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The hypoxia-inducible factor-1α (HIF-1α) is a key regulator of hypoxic stress under physiological and pathological conditions. HIF-1α protein stability is tightly regulated by the ubiquitin-proteasome system (UPS) and autophagy in normoxia, hypoxia, and the tumor environment to mediate the hypoxic response. However, the mechanisms of how the UPS and autophagy interplay for HIF-1α proteostasis remain unclear. Here, we found a HIF-1α species propionylated at lysine (K) 709 by p300/CREB binding protein (CBP). HIF-1α stability and the choice of degradation pathway were affected by HIF-1α propionylation. K709-propionylation prevented HIF-1α from degradation through the UPS, while activated chaperon-mediated autophagy (CMA) induced the degradation of propionylated and nonpropionylated HIF-1α. CMA contributed to HIF-1α degradation in both normoxia and hypoxia. Furthermore, the pan-cancer analysis showed that CMA had a significant positive correlation with the hypoxic signatures, whereas SIRT1, responsible for K709-depropionylation correlated negatively with them. Altogether, our results revealed a novel mechanism of HIF-1α distribution into two different degradation pathways. [BMB Reports 2023; 56(4): 252-257].
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia , Neoplasias , Humanos , Neoplasias/patologia , Complexo de Endopeptidases do Proteassoma , Hipóxia , Hipóxia CelularRESUMO
The C-terminal fragment of CABIN1 interacts with calcineurin and represses the transcriptional activity of the nuclear factor of activated T cells (NFAT). However, the specific sequences and mechanisms through which it binds to calcineurin are unclear. This study determined that decameric peptide (CABIN1 residues 2146-2155) is minimally required for binding to calcineurin. This peptide contains a unique "PPTP" C-terminal sequence and a "PxIxIT" N-terminal motif. Furthermore, p38MAPK phosphorylated the threonine residue of the "PPTP" sequence under physiological conditions, dramatically enhancing the peptide's binding affinity to calcineurin. Therefore, the CABIN1 peptide inhibited the calcineurin-NFAT pathway and the activation of T cells more efficiently than the VIVIT peptide without affecting calcineurin's phosphatase activity. The CABIN1 peptide could thus be a more potent calcineurin inhibitor and provide therapeutic opportunities for various diseases caused by the calcineurin-NFAT pathway.
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Calcineurina , Fatores de Transcrição NFATC , Calcineurina/metabolismo , Ativação Linfocitária , Fatores de Transcrição NFATC/metabolismo , Peptídeos/farmacologia , Linfócitos T/metabolismoRESUMO
The androgen receptor (AR) is an important therapeutic target for treating prostate cancer (PCa). Moreover, there is an increasing need for understanding the AR-independent progression of tumor cells such as neuroendocrine prostate cancer (NEPC). Menin, which is encoded by multiple endocrine neoplasia type 1 (MEN1), serves as a direct link between AR and the mixed-lineage leukemia (MLL) complex in PCa development by activating AR target genes through histone H3 lysine 4 methylation. Although menin is a critical component of AR signaling, its tumorigenic role in AR-independent PCa cells remains unknown. Here, we compared the role of menin in AR-positive and AR-negative PCa cells via RNAi-mediated or pharmacological inhibition of menin. We demonstrated that menin was involved in tumor cell growth and metastasis in PCa cells with low or deficient levels of AR. The inhibition of menin significantly diminished the growth of PCa cells and induced apoptosis, regardless of the presence of AR. Additionally, transcriptome analysis showed that the expression of many metastasis-associated genes was perturbed by menin inhibition in AR-negative DU145 cells. Furthermore, wound-healing assay results showed that menin promoted cell migration in AR-independent cellular contexts. Overall, these findings suggest a critical function of menin in tumorigenesis and provide a rationale for drug development against menin toward targeting high-risk metastatic PCa, especially those independent of AR.
