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1.
Nat Genet ; 53(8): 1177-1186, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34341563

RESUMO

Hereditary persistence of fetal hemoglobin (HPFH) ameliorates ß-hemoglobinopathies by inhibiting the developmental switch from γ-globin (HBG1/HBG2) to ß-globin (HBB) gene expression. Some forms of HPFH are associated with γ-globin promoter variants that either disrupt binding motifs for transcriptional repressors or create new motifs for transcriptional activators. How these variants sustain γ-globin gene expression postnatally remains undefined. We mapped γ-globin promoter sequences functionally in erythroid cells harboring different HPFH variants. Those that disrupt a BCL11A repressor binding element induce γ-globin expression by facilitating the recruitment of nuclear transcription factor Y (NF-Y) to a nearby proximal CCAAT box and GATA1 to an upstream motif. The proximal CCAAT element becomes dispensable for HPFH variants that generate new binding motifs for activators NF-Y or KLF1, but GATA1 recruitment remains essential. Our findings define distinct mechanisms through which transcription factors and their cis-regulatory elements activate γ-globin expression in different forms of HPFH, some of which are being recreated by therapeutic genome editing.


Assuntos
Fator de Ligação a CCAAT/genética , Hemoglobina Fetal/genética , Fator de Transcrição GATA1/genética , gama-Globinas/genética , Animais , Sítios de Ligação , Células COS , Sistemas CRISPR-Cas , Linhagem Celular , Chlorocebus aethiops , Células Eritroides , Edição de Genes/métodos , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo
2.
Phys Chem Chem Phys ; 23(32): 17576-17590, 2021 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-34369509

RESUMO

The rational design of selective histone deacetylase 2 (HDAC2) inhibitors is beneficial for the therapeutic treatment of liver cancer, though HDAC2 is highly homologous to HDAC8, which may lead to undesired side effects due to the pan-inhibition towards HDAC2 and HDAC8. To clarify the structural basis of selective inhibition towards HDAC2 over HDAC8, we utilized multiple in silico strategies, including sequence alignment, structural comparison, molecular docking, molecular dynamics simulations, free energy calculations, alanine scanning mutagenesis, pharmacophore modeling, protein contacts atlas analysis and QM/MM calculations to study the binding patterns of HDAC2/8 selective inhibitors. Through the whole process described above, it is found that although HDAC2 has conserved GLY154 and PHE210 that also exist within HDAC8, namely GLY151 and PHE208, the two isoforms exhibit diverse binding modes towards their inhibitors. Typically, HDAC2 inhibitors interact with the Zn2+ ions through the core chelate group, while HDAC8 inhibitors adopt a bent conformation within the HDAC8 pocket that inclines to be in contact with the Zn2+ ions through the terminal hydroxamic acid group. In summary, our data comprehensively elucidate the selectivity mechanism towards HDAC2 over HDAC8, which would guide the rational design of selective HDAC2 inhibitors for liver cancer treatment.


Assuntos
Histona Desacetilase 2/antagonistas & inibidores , Histona Desacetilase 2/metabolismo , Inibidores de Histona Desacetilases/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Desenho de Fármacos , Histona Desacetilase 2/química , Histona Desacetilase 2/genética , Inibidores de Histona Desacetilases/química , Histona Desacetilases/química , Histona Desacetilases/metabolismo , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mutagênese , Mutação , Ligação Proteica , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Termodinâmica
3.
Nat Commun ; 12(1): 4929, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389727

RESUMO

Synthetic metabolic pathways are a burden for engineered bacteria, but the underlying mechanisms often remain elusive. Here we show that the misregulated activity of the transcription factor Cra is responsible for the growth burden of glycerol overproducing E. coli. Glycerol production decreases the concentration of fructose-1,6-bisphoshate (FBP), which then activates Cra resulting in the downregulation of glycolytic enzymes and upregulation of gluconeogenesis enzymes. Because cells grow on glucose, the improper activation of gluconeogenesis and the concomitant inhibition of glycolysis likely impairs growth at higher induction of the glycerol pathway. We solve this misregulation by engineering a Cra-binding site in the promoter controlling the expression of the rate limiting enzyme of the glycerol pathway to maintain FBP levels sufficiently high. We show the broad applicability of this approach by engineering Cra-dependent regulation into a set of constitutive and inducible promoters, and use one of them to overproduce carotenoids in E. coli.


