Cell cycle-dependent gene networks for cell proliferation activated by nuclear CK2α complexes.

Nuclear expression of protein kinase CK2α is reportedly elevated in human carcinomas, but mechanisms underlying its variable localization in cells are poorly understood. This study demonstrates a functional connection between nuclear CK2 and gene expression in relation to cell proliferation. Growth stimulation of quiescent human normal fibroblasts and phospho-proteomic analysis identified a pool of CK2α that is highly phosphorylated at serine 7. Phosphorylated CK2α translocates into the nucleus, and this phosphorylation appears essential for nuclear localization and catalytic activity. Protein signatures associated with nuclear CK2 complexes reveal enrichment of apparently unique transcription factors and chromatin remodelers during progression through the G1 phase of the cell cycle. Chromatin immunoprecipitation-sequencing profiling demonstrated recruitment of CK2α to active gene loci, more abundantly in late G1 phase than in early G1, notably at transcriptional start sites of core histone genes, growth stimulus-associated genes, and ribosomal RNAs. Our findings reveal that nuclear CK2α complexes may be essential to facilitate progression of the cell cycle, by activating histone genes and triggering ribosomal biogenesis, specified in association with nuclear and nucleolar transcriptional regulators.

Context-dependent perturbations in chromatin folding and the transcriptome by cohesin and related factors.

Cohesin regulates gene expression through context-specific chromatin folding mechanisms such as enhancer-promoter looping and topologically associating domain (TAD) formation by cooperating with factors such as cohesin loaders and the insulation factor CTCF. We developed a computational workflow to explore how three-dimensional (3D) structure and gene expression are regulated collectively or individually by cohesin and related factors. The main component is CustardPy, by which multi-omics datasets are compared systematically. To validate our methodology, we generated 3D genome, transcriptome, and epigenome data before and after depletion of cohesin and related factors and compared the effects of depletion. We observed diverse effects on the 3D genome and transcriptome, and gene expression changes were correlated with the splitting of TADs caused by cohesin loss. We also observed variations in long-range interactions across TADs, which correlated with their epigenomic states. These computational tools and datasets will be valuable for 3D genome and epigenome studies.

EVI1 exerts distinct roles in AML via ERG and cyclin D1 promoting a chemoresistant and immune-suppressive environment.

Aberrant expression of ecotropic viral integration site-1 (EVI1+) is associated with very poor outcomes in acute myeloid leukemia (AML), mechanisms of which are only partially understood. Using the green fluorescent protein reporter system to monitor EVI1 promoter activity, we demonstrated that Evi1high KMT2A-MLLT1-transformed AML cells possess distinct features from Evi1low cells: the potential for aggressive disease independent of stem cell activity and resistance to cytotoxic chemotherapy, along with the consistent gene expression profiles. RNA sequencing and chromatin immunoprecipitation sequencing in EVI1-transformed AML cells and normal hematopoietic cells combined with functional screening by cell proliferation-related short hairpin RNAs revealed that the erythroblast transformation-specific transcription factor ERG (E26 transformation-specific [ETS]-related gene) and cyclin D1 were downstream targets and therapeutic vulnerabilities of EVI1+ AML. Silencing Erg in murine EVI1+ AML models severely impaired cell proliferation, chemoresistance, and leukemogenic capacity. Cyclin D1 is also requisite for efficient EVI1-AML development, associated with gene expression profiles related to chemokine production and interferon signature, and T- and natural killer-cell exhaustion phenotype, depending on the interferon gamma (IFN-γ)/STAT1 pathway but not on CDK4/CDK6. Inhibiting the IFN-γ/STAT1 pathway alleviated immune exhaustion and impaired EVI1-AML development. Overexpression of EVI1 and cyclin D1 was associated with IFN-γ signature and increased expression of chemokines, with increased exhaustion molecules in T cells also in human AML data sets. These data collectively suggest that ERG and cyclin D1 play pivotal roles in the biology of EVI1+ AML, where ERG contributes to aggressive disease nature and chemoresistance, and cyclin D1 leads to IFN-γ signature and exhausted T-cell phenotypes, which could potentially be targeted.

Large-scale multi-omics analysis suggests specific roles for intragenic cohesin in transcriptional regulation.

