding during mitotic exit, coinciding with enhancer-promoter loop establishment. YY1 degradation during the mitosis-to-G1-phase interval revealed a set of enhancer-promoter loops that require YY1 for establishment during G1-phase entry but not for maintenance in interphase, suggesting that cell cycle stage influences YY1's architectural function. Thus, as revealed here for YY1, chromatin architectural functions of transcription factors can vary in their interplay with CTCF and cohesin as well as by cell cycle stage.

Interspecies regulatory landscapes and elements revealed by novel joint systematic integration of human and mouse blood cell epigenomes.

Knowledge of locations and activities of cis-regulatory elements (CREs) is needed to decipher basic mechanisms of gene regulation and to understand the impact of genetic variants on complex traits. Previous studies identified candidate CREs (cCREs) using epigenetic features in one species, making comparisons difficult between species. In contrast, we conducted an interspecies study defining epigenetic states and identifying cCREs in blood cell types to generate regulatory maps that are comparable between species, using integrative modeling of eight epigenetic features jointly in human and mouse in our Validated Systematic Integration (VISION) Project. The resulting catalogs of cCREs are useful resources for further studies of gene regulation in blood cells, indicated by high overlap with known functional elements and strong enrichment for human genetic variants associated with blood cell phenotypes. The contribution of each epigenetic state in cCREs to gene regulation, inferred from a multivariate regression, was used to estimate epigenetic state regulatory potential (esRP) scores for each cCRE in each cell type, which were used to categorize dynamic changes in cCREs. Groups of cCREs displaying similar patterns of regulatory activity in human and mouse cell types, obtained by joint clustering on esRP scores, harbor distinctive transcription factor binding motifs that are similar between species. An interspecies comparison of cCREs revealed both conserved and species-specific patterns of epigenetic evolution. Finally, we show that comparisons of the epigenetic landscape between species can reveal elements with similar roles in regulation, even in the absence of genomic sequence alignment.

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.

let-7 miRNAs repress HIC2 to regulate BCL11A transcription and hemoglobin switching.

ABSTRACT: The switch from fetal hemoglobin (γ-globin, HBG) to adult hemoglobin (ß-globin, HBB) gene transcription in erythroid cells serves as a paradigm for a complex and clinically relevant developmental gene regulatory program. We previously identified HIC2 as a regulator of the switch by inhibiting the transcription of BCL11A, a key repressor of HBG production. HIC2 is highly expressed in fetal cells, but the mechanism of its regulation is unclear. Here we report that HIC2 developmental expression is controlled by microRNAs (miRNAs), as loss of global miRNA biogenesis through DICER1 depletion leads to upregulation of HIC2 and HBG messenger RNA. We identified the adult-expressed let-7 miRNA family as a direct posttranscriptional regulator of HIC2. Ectopic expression of let-7 in fetal cells lowered HIC2 levels, whereas inhibition of let-7 in adult erythroblasts increased HIC2 production, culminating in decommissioning of a BCL11A erythroid enhancer and reduced BCL11A transcription. HIC2 depletion in let-7-inhibited cells restored BCL11A-mediated repression of HBG. Together, these data establish that fetal hemoglobin silencing in adult erythroid cells is under the control of a miRNA-mediated inhibitory pathway (let-7 ⊣ HIC2 ⊣ BCL11A ⊣ HBG).

Interspecies regulatory landscapes and elements revealed by novel joint systematic integration of human and mouse blood cell epigenomes.

Knowledge of locations and activities of cis -regulatory elements (CREs) is needed to decipher basic mechanisms of gene regulation and to understand the impact of genetic variants on complex traits. Previous studies identified candidate CREs (cCREs) using epigenetic features in one species, making comparisons difficult between species. In contrast, we conducted an interspecies study defining epigenetic states and identifying cCREs in blood cell types to generate regulatory maps that are comparable between species, using integrative modeling of eight epigenetic features jointly in human and mouse in our V al i dated S ystematic I ntegrati on (VISION) Project. The resulting catalogs of cCREs are useful resources for further studies of gene regulation in blood cells, indicated by high overlap with known functional elements and strong enrichment for human genetic variants associated with blood cell phenotypes. The contribution of each epigenetic state in cCREs to gene regulation, inferred from a multivariate regression, was used to estimate epigenetic state Regulatory Potential (esRP) scores for each cCRE in each cell type, which were used to categorize dynamic changes in cCREs. Groups of cCREs displaying similar patterns of regulatory activity in human and mouse cell types, obtained by joint clustering on esRP scores, harbored distinctive transcription factor binding motifs that were similar between species. An interspecies comparison of cCREs revealed both conserved and species-specific patterns of epigenetic evolution. Finally, we showed that comparisons of the epigenetic landscape between species can reveal elements with similar roles in regulation, even in the absence of genomic sequence alignment.