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Neoplasias da Próstata , Receptores Androgênicos , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Masculino , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Transdução de Sinais , Fatores de TranscriçãoRESUMO
Sox2 is a core transcription factor in embryonic stem cells (ESCs), and O-GlcNAcylation is a type of post-translational modification of nuclear-cytoplasmic proteins. Although both factors play important roles in the maintenance and differentiation of ESCs and the serine 248 (S248) and threonine 258 (T258) residues of Sox2 are modified by O-GlcNAcylation, the function of Sox2 O-GlcNAcylation is unclear. Here, we show that O-GlcNAcylation of Sox2 at T258 regulates mouse ESC self-renewal and early cell fate. ESCs in which wild-type Sox2 was replaced with the Sox2 T258A mutant exhibited reduced self-renewal, whereas ESCs with the Sox2 S248A point mutation did not. ESCs with the Sox2 T258A mutation heterologously introduced using the CRISPR/Cas9 system, designated E14-Sox2TA/WT, also exhibited reduced self-renewal. RNA sequencing analysis under self-renewal conditions showed that upregulated expression of early differentiation genes, rather than a downregulated expression of self-renewal genes, was responsible for the reduced self-renewal of E14-Sox2TA/WT cells. There was a significant decrease in ectodermal tissue and a marked increase in cartilage tissue in E14-Sox2TA/WT-derived teratomas compared with normal E14 ESC-derived teratomas. RNA sequencing of teratomas revealed that genes related to brain development had generally downregulated expression in the E14-Sox2TA/WT-derived teratomas. Our findings using the Sox2 T258A mutant suggest that Sox2 T258 O-GlcNAc has a positive effect on ESC self-renewal and plays an important role in the proper development of ectodermal lineage cells. Overall, our study directly links O-GlcNAcylation and early cell fate decisions.
Assuntos
Autorrenovação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Treonina/metabolismo , Alelos , Animais , Diferenciação Celular/genética , Linhagem da Célula , Autorrenovação Celular/genética , Células Cultivadas , Imunofluorescência , Edição de Genes , Regulação da Expressão Gênica , Glicosilação , Camundongos , Mutação , Processamento de Proteína Pós-Traducional , Fatores de Transcrição SOXB1/genética , Teratoma/etiologia , Teratoma/metabolismo , Teratoma/patologiaRESUMO
2-oxoglutarate and iron-dependent oxygenase domain-containing protein 1 (OGFOD1) expression is upregulated in a variety of cancers and has been related to poor prognosis. However, despite this significance to cancer progression, the precise oncogenic mechanism of OGFOD1 is not understood. We demonstrated that OGFOD1 plays a role in enhancing the transcriptional activity of RNA polymerase II in breast cancer cells. OGFOD1 directly binds to the C-terminal domain of RNA polymerase II to alter phosphorylation status. The elimination of OGFOD1 resulted in decreased tumor development. Additionally, cell cycle-dependent kinase 7 and cell cycle-dependent kinase 9, critical enzymes for activating RNA polymerase II, phosphorylated serine 256 of OGFOD1, whereas a non-phosphorylated mutant OGFOD1 failed to enhance transcriptional activation and tumor growth. Consequently, OGFOD1 helps promote tumor growth by enhancing RNA polymerase II, whereas simultaneous phosphorylation of OGFOD1 by CDK enzymes is essential in stimulating RNA polymerase II-mediated transcription both in vitro and in vivo, and expression of target genes.
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Acetylation is the most studied histone acyl modification and has been recognized as a fundamental player in metabolic gene regulation, whereas other short-chain acyl modifications have only been recently identified, and little is known about their dynamics or molecular functions at the intersection of metabolism and epigenetic gene regulation. In this study, we aimed to understand the link between nonacetyl histone acyl modification, metabolic transcriptional regulation, and cellular adaptation. Using antibodies specific for butyrylated, propionylated, and crotonylated H3K23, we analyzed dynamic changes of H3K23 acylation upon various metabolic challenges. Here, we show that H3K23 modifications were highly responsive and reversibly regulated by nutrient availability. These modifications were commonly downregulated by the depletion of glucose and recovered based on glucose or fatty acid availability. Depletion of metabolic enzymes, namely, ATP citrate lyase, carnitine acetyltransferase, and acetyl-CoA synthetase, which are involved in Ac-CoA synthesis, resulted in global loss of H3K23 butyrylation, crotonylation, propionylation, and acetylation, with a profound impact on gene expression and cellular metabolic states. Our data indicate that Ac-CoA/CoA and central metabolic inputs are important for the maintenance of histone acylation. Additionally, genome-wide analysis revealed that acyl modifications are associated with gene activation. Our study shows that histone acylation acts as an immediate and reversible metabolic sensor enabling cellular adaptation to metabolic stress by reprogramming gene expression.