Cohesin, an essential protein complex for chromosome segregation, regulates transcription through a variety of mechanisms. It is not a trivial task to assign diverse cohesin functions. Moreover, the context-specific roles of cohesin-mediated interactions, especially on intragenic regions, have not been thoroughly investigated. Here we perform a comprehensive characterization of cohesin binding sites in several human cell types. We integrate epigenomic, transcriptomic and chromatin interaction data to explore the context-specific functions of intragenic cohesin related to gene activation. We identify a specific subset of cohesin binding sites, decreased intragenic cohesin sites (DICs), which are negatively correlated with transcriptional regulation. A subgroup of DICs is enriched with enhancer markers and RNA polymerase II, while the others are more correlated to chromatin architecture. DICs are observed in various cell types, including cells from patients with cohesinopathy. We also implement machine learning to our data and identified genomic features for isolating DICs from all cohesin sites. These results suggest a previously unidentified function of cohesin on intragenic regions for transcriptional regulation.

Transcription-dependent cohesin repositioning rewires chromatin loops in cellular senescence.

Senescence is a state of stable proliferative arrest, generally accompanied by the senescence-associated secretory phenotype, which modulates tissue homeostasis. Enhancer-promoter interactions, facilitated by chromatin loops, play a key role in gene regulation but their relevance in senescence remains elusive. Here, we use Hi-C to show that oncogenic RAS-induced senescence in human diploid fibroblasts is accompanied by extensive enhancer-promoter rewiring, which is closely connected with dynamic cohesin binding to the genome. We find de novo cohesin peaks often at the 3' end of a subset of active genes. RAS-induced de novo cohesin peaks are transcription-dependent and enriched for senescence-associated genes, exemplified by IL1B, where de novo cohesin binding is involved in new loop formation. Similar IL1B induction with de novo cohesin appearance and new loop formation are observed in terminally differentiated macrophages, but not TNFα-treated cells. These results suggest that RAS-induced senescence represents a cell fate determination-like process characterised by a unique gene expression profile and 3D genome folding signature, mediated in part through cohesin redistribution on chromatin.

Missense Mutations in NKAP Cause a Disorder of Transcriptional Regulation Characterized by Marfanoid Habitus and Cognitive Impairment.

NKAP is a ubiquitously expressed nucleoplasmic protein that is currently known as a transcriptional regulatory molecule via its interaction with HDAC3 and spliceosomal proteins. Here, we report a disorder of transcriptional regulation due to missense mutations in the X chromosome gene, NKAP. These mutations are clustered in the C-terminal region of NKAP where NKAP interacts with HDAC3 and post-catalytic spliceosomal complex proteins. Consistent with a role for the C-terminal region of NKAP in embryogenesis, nkap mutant zebrafish with a C-terminally truncated NKAP demonstrate severe developmental defects. The clinical features of affected individuals are highly conserved and include developmental delay, hypotonia, joint contractures, behavioral abnormalities, Marfanoid habitus, and scoliosis. In affected cases, transcriptome analysis revealed the presence of a unique transcriptome signature, which is characterized by the downregulation of long genes with higher exon numbers. These observations indicate the critical role of NKAP in transcriptional regulation and demonstrate that perturbations of the C-terminal region lead to developmental defects in both humans and zebrafish.

Cornelia de Lange syndrome, related disorders, and the Cohesin complex: Abstracts from the 8th biennial scientific and educational symposium 2018.

Cornelia de Lange Syndrome (CdLS), due to mutations in genes of the cohesin protein complex, is described as a disorder of transcriptional regulation. Phenotypes in this expanding field include short stature, microcephaly, intellectual disability, variable facial features and organ involvement, resulting in overlapping presentations, including established syndromes and newly described conditions. Individuals with all forms of CdLS have multifaceted complications, including neurodevelopmental, feeding, craniofacial, and communication. Coping mechanisms and management of challenging behaviors in CdLS, disruption of normal behaviors, and how behavior molds the life of the individual within the family is now better understood. Some psychotropic medications are known to be effective for behavior. Other medications, for example, Indomethacin, are being investigated for effects on gene expression, fetal brain tissue, brain morphology and function in Drosophila, mice, and human fibroblasts containing CdLS-related mutations. Developmental studies have clarified the origin of cardiac defects and role of placenta in CdLS. Chromosome architecture and cohesin complex structure are elucidated, leading to a better understanding of regulatory aspects and controls. As examples, when mutations are present, the formation of loop domains by cohesin, facilitating enhancer-promotor interactions, can be eliminated, and embryologically, the nuclear structure of zygotes is disrupted. Several important genes are now known to interact with cohesin, including Brca2. The following abstracts are from the 8th Cornelia de Lange Syndrome Scientific and Educational Symposium, held in June 2018, Minneapolis, MN, before the CdLS Foundation National Meeting, AMA CME credits provided by GBMC, Baltimore, MD. All studies have been approved by an ethics committee.