Assuntos
Escherichia coli/genética , Glicólise/genética , Engenharia Metabólica/métodos , Metabolômica/métodos , Proteômica/métodos , Transcrição Genética , Algoritmos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação/genética , Carotenoides/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Modelos Genéticos , Regiões Promotoras Genéticas/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
Cancer Sci ; 112(9): 3835-3845, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34219323

RESUMO

Tumor-induced angiogenesis is important for further progression of solid tumors. The initiation of tumor angiogenesis is dictated by a shift in the balance between proangiogenic and antiangiogenic gene expression programs. However, the potential mechanism controlling the expression of angiogenesis-related genes in the tumor cells, especially the process mediated by RNA-binding protein (RBP) remains unclear. SAMD4A is a conserved RBP across fly to mammals, and is believed to play an important role in controlling gene translation and stability. In this study, we identified the potential role of SAMD4A in modulating angiogenesis-related gene expression and tumor progression in breast cancer. SAMD4A expression was repressed in breast cancer tissues and cells and low SAMD4A expression in human breast tumor samples was strongly associated with poor survival of patients. Overexpression of SAMD4A inhibited breast tumor angiogenesis and caner progression, whereas knockdown of SAMD4A demonstrated a reversed effect. Mechanistically, SAMD4A was found to specifically destabilize the proangiogenic gene transcripts, including C-X-C motif chemokine ligand 5 (CXCL5), endoglin (ENG), interleukin 1ß (IL1ß), and angiopoietin 1 (ANGPT1), by directly interacting with the stem-loop structure in the 3' untranslated region (3'UTR) of these mRNAs through its sterile alpha motif (SAM) domain, resulting in the imbalance of angiogenic genes expression. Collectively, our results suggest that SAMD4A is a novel breast tumor suppressor that inhibits tumor angiogenesis by specifically downregulating the expression of proangiogenic genes, which might be a potential antiangiogenic target for breast cancer therapy.


Assuntos
Neoplasias da Mama/irrigação sanguínea , Regulação Neoplásica da Expressão Gênica , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Progressão da Doença , Feminino , Células HEK293 , Humanos , Células MCF-7 , Glândulas Mamárias Humanas/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/genética , Transfecção , Carga Tumoral/genética , Proteínas Supressoras de Tumor/genética , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Nat Commun ; 12(1): 4170, 2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34234130

RESUMO

Genome organization is driven by forces affecting transcriptional state, but the relationship between transcription and genome architecture remains unclear. Here, we identified the Drosophila transcription factor Motif 1 Binding Protein (M1BP) in physical association with the gypsy chromatin insulator core complex, including the universal insulator protein CP190. M1BP is required for enhancer-blocking and barrier activities of the gypsy insulator as well as its proper nuclear localization. Genome-wide, M1BP specifically colocalizes with CP190 at Motif 1-containing promoters, which are enriched at topologically associating domain (TAD) borders. M1BP facilitates CP190 chromatin binding at many shared sites and vice versa. Both factors promote Motif 1-dependent gene expression and transcription near TAD borders genome-wide. Finally, loss of M1BP reduces chromatin accessibility and increases both inter- and intra-TAD local genome compaction. Our results reveal physical and functional interaction between CP190 and M1BP to activate transcription at TAD borders and mediate chromatin insulator-dependent genome organization.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Animais , Animais Geneticamente Modificados , Linhagem Celular , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Proteínas de Drosophila/genética , Técnicas de Silenciamento de Genes , Genoma de Inseto , Elementos Isolantes/genética , Masculino , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , RNA-Seq , Proteínas Repressoras/genética , Fatores de Transcrição/genética
6.
Nat Commun ; 12(1): 4227, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244482

RESUMO

Glycine decarboxylase (GLDC) is a key enzyme of glycine cleavage system that converts glycine into one-carbon units. GLDC is commonly up-regulated and plays important roles in many human cancers. Whether and how GLDC is regulated by post-translational modifications is unknown. Here we report that mechanistic target of rapamycin complex 1 (mTORC1) signal inhibits GLDC acetylation at lysine (K) 514 by inducing transcription of the deacetylase sirtuin 3 (SIRT3). Upon inhibition of mTORC1, the acetyltransferase acetyl-CoA acetyltransferase 1 (ACAT1) catalyzes GLDC K514 acetylation. This acetylation of GLDC impairs its enzymatic activity. In addition, this acetylation of GLDC primes for its K33-linked polyubiquitination at K544 by the ubiquitin ligase NF-X1, leading to its degradation by the proteasomal pathway. Finally, we find that GLDC K514 acetylation inhibits glycine catabolism, pyrimidines synthesis and glioma tumorigenesis. Our finding reveals critical roles of post-translational modifications of GLDC in regulation of its enzymatic activity, glycine metabolism and tumorigenesis, and provides potential targets for therapeutics of cancers such as glioma.