Genome folding principles revealed in condensin-depleted mitotic chromosomes.

During mitosis, condensin activity interferes with interphase chromatin structures. Here, we generated condensin-free mitotic chromosomes to investigate genome folding principles. Co-depletion of condensin I and II, but neither alone, triggered mitotic chromosome compartmentalization in ways that differ from interphase. Two distinct euchromatic compartments, indistinguishable in interphase, rapidly emerged upon condensin loss with different interaction preferences and dependence on H3K27ac. Constitutive heterochromatin gradually self-aggregated and co-compartmentalized with the facultative heterochromatin, contrasting with their separation during interphase. While topologically associating domains (TADs) and CTCF/cohesin mediated structural loops remained undetectable, cis-regulatory element contacts became apparent, providing an explanation for their quick re-establishment during mitotic exit. 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, re-established constitutive heterochromatin compartments normally. In sum, "clean-slate" condensing-deficient mitotic chromosomes illuminate mechanisms of genome compartmentalization not revealed in interphase cells.

Molecular basis of polycomb group protein-mediated fetal hemoglobin repression.

The switch from fetal hemoglobin (HbF) to adult hemoglobin (HbA) is a paradigm for developmental gene expression control with relevance to sickle cell disease and ß-thalassemia. Polycomb repressive complex (PRC) proteins regulate this switch, and an inhibitor of PRC2 has entered a clinical trial for HbF activation. Yet, how PRC complexes function in this process, their target genes, and relevant subunit composition are unknown. Here, we identified the PRC1 subunit BMI1 as a novel HbF repressor. We uncovered the RNA binding proteins LIN28B, IGF2BP1, and IGF2BP3 genes as direct BMI1 targets, and demonstrate that they account for the entirety of BMI1's effect on HbF regulation. BMI1 functions as part of the canonical PRC1 (cPRC1) subcomplex as revealed by the physical and functional dissection of BMI1 protein partners. Lastly, we demonstrate that BMI1/cPRC1 acts in concert with PRC2 to repress HbF through the same target genes. Our study illuminates how PRC silences HbF, highlighting an epigenetic mechanism involved in hemoglobin switching.

Gene silencing dynamics are modulated by transiently active regulatory elements.

Transcriptional enhancers have been extensively characterized, but cis-regulatory elements involved in acute gene repression have received less attention. Transcription factor GATA1 promotes erythroid differentiation by activating and repressing distinct gene sets. Here, we study the mechanism by which GATA1 silences the proliferative gene Kit during murine erythroid cell maturation and define stages from initial loss of activation to heterochromatinization. We find that GATA1 inactivates a potent upstream enhancer but concomitantly creates a discrete intronic regulatory region marked by H3K27ac, short noncoding RNAs, and de novo chromatin looping. This enhancer-like element forms transiently and serves to delay Kit silencing. The element is ultimately erased via the FOG1/NuRD deacetylase complex, as revealed by the study of a disease-associated GATA1 variant. Hence, regulatory sites can be self-limiting by dynamic co-factor usage. Genome-wide analyses across cell types and species uncover transiently active elements at numerous genes during repression, suggesting that modulation of silencing kinetics is widespread.

Snapshot: a package for clustering and visualizing epigenetic history during cell differentiation.

BACKGROUND: Epigenetic modification of chromatin plays a pivotal role in regulating gene expression during cell differentiation. The scale and complexity of epigenetic data pose significant challenges for biologists to identify the regulatory events controlling cell differentiation. RESULTS: To reduce the complexity, we developed a package, called Snapshot, for clustering and visualizing candidate cis-regulatory elements (cCREs) based on their epigenetic signals during cell differentiation. This package first introduces a binarized indexing strategy for clustering the cCREs. It then provides a series of easily interpretable figures for visualizing the signal and epigenetic state patterns of the cCREs clusters during the cell differentiation. It can also use different hierarchies of cell types to highlight the epigenetic history specific to any particular cell lineage. We demonstrate the utility of Snapshot using data from a consortium project for ValIdated Systematic IntegratiON (VISION) of epigenomic data in hematopoiesis. CONCLUSION: The package Snapshot can identify all distinct clusters of genomic locations with unique epigenetic signal patterns during cell differentiation. It outperforms other methods in terms of interpreting and reproducing the identified cCREs clusters. The package of Snapshot is available at GitHub: https://github.com/guanjue/Snapshot .

CTCF blocks antisense transcription initiation at divergent promoters.