Temporal Regulation of ESCO2 Degradation by the MCM Complex, the CUL4-DDB1-VPRBP Complex, and the Anaphase-Promoting Complex.

Sister chromatid cohesion, mediated by cohesin, is required for accurate chromosome segregation [1, 2]. This process requires acetylation of cohesin subunit SMC3 by evolutionarily conserved cohesin acetyltransferases: Eco1 in budding yeast; XEco1 and XEco2 in Xenopus; and ESCO1 and ESCO2 in human [3-10]. Eco1 is recruited to chromatin through physical interaction with PCNA [11] and is degraded by the Skp1/Cul1/F-box protein complex after DNA replication to prevent ectopic cohesion formation [12]. In contrast, XEco2 recruitment to chromatin requires prereplication complex formation [13] and is degraded by the anaphase-promoting complex (APC) [14]. In human, whereas ESCO1 is expressed throughout the cell cycle, ESCO2 is detectable in S phase and is degraded after DNA replication [6, 15]. Although PDS5, a cohesin regulator, preferentially promotes ESCO1-dependent SMC3 acetylation [16], little is known about the molecular basis of the temporal regulation of ESCO2. Here, we show that ESCO2 is recruited to chromatin before PCNA accumulation. Whereas no interaction between PCNA and ESCO proteins is observed, ESCO2, but not ESCO1, interacts with the MCM complex through a unique ESCO2 domain. Interestingly, the interaction is required to protect ESCO2 from proteasomal degradation and is attenuated in late S phase. We also found that ESCO2 physically interacts with the CUL4-DDB1-VPRBP E3 ubiquitin ligase complex in late S phase and that post-replicative ESCO2 degradation requires the complex as well as APC. Thus, we propose that the MCM complex couples ESCO2 with DNA replication and that the CUL4-DDB1-VPRBP complex promotes post-replicative ESCO2 degradation, presumably to suppress cohesion formation during mitosis.

Decreased cohesin in the brain leads to defective synapse development and anxiety-related behavior.

Abnormal epigenetic regulation can cause the nervous system to develop abnormally. Here, we sought to understand the mechanism by which this occurs by investigating the protein complex cohesin, which is considered to regulate gene expression and, when defective, is associated with higher-level brain dysfunction and the developmental disorder Cornelia de Lange syndrome (CdLS). We generated conditional Smc3-knockout mice and observed greater dendritic complexity and larger numbers of immature synapses in the cerebral cortex of Smc3+/- mice. Smc3+/- mice also exhibited more anxiety-related behavior, which is a symptom of CdLS. Further, a gene ontology analysis after RNA-sequencing suggested the enrichment of immune processes, particularly the response to interferons, in the Smc3+/- mice. Indeed, fewer synapses formed in their cortical neurons, and this phenotype was rescued by STAT1 knockdown. Thus, low levels of cohesin expression in the developing brain lead to changes in gene expression that in turn lead to a specific and abnormal neuronal and behavioral phenotype.

DNMT3A R882 mutants interact with polycomb proteins to block haematopoietic stem and leukaemic cell differentiation.