Assuntos
Carcinogênese/genética , Glioma/genética , Glicina Desidrogenase (Descarboxilante)/metabolismo , Glicina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Acetil-CoA C-Acetiltransferase/metabolismo , Acetilação , Animais , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Glioma/metabolismo , Glioma/patologia , Células HEK293 , Humanos , Masculino , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Proteólise , Pirimidinas/biossíntese , Proteínas Repressoras/metabolismo , Sirtuína 3/genética , Sirtuína 3/metabolismo , Ativação Transcricional , Ubiquitinação/genética , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Nat Commun ; 12(1): 4090, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215745

RESUMO

The transition from pluripotent to somatic states marks a critical event in mammalian development, but remains largely unresolved. Here we report the identification of SS18 as a regulator for pluripotent to somatic transition or PST by CRISPR-based whole genome screens. Mechanistically, SS18 forms microscopic condensates in nuclei through a C-terminal intrinsically disordered region (IDR) rich in tyrosine, which, once mutated, no longer form condensates nor rescue SS18-/- defect in PST. Yet, the IDR alone is not sufficient to rescue the defect even though it can form condensates indistinguishable from the wild type protein. We further show that its N-terminal 70aa is required for PST by interacting with the Brg/Brahma-associated factor (BAF) complex, and remains functional even swapped onto unrelated IDRs or even an artificial 24 tyrosine polypeptide. Finally, we show that SS18 mediates BAF assembly through phase separation to regulate PST. These studies suggest that SS18 plays a role in the pluripotent to somatic interface and undergoes liquid-liquid phase separation through a unique tyrosine-based mechanism.


Assuntos
Transição de Fase , Células-Tronco Pluripotentes/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Animais , Núcleo Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Feminino , Células HEK293 , Humanos , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Tirosina
8.
Int J Mol Sci ; 22(11)2021 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-34204139

RESUMO

The prohibitin (PHB)-binding compound fluorizoline as well as PHB-downregulation activate the integrated stress response (ISR) in HEK293T and U2OS human cell lines. This activation is denoted by phosphorylation of eIF2α and increases in ATF4, ATF3, and CHOP protein levels. The blockage of the activation of the ISR by overexpression of GRP78, as well as an increase in IRE1 activity, indicate the presence of ER stress after fluorizoline treatment. The inhibition of the ER stress response in HEK293T and U2OS led to increased sensitivity to fluorizoline-induced apoptosis, indicating a pro-survival role of this pathway after fluorizoline treatment in these cell lines. Fluorizoline induced an increase in calcium concentration in the cytosol and the mitochondria. Finally, two different calcium chelators reduced fluorizoline-induced apoptosis in U2OS cells. Thus, we have found that fluorizoline causes increased ER stress and activation of the integrated stress response, which in HEK293T and U2OS cells are protective against fluorizoline-induced apoptosis.


Assuntos
Apoptose , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Tiazóis/farmacologia , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Linhagem Celular Tumoral , Respiração Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Células HEK293 , Homeostase/efeitos dos fármacos , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/efeitos dos fármacos
9.
Nat Commun ; 12(1): 4439, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34290235

