RESUMO
Despite advances in defining diverse somatic mutations that cause myeloid malignancies, a significant heritable component for these cancers remains largely unexplained. Here, we perform rare variant association studies in a large population cohort to identify inherited predisposition genes for these blood cancers. CTR9, which encodes a key component of the PAF1 transcription elongation complex, is among the significant genes identified. The risk variants found in the cases cause loss of function and result in a â¼10-fold increased odds of acquiring a myeloid malignancy. Partial CTR9 loss of function expands human hematopoietic stem cells (HSCs) by increased super elongation complex-mediated transcriptional activity, which thereby increases the expression of key regulators of HSC self-renewal. By following up on insights from a human genetic study examining inherited predisposition to the myeloid malignancies, we define a previously unknown antagonistic interaction between the PAF1 and super elongation complexes. These insights could enable targeted approaches for blood cancer prevention.
Assuntos
Neoplasias Hematológicas , Fosfoproteínas , Elongação da Transcrição Genética , Fatores de Transcrição , Humanos , Neoplasias Hematológicas/genética , Células-Tronco Hematopoéticas/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Fosfoproteínas/genéticaRESUMO
Metazoan gene expression regulation involves pausing of RNA polymerase (Pol II) in the promoter-proximal region of genes and is stabilized by DSIF and NELF. Upon depletion of elongation factors, NELF appears to accompany elongating Pol II past pause sites; however, prior work indicates that NELF prevents Pol II elongation. Here, we report cryoelectron microscopy structures of Pol II-DSIF-NELF complexes with NELF in two distinct conformations corresponding to paused and poised states. The paused NELF state supports Pol II stalling, whereas the poised NELF state enables transcription elongation as it does not support a tilted RNA-DNA hybrid. Further, the poised NELF state can accommodate TFIIS binding to Pol II, allowing for Pol II reactivation at paused or backtracking sites. Finally, we observe that the NELF-A tentacle interacts with the RPB2 protrusion and is necessary for pausing. Our results define how NELF can support pausing, reactivation, and elongation by Pol II.
Assuntos
Proteínas Nucleares , RNA Polimerase II , Animais , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Microscopia Crioeletrônica , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The MYCN oncoprotein binds active promoters in a heterodimer with its partner protein MAX. MYCN also interacts with the nuclear exosome, a 3'-5' exoribonuclease complex, suggesting a function in RNA metabolism. Here, we show that MYCN forms stable high-molecular-weight complexes with the exosome and multiple RNA-binding proteins. MYCN binds RNA in vitro and in cells via a conserved sequence termed MYCBoxI. In cells, MYCN associates with thousands of intronic transcripts together with the ZCCHC8 subunit of the nuclear exosome targeting complex and enhances their processing. Perturbing exosome function results in global re-localization of MYCN from promoters to intronic RNAs. On chromatin, MYCN is then replaced by the MNT(MXD6) repressor protein, inhibiting MYCN-dependent transcription. RNA-binding-deficient alleles show that RNA-binding limits MYCN's ability to activate cell growth-related genes but is required for MYCN's ability to promote progression through S phase and enhance the stress resilience of neuroblastoma cells.
Assuntos
Proteína Proto-Oncogênica N-Myc , Proteínas Nucleares , Proteínas Oncogênicas , Proteínas de Ligação a RNA , Proteína Proto-Oncogênica N-Myc/metabolismo , Proteína Proto-Oncogênica N-Myc/genética , Humanos , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Oncogênicas/metabolismo , Proteínas Oncogênicas/genética , Regiões Promotoras Genéticas , Linhagem Celular Tumoral , Neuroblastoma/metabolismo , Neuroblastoma/genética , Neuroblastoma/patologia , Exossomos/metabolismo , Exossomos/genética , Íntrons , Ligação Proteica , Núcleo Celular/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Regulação Neoplásica da Expressão Gênica , RNA/metabolismo , RNA/genética , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Proliferação de CélulasRESUMO
During gene transcription, RNA polymerase II (RNA Pol II) passes nucleosomes with the help of various elongation factors. Here, we show that RNA Pol II achieves efficient nucleosome passage when the human elongation factors DSIF, PAF1 complex (PAF), RTF1, SPT6, and TFIIS are present. The cryo-EM structure of an intermediate of the nucleosome passage shows a partially unraveled hexasome that lacks the proximal H2A-H2B dimer and interacts with the RNA Pol II jaw, DSIF, and the CTR9trestle helix. RNA Pol II adopts a backtracked state with the RNA 3' end dislodged from the active site and bound in the RNA Pol II pore. Additional structures and biochemical data show that human TFIIS enters the RNA Pol II pore and stimulates the cleavage of the backtracked RNA and nucleosome passage.