Despite the clinical impact of DNMT3A mutation on acute myeloid leukaemia, the molecular mechanisms regarding how this mutation causes leukaemogenesis in vivo are largely unknown. Here we show that, in murine transplantation experiments, recipients transplanted with DNMT3A mutant-transduced cells exhibit aberrant haematopoietic stem cell (HSC) accumulation. Differentiation-associated genes are downregulated without accompanying changes in methylation status of their promoter-associated CpG islands in DNMT3A mutant-transduced stem/progenitor cells, representing a DNA methylation-independent role of mutated DNMT3A. DNMT3A R882H also promotes monoblastic transformation in vitro in combination with HOXA9. Molecularly, the DNMT3A mutant interacts with polycomb repressive complex 1 (PRC1), causing transcriptional silencing, revealing a DNA methylation-independent role of DNMT3A mutation. Suppression of PRC1 impairs aberrant HSC accumulation and monoblastic transformation. From our data, it is shown that DNMT3A mutants can block the differentiation of HSCs and leukaemic cells via PRC1. This interaction could be targetable in DNMT3A-mutated leukaemias.

Condensin targets and reduces unwound DNA structures associated with transcription in mitotic chromosome condensation.

Chromosome condensation is a hallmark of mitosis in eukaryotes and is a prerequisite for faithful segregation of genetic material to daughter cells. Here we show that condensin, which is essential for assembling condensed chromosomes, helps to preclude the detrimental effects of gene transcription on mitotic condensation. ChIP-seq profiling reveals that the fission yeast condensin preferentially binds to active protein-coding genes in a transcription-dependent manner during mitosis. Pharmacological and genetic attenuation of transcription largely rescue bulk chromosome segregation defects observed in condensin mutants. We also demonstrate that condensin is associated with and reduces unwound DNA segments generated by transcription, providing a direct link between an in vitro activity of condensin and its in vivo function. The human condensin isoform condensin I also binds to unwound DNA regions at the transcription start sites of active genes, implying that our findings uncover a fundamental feature of condensin complexes.

The Deubiquitinating Enzyme USP7 Regulates Androgen Receptor Activity by Modulating Its Binding to Chromatin.

The androgen receptor (AR), a nuclear receptor superfamily transcription factor, plays a key role in prostate cancer. AR signaling is the principal target for prostate cancer treatment, but current androgen-deprivation therapies cannot completely abolish AR signaling because of the heterogeneity of prostate cancers. Therefore, unraveling the mechanism of AR reactivation in androgen-depleted conditions can identify effective prostate cancer therapeutic targets. Increasing evidence indicates that AR activity is mediated by the interplay of modifying/demodifying enzymatic co-regulators. To better understand the mechanism of AR transcriptional activity regulation, we used antibodies against AR for affinity purification and identified the deubiquitinating enzyme ubiquitin-specific protease 7, USP7 as a novel AR co-regulator in prostate cancer cells. We showed that USP7 associates with AR in an androgen-dependent manner and mediates AR deubiquitination. Sequential ChIP assays indicated that USP7 forms a complex with AR on androgen-responsive elements of target genes upon stimulation with the androgen 5α-dihydrotestosterone. Further investigation indicated that USP7 is necessary to facilitate androgen-activated AR binding to chromatin. Transcriptome profile analysis of USP7-knockdown LNCaP cells also revealed the essential role of USP7 in the expression of a subset of androgen-responsive genes. Hence, inhibition of USP7 represents a compelling therapeutic strategy for the treatment of prostate cancer.

Esco1 Acetylates Cohesin via a Mechanism Different from That of Esco2.

Sister chromatid cohesion is mediated by cohesin and is essential for accurate chromosome segregation. The cohesin subunits SMC1, SMC3, and Rad21 form a tripartite ring within which sister chromatids are thought to be entrapped. This event requires the acetylation of SMC3 and the association of sororin with cohesin by the acetyltransferases Esco1 and Esco2 in humans, but the functional mechanisms of these acetyltransferases remain elusive. Here, we showed that Esco1 requires Pds5, a cohesin regulatory subunit bound to Rad21, to form cohesion via SMC3 acetylation and the stabilization of the chromatin association of sororin, whereas Esco2 function was not affected by Pds5 depletion. Consistent with the functional link between Esco1 and Pds5, Pds5 interacted exclusively with Esco1, and this interaction was dependent on a unique and conserved Esco1 domain. Crucially, this interaction was essential for SMC3 acetylation and sister chromatid cohesion. Esco1 localized to cohesin localization sites on chromosomes throughout interphase in a manner that required the Esco1-Pds5 interaction, and it could acetylate SMC3 before and after DNA replication. These results indicate that Esco1 acetylates SMC3 via a mechanism different from that of Esco2. We propose that, by interacting with a unique domain of Esco1, Pds5 recruits Esco1 to chromatin-bound cohesin complexes to form cohesion. Furthermore, Esco1 acetylates SMC3 independently of DNA replication.