RESUMO

The α- and ß-globin loci harbor developmentally expressed genes, which are silenced throughout post-natal life. Reactivation of these genes may offer therapeutic approaches for the hemoglobinopathies, the most common single gene disorders. Here, we address mechanisms regulating the embryonically expressed α-like globin, termed ζ-globin. We show that in embryonic erythroid cells, the ζ-gene lies within a ~65 kb sub-TAD (topologically associating domain) of open, acetylated chromatin and interacts with the α-globin super-enhancer. By contrast, in adult erythroid cells, the ζ-gene is packaged within a small (~10 kb) sub-domain of hypoacetylated, facultative heterochromatin within the acetylated sub-TAD and that it no longer interacts with its enhancers. The ζ-gene can be partially re-activated by acetylation and inhibition of histone de-acetylases. In addition to suggesting therapies for severe α-thalassemia, these findings illustrate the general principles by which reactivation of developmental genes may rescue abnormalities arising from mutations in their adult paralogues.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Ativação Transcricional , Globinas zeta/genética , Acetilação , Animais , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Células Eritroides/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Humanos , Camundongos , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional/efeitos dos fármacos , alfa-Globinas/genética
10.
Sci Transl Med ; 13(604)2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-34321320

RESUMO

Accumulation of the parkin-interacting substrate (PARIS; ZNF746), due to inactivation of parkin, contributes to Parkinson's disease (PD) through repression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α; PPARGC1A) activity. Here, we identify farnesol as an inhibitor of PARIS. Farnesol promoted the farnesylation of PARIS, preventing its repression of PGC-1α via decreasing PARIS occupancy on the PPARGC1A promoter. Farnesol prevented dopaminergic neuronal loss and behavioral deficits via farnesylation of PARIS in PARIS transgenic mice, ventral midbrain transduction of AAV-PARIS, adult conditional parkin KO mice, and the α-synuclein preformed fibril model of sporadic PD. PARIS farnesylation is decreased in the substantia nigra of patients with PD, suggesting that reduced farnesylation of PARIS may play a role in PD. Thus, farnesol may be beneficial in the treatment of PD by enhancing the farnesylation of PARIS and restoring PGC-1α activity.


Assuntos
Doença de Parkinson , Animais , Dopamina , Camundongos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Prenilação , Proteínas Repressoras/metabolismo , Substância Negra/metabolismo
11.
Methods Mol Biol ; 2328: 203-214, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34251628

RESUMO

Plants use different regulatory modules in response to changes in their surroundings. With the transcriptomic approaches governing all research areas, an integrative, fast, and sensitive approach toward validating genes of interest becomes a critical step prior to functional studies in planta. This chapter describes a detailed method for a quantitative analysis of transcriptional readouts of defense response genes using tobacco leaves as a transient system. The method uses Luciferase reporter assays to monitor activities of defense pathway promoters. Under normal conditions, the JASMONATE ZIM-DOMAIN (JAZ) proteins repress defense genes by preventing their expression. Here, we will provide a detailed protocol on the use of a dual-luciferase system to analyze activities of various defense response promoters simultaneously. We will use two well-characterized modules from the Jasmonic acid (JA) defense pathway; the JAZ3 repressor protein and the promoters of three of JA responsive genes, MYC2, 3 and 4. This assay revealed not only differences in promoter strength but also provided quantitative insights on the JAZ3 repression of MYCs in a quantitative manner.


Assuntos
Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Folhas de Planta/metabolismo , Proteínas Repressoras/metabolismo , Tabaco/metabolismo , Agrobacterium tumefaciens/metabolismo , Primers do DNA , Genes myc/genética , Luciferases/genética , Plantas Geneticamente Modificadas/genética , Regiões Promotoras Genéticas , Domínios Proteicos/genética , Proteínas Repressoras/genética , Tabaco/genética
12.
Nat Commun ; 12(1): 4626, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34330913

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer that has remained clinically challenging to manage. Here we employ an RNAi-based in vivo functional genomics platform to determine epigenetic vulnerabilities across a panel of patient-derived PDAC models. Through this, we identify protein arginine methyltransferase 1 (PRMT1) as a critical dependency required for PDAC maintenance. Genetic and pharmacological studies validate the role of PRMT1 in maintaining PDAC growth. Mechanistically, using proteomic and transcriptomic analyses, we demonstrate that global inhibition of asymmetric arginine methylation impairs RNA metabolism, which includes RNA splicing, alternative polyadenylation, and transcription termination. This triggers a robust downregulation of multiple pathways involved in the DNA damage response, thereby promoting genomic instability and inhibiting tumor growth. Taken together, our data support PRMT1 as a compelling target in PDAC and informs a mechanism-based translational strategy for future therapeutic development.Statement of significancePDAC is a highly lethal cancer with limited therapeutic options. This study identified and characterized PRMT1-dependent regulation of RNA metabolism and coordination of key cellular processes required for PDAC tumor growth, defining a mechanism-based translational hypothesis for PRMT1 inhibitors.