Assuntos
Nucleossomos , RNA Polimerase II , Núcleo Celular/metabolismo , Humanos , Nucleossomos/genética , RNA , RNA Polimerase II/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Fatores de Elongação da Transcrição/metabolismoRESUMO
The MYCN oncoprotein drives the development of numerous neuroendocrine and pediatric tumors. Here we show that MYCN interacts with the nuclear RNA exosome, a 3'-5' exoribonuclease complex, and recruits the exosome to its target genes. In the absence of the exosome, MYCN-directed elongation by RNA polymerase II (RNAPII) is slow and non-productive on a large group of cell-cycle-regulated genes. During the S phase of MYCN-driven tumor cells, the exosome is required to prevent the accumulation of stalled replication forks and of double-strand breaks close to the transcription start sites. Upon depletion of the exosome, activation of ATM causes recruitment of BRCA1, which stabilizes nuclear mRNA decapping complexes, leading to MYCN-dependent transcription termination. Disruption of mRNA decapping in turn activates ATR, indicating transcription-replication conflicts. We propose that exosome recruitment by MYCN maintains productive transcription elongation during S phase and prevents transcription-replication conflicts to maintain the rapid proliferation of neuroendocrine tumor cells.
Assuntos
Núcleo Celular/enzimologia , Proliferação de Células , Replicação do DNA , Exossomos/enzimologia , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/enzimologia , RNA Polimerase II/metabolismo , Transcrição Gênica , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/genética , Quebras de DNA de Cadeia Dupla , Exorribonucleases/genética , Exorribonucleases/metabolismo , Exossomos/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Masculino , Camundongos , Proteína Proto-Oncogênica N-Myc/genética , Células NIH 3T3 , Neuroblastoma/genética , Neuroblastoma/patologia , Regiões Promotoras Genéticas , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Polimerase II/genética , Terminação da Transcrição GenéticaRESUMO
The influence of genome organization on transcription is central to our understanding of cell type specification. Higher-order genome organization is established through short- and long-range DNA interactions. Coordination of these interactions, from single atoms to entire chromosomes, plays a fundamental role in transcriptional control of gene expression. Loss of this coupling can result in disease. Analysis of transcriptional regulation typically involves disparate experimental approaches, from structural studies that define angstrom-level interactions to cell-biological and genomic approaches that assess mesoscale relationships. Thus, to fully understand the mechanisms that regulate gene expression, it is critical to integrate the findings gained across these distinct size scales. In this review, I illustrate fundamental ways in which cells regulate transcription in the context of genome organization.
Assuntos
Pareamento de Bases/genética , Cromossomos/genética , Transcrição Gênica/genética , Animais , Regulação da Expressão Gênica/genética , Humanos , Elementos Reguladores de Transcrição/genéticaRESUMO
The super elongation complex (SEC) contains the positive transcription elongation factor b (P-TEFb) and the subcomplex ELL2-EAF1, which stimulates RNA polymerase II (RNA Pol II) elongation. Here, we report the cryoelectron microscopy (cryo-EM) structure of ELL2-EAF1 bound to a RNA Pol II elongation complex at 2.8 Å resolution. The ELL2-EAF1 dimerization module directly binds the RNA Pol II lobe domain, explaining how SEC delivers P-TEFb to RNA Pol II. The same site on the lobe also binds the initiation factor TFIIF, consistent with SEC binding only after the transition from transcription initiation to elongation. Structure-guided functional analysis shows that the stimulation of RNA elongation requires the dimerization module and the ELL2 linker that tethers the module to the RNA Pol II protrusion. Our results show that SEC stimulates elongation allosterically and indicate that this stimulation involves stabilization of a closed conformation of the RNA Pol II active center cleft.