Transcription at most promoters is divergent, initiating at closely spaced oppositely oriented core promoters to produce sense transcripts along with often unstable upstream antisense transcripts (uasTrx). How antisense transcription is regulated and to what extent it is coordinated with sense transcription is not well understood. Here, by combining acute degradation of the multi-functional transcription factor CTCF and nascent transcription measurements, we find that CTCF specifically suppresses antisense but not sense transcription at hundreds of divergent promoters. Primary transcript RNA-FISH shows that CTCF lowers burst fraction but not burst intensity of uasTrx and that co-bursting of sense and antisense transcripts is disfavored. Genome editing, chromatin conformation studies and high-resolution transcript mapping revealed that precisely positioned CTCF directly suppresses the initiation of uasTrx, in a manner independent of its architectural function. In sum, CTCF shapes the transcriptional landscape in part by suppressing upstream antisense transcription.

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.

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.

Dual function NFI factors control fetal hemoglobin silencing in adult erythroid cells.

The mechanisms by which the fetal-type ß-globin-like genes HBG1 and HBG2 are silenced in adult erythroid precursor cells remain a fundamental question in human biology and have therapeutic relevance to sickle cell disease and ß-thalassemia. Here, we identify via a CRISPR-Cas9 genetic screen two members of the NFI transcription factor family-NFIA and NFIX-as HBG1/2 repressors. NFIA and NFIX are expressed at elevated levels in adult erythroid cells compared with fetal cells, and function cooperatively to repress HBG1/2 in cultured cells and in human-to-mouse xenotransplants. Genomic profiling, genome editing and DNA binding assays demonstrate that the potent concerted activity of NFIA and NFIX is explained in part by their ability to stimulate the expression of BCL11A, a known silencer of the HBG1/2 genes, and in part by directly repressing the HBG1/2 genes. Thus, NFI factors emerge as versatile regulators of the fetal-to-adult switch in ß-globin production.

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.

CTCF and transcription influence chromatin structure re-configuration after mitosis.

During mitosis, transcription is globally attenuated and chromatin architecture is dramatically reconfigured. We exploited the M- to G1-phase progression to interrogate the contributions of the architectural factor CTCF and the process of transcription to genome re-sculpting in newborn nuclei. Depletion of CTCF during the M- to G1-phase transition alters short-range compartmentalization after mitosis. Chromatin domain boundary re-formation is impaired upon CTCF loss, but a subset of boundaries, characterized by transitions in chromatin states, is established normally. Without CTCF, structural loops fail to form, leading to illegitimate contacts between cis-regulatory elements (CREs). Transient CRE contacts that are normally resolved after telophase persist deeply into G1-phase in CTCF-depleted cells. CTCF loss-associated gains in transcription are often linked to increased, normally illegitimate enhancer-promoter contacts. In contrast, at genes whose expression declines upon CTCF loss, CTCF seems to function as a conventional transcription activator, independent of its architectural role. CTCF-anchored structural loops facilitate formation of CRE loops nested within them, especially those involving weak CREs. Transcription inhibition does not significantly affect global architecture or transcription start site-associated boundaries. However, ongoing transcription contributes considerably to the formation of gene domains, regions of enriched contacts along gene bodies. Notably, gene domains emerge in ana/telophase prior to completion of the first round of transcription, suggesting that epigenetic features in gene bodies contribute to genome reconfiguration prior to transcription. The focus on the de novo formation of nuclear architecture during G1 entry yields insights into the contributions of CTCF and transcription to chromatin architecture dynamics during the mitosis to G1-phase progression.

S3V2-IDEAS: a package for normalizing, denoising and integrating epigenomic datasets across different cell types.

SUMMARY: Epigenetic modifications reflect key aspects of transcriptional regulation, and many epigenomic datasets have been generated under different biological contexts to provide insights into regulatory processes. However, the technical noise in epigenomic datasets and the many dimensions (features) examined make it challenging to effectively extract biologically meaningful inferences from these datasets. We developed a package that reduces noise while normalizing the epigenomic data by a novel normalization method, followed by integrative dimensional reduction by learning and assigning epigenetic states. This package, called S3V2-IDEAS, can be used to identify epigenetic states for multiple features, or identify discretized signal intensity levels and a master peak list across different cell types for a single feature. We illustrate the outputs and performance of S3V2-IDEAS using 137 epigenomics datasets from the VISION project that provides ValIdated Systematic IntegratiON of epigenomic data in hematopoiesis. AVAILABILITY AND IMPLEMENTATION: S3V2-IDEAS pipeline is freely available as open source software released under an MIT license at: https://github.com/guanjue/S3V2_IDEAS_ESMP. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.