Assuntos
Carcinoma Ductal Pancreático/genética , Dano ao DNA , Neoplasias Pancreáticas/genética , Proteína-Arginina N-Metiltransferases/genética , RNA/genética , Proteínas Repressoras/genética , Animais , Biocatálise/efeitos dos fármacos , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/prevenção & controle , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/prevenção & controle , Proteína-Arginina N-Metiltransferases/metabolismo , RNA/metabolismo , Interferência de RNA , Proteínas Repressoras/metabolismo , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
13.
J Biol Chem ; 297(1): 100866, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34118234

RESUMO

Genetically encoded fluorescent H2O2 probes continue to advance the field of redox biology. Here, we compare the previously established peroxiredoxin-based H2O2 probe roGFP2-Tsa2ΔCR with the newly described OxyR-based H2O2 probe HyPer7, using yeast as the model system. Although not as sensitive as roGFP2-Tsa2ΔCR, HyPer7 is much improved relative to earlier HyPer versions, most notably by ratiometric pH stability. The most striking difference between the two probes is the dynamics of intracellular probe reduction. HyPer7 is rapidly reduced, predominantly by the thioredoxin system, whereas roGFP2-Tsa2ΔCR is reduced more slowly, predominantly by the glutathione system. We discuss the pros and cons of each probe and suggest that future side-by-side measurements with both probes may provide information on the relative activity of the two major cellular reducing systems.


Assuntos
Técnicas Biossensoriais/métodos , Proteínas de Escherichia coli/metabolismo , Peróxido de Hidrogênio/análise , Peroxirredoxinas/metabolismo , Proteínas Repressoras/metabolismo , Técnicas Biossensoriais/normas , Proteínas de Escherichia coli/genética , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Peróxido de Hidrogênio/metabolismo , Peroxidases/genética , Peroxidases/metabolismo , Peroxirredoxinas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
14.
Mol Cell Biol ; 41(8): e0038220, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34060915

RESUMO

The long noncoding RNA XIST is the master regulator for the process of X chromosome inactivation (XCI) in mammalian females. Here, we report the existence of a hitherto-uncharacterized cis regulatory element (cRE) within the first exon of human XIST, which determines the transcriptional status of XIST during the initiation and maintenance phases of XCI. In the initiation phase, pluripotency factors bind to this cRE and keep XIST repressed. In the maintenance phase of XCI, the cRE is enriched for CTCF, which activates XIST transcription. By employing a CRISPR-dCas9-KRAB-based interference strategy, we demonstrate that binding of CTCF to the newly identified cRE is critical for regulating XIST in a YY1-dependent manner. Collectively, our study uncovers the combinatorial effect of multiple transcriptional regulators influencing XIST expression during the initiation and maintenance phases of XCI.


Assuntos
Fator de Ligação a CCCTC/metabolismo , Células-Tronco Embrionárias/metabolismo , RNA Longo não Codificante/genética , Inativação do Cromossomo X/fisiologia , Fator de Ligação a CCCTC/genética , Humanos , Sequências Reguladoras de Ácido Nucleico/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Inativação do Cromossomo X/genética
15.
Nucleic Acids Res ; 49(12): 6788-6803, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34133717

RESUMO

Speckle-type Poz protein (SPOP), an E3 ubiquitin ligase adaptor, is the most frequently mutated gene in prostate cancer. The SPOP-mutated subtype of prostate cancer shows high genomic instability, but the underlying mechanisms causing this phenotype are still largely unknown. Here, we report that upon DNA damage, SPOP is phosphorylated at Ser119 by the ATM serine/threonine kinase, which potentiates the binding of SPOP to homeodomain-interacting protein kinase 2 (HIPK2), resulting in a nondegradative ubiquitination of HIPK2. This modification subsequently increases the phosphorylation activity of HIPK2 toward HP1γ, and then promotes the dissociation of HP1γ from trimethylated (Lys9) histone H3 (H3K9me3) to initiate DNA damage repair. Moreover, the effect of SPOP on the HIPK2-HP1γ axis is abrogated by prostate cancer-associated SPOP mutations. Our findings provide new insights into the molecular mechanism of SPOP mutations-driven genomic instability in prostate cancer.