Assuntos
Fator B de Elongação Transcricional Positiva/ultraestrutura , RNA Polimerase II/genética , Fatores de Transcrição/genética , Fatores de Elongação da Transcrição/genética , Regulação Alostérica/genética , Núcleo Celular/genética , Núcleo Celular/ultraestrutura , Microscopia Crioeletrônica , Humanos , Estrutura Molecular , Complexos Multiproteicos/genética , Complexos Multiproteicos/ultraestrutura , Fator B de Elongação Transcricional Positiva/genética , Ligação Proteica/genética , Conformação Proteica , RNA Polimerase II/ultraestrutura , Elongação da Transcrição Genética , Fatores de Transcrição/ultraestrutura , Transcrição Gênica/genética , Fatores de Elongação da Transcrição/ultraestruturaRESUMO
The MYC oncoprotein globally affects the function of RNA polymerase II (RNAPII). The ability of MYC to promote transcription elongation depends on its ubiquitylation. Here, we show that MYC and PAF1c (polymerase II-associated factor 1 complex) interact directly and mutually enhance each other's association with active promoters. PAF1c is rapidly transferred from MYC onto RNAPII. This transfer is driven by the HUWE1 ubiquitin ligase and is required for MYC-dependent transcription elongation. MYC and HUWE1 promote histone H2B ubiquitylation, which alters chromatin structure both for transcription elongation and double-strand break repair. Consistently, MYC suppresses double-strand break accumulation in active genes in a strictly PAF1c-dependent manner. Depletion of PAF1c causes transcription-dependent accumulation of double-strand breaks, despite widespread repair-associated DNA synthesis. Our data show that the transfer of PAF1c from MYC onto RNAPII efficiently couples transcription elongation with double-strand break repair to maintain the genomic integrity of MYC-driven tumor cells.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/metabolismo , Elongação da Transcrição Genética , ATPases Associadas a Diversas Atividades Celulares/genética , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Linhagem Celular Tumoral , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-myc/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
In response to stress, human cells coordinately downregulate transcription and translation of housekeeping genes. To downregulate transcription, the negative elongation factor (NELF) is recruited to gene promoters impairing RNA polymerase II elongation. Here we report that NELF rapidly forms nuclear condensates upon stress in human cells. Condensate formation requires NELF dephosphorylation and SUMOylation induced by stress. The intrinsically disordered region (IDR) in NELFA is necessary for nuclear NELF condensation and can be functionally replaced by the IDR of FUS or EWSR1 protein. We find that biomolecular condensation facilitates enhanced recruitment of NELF to promoters upon stress to drive transcriptional downregulation. Importantly, NELF condensation is required for cellular viability under stressful conditions. We propose that stress-induced NELF condensates reported here are nuclear counterparts of cytosolic stress granules. These two stress-inducible condensates may drive the coordinated downregulation of transcription and translation, likely forming a critical node of the stress survival strategy.
Assuntos
Resposta ao Choque Térmico/genética , Proteínas Intrinsicamente Desordenadas/genética , Processamento de Proteína Pós-Traducional , RNA Polimerase II/genética , Transcrição Gênica , Fatores de Elongação da Transcrição/genética , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Cromatina/química , Cromatina/metabolismo , Células Clonais , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Genes Reporter , Células HEK293 , Células HeLa , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Fosforilação , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Transdução de Sinais , Estresse Fisiológico , Sumoilação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/metabolismo , Proteína Vermelha FluorescenteRESUMO
The MYC oncoprotein binds to promoter-proximal regions of virtually all transcribed genes and enhances RNA polymerase II (Pol II) function, but its precise mode of action is poorly understood. Using mass spectrometry of both MYC and Pol II complexes, we show here that MYC controls the assembly of Pol II with a small set of transcription elongation factors that includes SPT5, a subunit of the elongation factor DSIF. MYC directly binds SPT5, recruits SPT5 to promoters, and enables the CDK7-dependent transfer of SPT5 onto Pol II. Consistent with known functions of SPT5, MYC is required for fast and processive transcription elongation. Intriguingly, the high levels of MYC that are expressed in tumors sequester SPT5 into non-functional complexes, thereby decreasing the expression of growth-suppressive genes. Altogether, these results argue that MYC controls the productive assembly of processive Pol II elongation complexes and provide insight into how oncogenic levels of MYC permit uncontrolled cellular growth.