Germline gain-of-function mutations in AFF4 cause a developmental syndrome functionally linking the super elongation complex and cohesin.

Transcriptional elongation is critical for gene expression regulation during embryogenesis. The super elongation complex (SEC) governs this process by mobilizing paused RNA polymerase II (RNAP2). Using exome sequencing, we discovered missense mutations in AFF4, a core component of the SEC, in three unrelated probands with a new syndrome that phenotypically overlaps Cornelia de Lange syndrome (CdLS) that we have named CHOPS syndrome (C for cognitive impairment and coarse facies, H for heart defects, O for obesity, P for pulmonary involvement and S for short stature and skeletal dysplasia). Transcriptome and chromatin immunoprecipitation sequencing (ChIP-seq) analyses demonstrated similar alterations of genome-wide binding of AFF4, cohesin and RNAP2 in CdLS and CHOPS syndrome. Direct molecular interaction of the SEC, cohesin and RNAP2 was demonstrated. These data support a common molecular pathogenesis for CHOPS syndrome and CdLS caused by disturbance of transcriptional elongation due to alterations in genome-wide binding of AFF4 and cohesin.

Dimeric combinations of MafB, cFos and cJun control the apoptosis-survival balance in limb morphogenesis.

Apoptosis is an important mechanism for sculpting morphology. However, the molecular cascades that control apoptosis in developing limb buds remain largely unclear. Here, we show that MafB was specifically expressed in apoptotic regions of chick limb buds, and MafB/cFos heterodimers repressed apoptosis, whereas MafB/cJun heterodimers promoted apoptosis for sculpting the shape of the limbs. Chromatin immunoprecipitation sequencing in chick limb buds identified potential target genes and regulatory elements controlled by Maf and Jun. Functional analyses revealed that expression of p63 and p73, key components known to arrest the cell cycle, was directly activated by MafB and cJun. Our data suggest that dimeric combinations of MafB, cFos and cJun in developing chick limb buds control the number of apoptotic cells, and that MafB/cJun heterodimers lead to apoptosis via activation of p63 and p73.

Chromatin immunoprecipitation protocol for mammalian cells.

The genomic approach (ChIP-seq) we introduce here is now a widely used powerful tool to explore protein-DNA interaction at genome-wide level in high resolution. This technology opens up the way to understand how local event mediated by protein-protein or protein-DNA interactions lead to the dynamic changes of overall chromosome structure and how variety of proteins make a regulatory network for the faithful execution of various chromosomal functions (i.e., transcription, replication, recombination, repair, and partition).

Loss-of-function HDAC8 mutations cause a phenotypic spectrum of Cornelia de Lange syndrome-like features, ocular hypertelorism, large fontanelle and X-linked inheritance.

Cornelia de Lange syndrome (CdLS) is a multisystem genetic disorder with distinct facies, growth failure, intellectual disability, distal limb anomalies, gastrointestinal and neurological disease. Mutations in NIPBL, encoding a cohesin regulatory protein, account for >80% of cases with typical facies. Mutations in the core cohesin complex proteins, encoded by the SMC1A, SMC3 and RAD21 genes, together account for ∼5% of subjects, often with atypical CdLS features. Recently, we identified mutations in the X-linked gene HDAC8 as the cause of a small number of CdLS cases. Here, we report a cohort of 38 individuals with an emerging spectrum of features caused by HDAC8 mutations. For several individuals, the diagnosis of CdLS was not considered prior to genomic testing. Most mutations identified are missense and de novo. Many cases are heterozygous females, each with marked skewing of X-inactivation in peripheral blood DNA. We also identified eight hemizygous males who are more severely affected. The craniofacial appearance caused by HDAC8 mutations overlaps that of typical CdLS but often displays delayed anterior fontanelle closure, ocular hypertelorism, hooding of the eyelids, a broader nose and dental anomalies, which may be useful discriminating features. HDAC8 encodes the lysine deacetylase for the cohesin subunit SMC3 and analysis of the functional consequences of the missense mutations indicates that all cause a loss of enzymatic function. These data demonstrate that loss-of-function mutations in HDAC8 cause a range of overlapping human developmental phenotypes, including a phenotypically distinct subgroup of CdLS.