Assuntos
Proteínas de Transporte/metabolismo , Instabilidade Genômica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Transporte/química , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/metabolismo , Dano ao DNA , Histonas/metabolismo , Humanos , Masculino , Mutação , Fosforilação , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/patologia , Proteínas Serina-Treonina Quinases/química , Serina/metabolismo , Ubiquitinação
16.
Nucleic Acids Res ; 49(12): 7075-7087, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34139006

RESUMO

In Pseudomonas aeruginosa the RNA chaperone Hfq and the catabolite repression control protein (Crc) govern translation of numerous transcripts during carbon catabolite repression. Here, Crc was shown to enhance Hfq-mediated translational repression of several mRNAs. We have developed a single-molecule fluorescence assay to quantitatively assess the cooperation of Hfq and Crc to form a repressive complex on a RNA, encompassing the translation initiation region and the proximal coding sequence of the P. aeruginosa amiE gene. The presence of Crc did not change the amiE RNA-Hfq interaction lifetimes, whereas it changed the equilibrium towards more stable repressive complexes. This observation is in accord with Cryo-EM analyses, which showed an increased compactness of the repressive Hfq/Crc/RNA assemblies. These biophysical studies revealed how Crc protein kinetically stabilizes Hfq/RNA complexes, and how the two proteins together fold a large segment of the mRNA into a more compact translationally repressive structure. In fact, the presence of Crc resulted in stronger translational repression in vitro and in a significantly reduced half-life of the target amiE mRNA in vivo. Although Hfq is well-known to act with small regulatory RNAs, this study shows how Hfq can collaborate with another protein to down-regulate translation of mRNAs that become targets for the degradative machinery.


Assuntos
Proteínas de Bactérias/metabolismo , Fator Proteico 1 do Hospedeiro/metabolismo , Biossíntese de Proteínas , Pseudomonas aeruginosa/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Motivos de Nucleotídeos , Pseudomonas aeruginosa/metabolismo , Estabilidade de RNA , RNA Mensageiro/química
17.
Nat Commun ; 12(1): 3720, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140524

RESUMO

Low levels of reactive oxygen species (ROS) are crucial for maintaining cancer stem cells (CSCs) and their ability to resist therapy, but the ROS regulatory mechanisms in CSCs remains to be explored. Here, we discover that prohibitin (PHB) specifically regulates mitochondrial ROS production in glioma stem-like cells (GSCs) and facilitates GSC radiotherapeutic resistance. We find that PHB is upregulated in GSCs and is associated with malignant gliomas progression and poor prognosis. PHB binds to peroxiredoxin3 (PRDX3), a mitochondrion-specific peroxidase, and stabilizes PRDX3 protein through the ubiquitin-proteasome pathway. Knockout of PHB dramatically elevates ROS levels, thereby inhibiting GSC self-renewal. Importantly, deletion or pharmacological inhibition of PHB potently slows tumor growth and sensitizes tumors to radiotherapy, thus providing significant survival benefits in GSC-derived orthotopic tumors and glioblastoma patient-derived xenografts. These results reveal a selective role of PHB in mitochondrial ROS regulation in GSCs and suggest that targeting PHB improves radiotherapeutic efficacy in glioblastoma.


Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Células-Tronco Neoplásicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Proteínas Repressoras/metabolismo , Adulto , Idoso , Animais , Astrocitoma/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Inativação de Genes , Glioblastoma/genética , Glioblastoma/patologia , Glioblastoma/radioterapia , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Gradação de Tumores , Peroxirredoxinas/metabolismo , Prognóstico , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Análise Serial de Tecidos , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Nat Commun ; 12(1): 4017, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34188048

RESUMO

The synchronization is a recurring phenomenon in neuroscience, ecology, human sciences, and biology. However, controlling synchronization in complex eukaryotic consortia on extended spatial-temporal scales remains a major challenge. Here, to address this issue we construct a minimal synthetic system that directly converts chemical signals into a coherent gene expression synchronized among eukaryotic communities through rate-dependent hysteresis. Guided by chemical rhythms, isolated colonies of yeast Saccharomyces cerevisiae oscillate in near-perfect synchrony despite the absence of intercellular coupling or intrinsic oscillations. Increased speed of chemical rhythms and incorporation of feedback in the system architecture can tune synchronization and precision of the cell responses in a growing cell collectives. This synchronization mechanism remain robust under stress in the two-strain consortia composed of toxin-sensitive and toxin-producing strains. The sensitive cells can maintain the spatial-temporal synchronization for extended periods under the rhythmic toxin dosages produced by killer cells. Our study provides a simple molecular framework for generating global coordination of eukaryotic gene expression through dynamic environment.