Assuntos
Proteínas Nucleares/genética , Proteínas Proto-Oncogênicas c-myc/genética , RNA Polimerase II/genética , Transcrição Gênica , Fatores de Elongação da Transcrição/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Quinases Ciclina-Dependentes/genética , Chaperonas de Histonas/genética , Humanos , Neoplasias/genética , Regiões Promotoras Genéticas , Quinase Ativadora de Quinase Dependente de CiclinaRESUMO
OBJECTIVE: The hallmark oncogene MYC drives the progression of most tumours, but direct inhibition of MYC by a small-molecule drug has not reached clinical testing. MYC is a transcription factor that depends on several binding partners to function. We therefore explored the possibility of targeting MYC via its interactome in pancreatic ductal adenocarcinoma (PDAC). DESIGN: To identify the most suitable targets among all MYC binding partners, we constructed a targeted shRNA library and performed screens in cultured PDAC cells and tumours in mice. RESULTS: Unexpectedly, many MYC binding partners were found to be important for cultured PDAC cells but dispensable in vivo. However, some were also essential for tumours in their natural environment and, among these, the ATPases RUVBL1 and RUVBL2 ranked first. Degradation of RUVBL1 by the auxin-degron system led to the arrest of cultured PDAC cells but not untransformed cells and to complete tumour regression in mice, which was preceded by immune cell infiltration. Mechanistically, RUVBL1 was required for MYC to establish oncogenic and immunoevasive gene expression identifying the RUVBL1/2 complex as a druggable vulnerability in MYC-driven cancer. CONCLUSION: One implication of our study is that PDAC cell dependencies are strongly influenced by the environment, so genetic screens should be performed in vitro and in vivo. Moreover, the auxin-degron system can be applied in a PDAC model, allowing target validation in living mice. Finally, by revealing the nuclear functions of the RUVBL1/2 complex, our study presents a pharmaceutical strategy to render pancreatic cancers potentially susceptible to immunotherapy.
Assuntos
ATPases Associadas a Diversas Atividades Celulares , Carcinoma Ductal Pancreático , DNA Helicases , Neoplasias Pancreáticas , Proteínas Proto-Oncogênicas c-myc , Animais , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Camundongos , Humanos , DNA Helicases/genética , DNA Helicases/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Linhagem Celular Tumoral , Proteínas de Transporte/metabolismo , Proteínas de Transporte/genéticaRESUMO
Metazoan gene regulation often involves the pausing of RNA polymerase II (Pol II) in the promoter-proximal region. Paused Pol II is stabilized by the protein complexes DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF). Here we report the cryo-electron microscopy structure of a paused transcription elongation complex containing Sus scrofa Pol II and Homo sapiens DSIF and NELF at 3.2 Å resolution. The structure reveals a tilted DNA-RNA hybrid that impairs binding of the nucleoside triphosphate substrate. NELF binds the polymerase funnel, bridges two mobile polymerase modules, and contacts the trigger loop, thereby restraining Pol II mobility that is required for pause release. NELF prevents binding of the anti-pausing transcription elongation factor IIS (TFIIS). Additionally, NELF possesses two flexible 'tentacles' that can contact DSIF and exiting RNA. These results define the paused state of Pol II and provide the molecular basis for understanding the function of NELF during promoter-proximal gene regulation.
Assuntos
Microscopia Crioeletrônica , Proteínas Nucleares/ultraestrutura , RNA Polimerase II/metabolismo , RNA Polimerase II/ultraestrutura , Elongação da Transcrição Genética , Fatores de Transcrição/ultraestrutura , Fatores de Elongação da Transcrição/ultraestrutura , Animais , DNA/genética , DNA/metabolismo , HIV-1/genética , Humanos , Modelos Moleculares , Movimento , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Conformação Proteica , Provírus/genética , RNA/genética , RNA/metabolismo , Sus scrofa , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/metabolismoRESUMO
Gene regulation involves activation of RNA polymerase II (Pol II) that is paused and bound by the protein complexes DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF). Here we show that formation of an activated Pol II elongation complex in vitro requires the kinase function of the positive transcription elongation factor b (P-TEFb) and the elongation factors PAF1 complex (PAF) and SPT6. The cryo-EM structure of an activated elongation complex of Sus scrofa Pol II and Homo sapiens DSIF, PAF and SPT6 was determined at 3.1 Å resolution and compared to the structure of the paused elongation complex formed by Pol II, DSIF and NELF. PAF displaces NELF from the Pol II funnel for pause release. P-TEFb phosphorylates the Pol II linker to the C-terminal domain. SPT6 binds to the phosphorylated C-terminal-domain linker and opens the RNA clamp formed by DSIF. These results provide the molecular basis for Pol II pause release and elongation activation.