Design, synthesis, and biological activity of NCC149 derivatives as histone deacetylase 8-selective inhibitors.

We recently discovered N-hydroxy-3-[1-(phenylthio)methyl-1H-1,2,3-triazol-4-yl]benzamide (NCC149) as a potent and selective histone deacetylase 8 (HDAC8) inhibitor from a 151-member triazole compound library using a click chemistry approach. In this work, we present a series of NCC149 derivatives bearing various aromatic linkers that were designed and synthesized as HDAC8-selective inhibitors. A series of in vitro assays were used to evaluate the newly synthesized compounds, four of which showed HDAC8 inhibitory activity similar to that of NCC149, and one of which displayed HDAC8 selectivity superior to that of NCC149. In addition, these top four compounds induced the increase of acetylated cohesin (an HDAC8 substrate) in HeLa cells in a dose-dependent manner, indicating inhibition of HDAC8 in the cells. While none of these compounds enhanced the acetylation of H3K9 (a substrate of HDAC1 and 2), only one compound refrained from increasing α-tubulin acetylation, a substrate of HDAC6, indicating that this compound is more selective for HDAC8 than the other derivatives. Furthermore, this HDAC8-selective inhibitor suppressed the growth of T-cell lymphoma cells more potently than did NCC149. These findings are useful for the further development of HDAC8-selective inhibitors.

Smc5/6-mediated regulation of replication progression contributes to chromosome assembly during mitosis in human cells.

The structural maintenance of chromosomes (SMC) proteins constitute the core of critical complexes involved in structural organization of chromosomes. In yeast, the Smc5/6 complex is known to mediate repair of DNA breaks and replication of repetitive genomic regions, including ribosomal DNA loci and telomeres. In mammalian cells, which have diverse genome structure and scale from yeast, the Smc5/6 complex has also been implicated in DNA damage response, but its further function in unchallenged conditions remains elusive. In this study, we addressed the behavior and function of Smc5/6 during the cell cycle. Chromatin fractionation, immunofluorescence, and live-cell imaging analyses indicated that Smc5/6 associates with chromatin during interphase but largely dissociates from chromosomes when they condense in mitosis. Depletion of Smc5 and Smc6 resulted in aberrant mitotic chromosome phenotypes that were accompanied by the abnormal distribution of topoisomerase IIα (topo IIα) and condensins and by chromosome segregation errors. Importantly, interphase chromatin structure indicated by the premature chromosome condensation assay suggested that Smc5/6 is required for the on-time progression of DNA replication and subsequent binding of topo IIα on replicated chromatids. These results indicate an essential role of the Smc5/6 complex in processing DNA replication, which becomes indispensable for proper sister chromatid assembly in mitosis.

Recurrent mutations in multiple components of the cohesin complex in myeloid neoplasms.

Cohesin is a multimeric protein complex that is involved in the cohesion of sister chromatids, post-replicative DNA repair and transcriptional regulation. Here we report recurrent mutations and deletions involving multiple components of the cohesin complex, including STAG2, RAD21, SMC1A and SMC3, in different myeloid neoplasms. These mutations and deletions were mostly mutually exclusive and occurred in 12.1% (19/157) of acute myeloid leukemia, 8.0% (18/224) of myelodysplastic syndromes, 10.2% (9/88) of chronic myelomonocytic leukemia, 6.3% (4/64) of chronic myelogenous leukemia and 1.3% (1/77) of classical myeloproliferative neoplasms. Cohesin-mutated leukemic cells showed reduced amounts of chromatin-bound cohesin components, suggesting a substantial loss of cohesin binding sites on chromatin. The growth of leukemic cell lines harboring a mutation in RAD21 (Kasumi-1 cells) or having severely reduced expression of RAD21 and STAG2 (MOLM-13 cells) was suppressed by forced expression of wild-type RAD21 and wild-type RAD21 and STAG2, respectively. These findings suggest a role for compromised cohesin functions in myeloid leukemogenesis.