Assuntos
Ciclo Celular/fisiologia , Regulação Fúngica da Expressão Gênica/genética , Saccharomyces cerevisiae/metabolismo , Fenômenos Bioquímicos , Expressão Gênica/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/genética
19.
Commun Biol ; 4(1): 757, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34145387

RESUMO

Although impaired keratinocyte migration is a recognized hallmark of chronic wounds, the molecular mechanisms underpinning impaired cell movement are poorly understood. Here, we demonstrate that both diabetic foot ulcers (DFUs) and venous leg ulcers (VLUs) exhibit global deregulation of cytoskeletal organization in genomic comparison to normal skin and acute wounds. Interestingly, we found that DFUs and VLUs exhibited downregulation of ArhGAP35, which serves both as an inactivator of RhoA and as a glucocorticoid repressor. Since chronic wounds exhibit elevated levels of cortisol and caveolin-1 (Cav1), we posited that observed elevation of Cav1 expression may contribute to impaired actin-cytoskeletal signaling, manifesting in aberrant keratinocyte migration. We showed that Cav1 indeed antagonizes ArhGAP35, resulting in increased activation of RhoA and diminished activation of Cdc42, which can be rescued by Cav1 disruption. Furthermore, we demonstrate that both inducible keratinocyte specific Cav1 knockout mice, and MßCD treated diabetic mice, exhibit accelerated wound closure. Taken together, our findings provide a previously unreported mechanism by which Cav1-mediated cytoskeletal organization prevents wound closure in patients with chronic wounds.


Assuntos
Caveolina 1/genética , Úlcera do Pé/patologia , Proteínas Ativadoras de GTPase/genética , Queratinócitos/metabolismo , Proteínas Repressoras/genética , Úlcera Varicosa/patologia , Cicatrização/fisiologia , Animais , Caveolina 1/metabolismo , Linhagem Celular , Movimento Celular/genética , Citoesqueleto/patologia , Pé Diabético/patologia , Regulação para Baixo/genética , Células Epiteliais/metabolismo , Epitélio/crescimento & desenvolvimento , Proteínas Ativadoras de GTPase/metabolismo , Glucocorticoides/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Repressoras/metabolismo , Cicatrização/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo
20.
Nat Cell Biol ; 23(6): 652-663, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34083785

RESUMO

Expression of exon-specific isoforms from alternatively spliced mRNA is a fundamental mechanism that substantially expands the proteome of a cell. However, conventional methods to assess alternative splicing are either consumptive and work-intensive or do not quantify isoform expression longitudinally at the protein level. Here, we therefore developed an exon-specific isoform expression reporter system (EXSISERS), which non-invasively reports the translation of exon-containing isoforms of endogenous genes by scarlessly excising reporter proteins from the nascent polypeptide chain through highly efficient, intein-mediated protein splicing. We applied EXSISERS to quantify the inclusion of the disease-associated exon 10 in microtubule-associated protein tau (MAPT) in patient-derived induced pluripotent stem cells and screened Cas13-based RNA-targeting effectors for isoform specificity. We also coupled cell survival to the inclusion of exon 18b of FOXP1, which is involved in maintaining pluripotency of embryonic stem cells, and confirmed that MBNL1 is a dominant factor for exon 18b exclusion. EXSISERS enables non-disruptive and multimodal monitoring of exon-specific isoform expression with high sensitivity and cellular resolution, and empowers high-throughput screening of exon-specific therapeutic interventions.


Assuntos
Processamento Alternativo , Fatores de Transcrição Forkhead/metabolismo , Ensaios de Triagem em Larga Escala , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteômica , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Repressoras/metabolismo , Proteínas tau/metabolismo , Sistemas CRISPR-Cas , Éxons , Fatores de Transcrição Forkhead/genética , Células HEK293 , Humanos , Isoformas de Proteínas , Proteoma , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas Repressoras/genética , Análise de Célula Única , Proteínas tau/genética
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