Assuntos
Microscopia Crioeletrônica , Proteínas Nucleares/ultraestrutura , RNA Polimerase II/metabolismo , RNA Polimerase II/ultraestrutura , Fatores de Transcrição/ultraestrutura , Fatores de Elongação da Transcrição/ultraestrutura , Animais , DNA/química , DNA/ultraestrutura , Humanos , Modelos Moleculares , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Fosfoproteínas/ultraestrutura , Fator B de Elongação Transcricional Positiva/metabolismo , RNA/química , RNA/ultraestrutura , Sus scrofa , Elongação da Transcrição Genética , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/metabolismoRESUMO
Topoisomerases are complex molecular machines that modulate DNA topology to maintain chromosome superstructure and integrity. Although capable of stand-alone activity in vitro, topoisomerases are frequently linked to larger pathways and systems that resolve specific DNA superstructures and intermediates arising from cellular processes such as DNA repair, transcription, replication and chromosome compaction. Topoisomerase activity is indispensible to cells, but requires the transient breakage of DNA strands. This property has been exploited, often for significant clinical benefit, by various exogenous agents that interfere with cell proliferation. Despite decades of study, surprising findings involving topoisomerases continue to emerge with respect to their cellular function, regulation and utility as therapeutic targets.
Assuntos
DNA Topoisomerases/metabolismo , Animais , Segregação de Cromossomos , DNA/química , DNA/metabolismo , Replicação do DNA , DNA Topoisomerases/química , Regulação da Expressão Gênica , Humanos , Inibidores da Topoisomerase/uso terapêuticoRESUMO
Chromatin-remodelling factors change nucleosome positioning and facilitate DNA transcription, replication, and repair. The conserved remodelling factor chromodomain-helicase-DNA binding protein 1(Chd1) can shift nucleosomes and induce regular nucleosome spacing. Chd1 is required for the passage of RNA polymerase IIthrough nucleosomes and for cellular pluripotency. Chd1 contains the DNA-binding domains SANT and SLIDE, a bilobal motor domain that hydrolyses ATP, and a regulatory double chromodomain. Here we report the cryo-electron microscopy structure of Chd1 from the yeast Saccharomyces cerevisiae bound to a nucleosome at a resolution of 4.8 Å. Chd1 detaches two turns of DNA from the histone octamer and binds between the two DNA gyres in a state poised for catalysis. The SANT and SLIDE domains contact detached DNA around superhelical location (SHL) -7 of the first DNA gyre. The ATPase motor binds the second DNA gyre at SHL +2 and is anchored to the N-terminal tail of histone H4, as seen in a recent nucleosome-Snf2 ATPase structure. Comparisons with published results reveal that the double chromodomain swings towards nucleosomal DNA at SHL +1, resulting in ATPase closure. The ATPase can then promote translocation of DNA towards the nucleosome dyad, thereby loosening the first DNA gyre and remodelling the nucleosome. Translocation may involve ratcheting of the two lobes of the ATPase, which is trapped in a pre- or post-translocation state in the absence or presence, respectively, of transition state-mimicking compounds.
Assuntos
Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/ultraestrutura , Microscopia Crioeletrônica , DNA/química , DNA/metabolismo , Proteínas de Ligação a DNA/química , Ativação Enzimática , Histonas/metabolismo , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Nucleossomos/química , Ligação Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
Precise control of supercoiling homeostasis is critical to DNA-dependent processes such as gene expression, replication, and damage response. Topoisomerases are central regulators of DNA supercoiling commonly thought to act independently in the recognition and modulation of chromosome superstructure; however, recent evidence has indicated that cells tightly regulate topoisomerase activity to support chromosome dynamics, transcriptional response, and replicative events. How topoisomerase control is executed and linked to the internal status of a cell is poorly understood. To investigate these connections, we determined the structure of Escherichia coli gyrase, a type IIA topoisomerase bound to YacG, a recently identified chromosomally encoded inhibitor protein. Phylogenetic analyses indicate that YacG is frequently associated with coenzyme A (CoA) production enzymes, linking the protein to metabolism and stress. The structure, along with supporting solution studies, shows that YacG represses gyrase by sterically occluding the principal DNA-binding site of the enzyme. Unexpectedly, YacG acts by both engaging two spatially segregated regions associated with small-molecule inhibitor interactions (fluoroquinolone antibiotics and the newly reported antagonist GSK299423) and remodeling the gyrase holoenzyme into an inactive, ATP-trapped configuration. This study establishes a new mechanism for the protein-based control of topoisomerases, an approach that may be used to alter supercoiling levels for responding to changes in cellular state.
Assuntos
DNA Girase/metabolismo , Escherichia coli/enzimologia , Modelos Moleculares , DNA Bacteriano/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Ligação Proteica , Estrutura Quaternária de ProteínaRESUMO
Chromosome partitioning in Escherichia coli is assisted by two interacting proteins, topoisomerase (topo) IV and MukB. MukB stimulates the relaxation of negative supercoils by topo IV; to understand the mechanism of their action and to define this functional interplay, we determined the crystal structure of a minimal MukB-topo IV complex to 2.3 Å resolution. The structure shows that the so-called 'hinge' region of MukB forms a heterotetrameric assembly with a C-terminal DNA binding domain (CTD) on topo IV's ParC subunit. Biochemical studies show that the hinge stimulates topo IV by competing for a site on the CTD that normally represses activity on negatively supercoiled DNA, while complementation tests using mutants implicated in the interaction reveal that the cellular dependency on topo IV derives from a joint need for both strand passage and MukB binding. Interestingly, the configuration of the MukB·topo IV complex sterically disfavours intradimeric interactions, indicating that the proteins may form oligomeric arrays with one another, and suggesting a framework by which MukB and topo IV may collaborate during daughter chromosome disentanglement.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , DNA Topoisomerase IV/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Cromossômicas não Histona/química , DNA Topoisomerase IV/química , DNA Bacteriano/metabolismo , DNA Super-Helicoidal/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Modelos Moleculares , Ligação Proteica , Conformação ProteicaRESUMO
The eukaryotic microrchidia (MORC) protein family are DNA gyrase, Hsp90, histidine kinase, MutL (GHKL)-type ATPases involved in gene expression regulation and chromatin compaction. The molecular mechanisms underlying these activities are incompletely understood. Here we studied the full-length human MORC2 protein biochemically. We identified a DNA binding site in the C-terminus of the protein, and we observe that this region is heavily phosphorylated in cells. Phosphorylation of MORC2 reduces its affinity for DNA and appears to exclude the protein from the nucleus. We observe that DNA binding by MORC2 reduces its ATPase activity and that MORC2 can topologically entrap multiple DNA substrates between its N-terminal GHKL and C-terminal coiled coil 3 dimerization domains. Finally, we observe that the MORC2 C-terminal DNA binding region is required for gene silencing in cells. Together, our data provide a model to understand how MORC2 engages with DNA substrates to mediate gene silencing.
RESUMO
Mutations in the methyl-DNA-binding protein MECP2 cause the neurodevelopmental disorder Rett syndrome (RTT). How MECP2 contributes to transcriptional regulation in normal and disease states is unresolved; it has been reported to be an activator and a repressor. We describe here the first integrated CUT&Tag, transcriptome, and proteome analyses using human neurons with wild-type (WT) and mutant MECP2 molecules. MECP2 occupies CpG-rich promoter-proximal regions in over four thousand genes in human neurons, including a plethora of autism risk genes, together with RNA polymerase II (RNA Pol II). MECP2 directly interacts with RNA Pol II, and genes occupied by both proteins showed reduced expression in neurons with MECP2 patient mutations. We conclude that MECP2 acts as a positive cofactor for RNA Pol II gene expression at many neuronal genes that harbor CpG islands in promoter-proximal regions and that RTT is due, in part, to the loss of gene activity of these genes in neurons.
Assuntos
Proteína 2 de Ligação a Metil-CpG , Neurônios , RNA Polimerase II , Transcrição Gênica , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Proteína 2 de Ligação a Metil-CpG/genética , Humanos , Neurônios/metabolismo , Regiões Promotoras Genéticas , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Ilhas de CpG/genética , Mutação , Regulação da Expressão Gênica/genéticaRESUMO
In contrast to the current state of knowledge in the field of eukaryotic chromosome segregation, relatively little is known about the mechanisms coordinating the appropriate segregation of bacterial chromosomes. In Escherichia coli, the MukB/E/F complex and topoisomerase IV (Topo IV) are both crucial players in this process. Topo IV removes DNA entanglements following the replication of the chromosome, whereas MukB, a member of the structural maintenance of chromosomes protein family, serves as a bacterial condensin. We demonstrate here a direct physical interaction between the dimerization domain of MukB and the C-terminal domain of the ParC subunit of Topo IV. In addition, we find that MukB alters the activity of Topo IV in vitro. Finally, we isolate a MukB mutant, D692A, that is deficient in its interaction with ParC and show that this mutant fails to rescue the temperature-sensitive growth phenotype of a mukB(-) strain. These results show that MukB and Topo IV are linked physically and functionally and indicate that the activities of these proteins are not limited to chromosome segregation but likely also play a key role in the control of higher-order bacterial chromosome structure.