Assuntos
Adaptação Biológica , Metabolismo Energético , Histonas/metabolismo , Acetilcoenzima A/metabolismo , Acilação , Animais , Linhagem Celular , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina , Coenzima A/metabolismo , Epigênese Genética , Cromatografia Gasosa-Espectrometria de Massas , Regulação da Expressão Gênica , Glucose/metabolismo , Humanos , Metaboloma , Metabolômica/métodos , Camundongos , Estresse Fisiológico , TranscriptomaRESUMO
Octamer-binding transcription factor 4 (Oct4) plays an important role in maintaining pluripotency in embryonic stem cells and is closely related to the malignancies of various cancers. Although posttranslational modifications of Oct4 have been widely studied, most of these have not yet been fully characterized, especially in cancer. In this study, we investigated the role of phosphorylation of serine 236 of OCT4 [OCT4 (S236)] in human germ cell tumors (GCTs). OCT4 was phosphorylated at S236 in a cell cycle-dependent manner in a patient sample and GCT cell lines. The substitution of endogenous OCT4 by a mimic of phosphorylated OCT4 with a serine-to-aspartate mutation at S236 (S236D) resulted in tumor cell differentiation, growth retardation, and inhibition of tumor sphere formation. GCT cells expressing OCT4 S236D instead of endogenous OCT4 were similar to cells with OCT4 depletion at the mRNA transcript level as well as in the phenotype. OCT4 S236D also induced tumor cell differentiation and growth retardation in mouse xenograft experiments. Inhibition of protein phosphatase 1 by chemicals or short hairpin RNAs increased phosphorylation at OCT4 (S236) and resulted in the differentiation of GCTs. These results reveal the role of OCT4 (S236) phosphorylation in GCTs and suggest a new strategy for suppressing OCT4 in cancer.
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BACKGROUND: Streptomyces seoulensis has contributed to the discovery and initiation of extensive research into nickel superoxide dismutase (NiSOD), a unique type of superoxide dismutase found in actinomycetes. Still so far, there is no information about whole genome sequence of this strain. OBJECTIVE: To investigate complete genome sequence and perform bioinformatic analyses for genomic functions related with nickel-associated genes. METHODS: DNA was extracted using the Wizard Genomic DNA Purification Kit then sequenced using a Pacific Biosciences SMRT cell 8Pac V3 DNA Polymerase Binding Kit P6 with the PacBiov2 RSII platform. We assembled the PacBio long-reads with the HGAP3 pipeline. RESULTS: We obtained complete genome sequence of S. seoulensis, which comprises a 6,339,363 bp linear chromosome. While analyzing the genome to annotate the genomic function, we discovered the nickel-associated genes. We observed that the sodN gene encoding for NiSOD is located adjacent to the sodX gene, which encodes for the nickel-type superoxide dismutase maturation protease. In addition, several nickel-associated genes and gene clusters-nickel-responsive regulator, nickel uptake transporter, nickel-iron [NiFe]-hydrogenase and other putative genes were also detected. Strain specific genes were discovered through a comparative analysis of S. coelicolor and S. griseus. Further bioinformatic analyses revealed that this strain encodes at least 22 putative biosynthetic gene clusters, thereby implying that S. seoulensis has the potential to produce novel bioactive compounds. CONCLUSION: We annotated the genome and determined nickel-associated genes and gene clusters and discovered biosynthetic gene clusters for secondary metabolites implying that S. seoulensis produces novel types of bioactive compounds.
Assuntos
Genoma Bacteriano , Streptomyces/genética , Superóxido Dismutase/genética , Genômica , Família Multigênica , Níquel/metabolismo , Sequenciamento Completo do GenomaRESUMO
Constitutive heterochromatin undergoes a dynamic clustering and spatial reorganization during myogenic differentiation. However the detailed mechanisms and its role in cell differentiation remain largely elusive. Here, we report the identification of a muscle-specific long non-coding RNA, ChRO1, involved in constitutive heterochromatin reorganization. ChRO1 is induced during terminal differentiation of myoblasts, and is specifically localized to the chromocenters in myotubes. ChRO1 is required for efficient cell differentiation, with global impacts on gene expression. It influences DNA methylation and chromatin compaction at peri/centromeric regions. Inhibition of ChRO1 leads to defects in the spatial fusion of chromocenters, and mislocalization of H4K20 trimethylation, Suv420H2, HP1, MeCP2 and cohesin. In particular, ChRO1 specifically associates with ATRX/DAXX/H3.3 complex at chromocenters to promote H3.3 incorporation and transcriptional induction of satellite repeats, which is essential for chromocenter clustering. Thus, our results unveil a mechanism involving a lncRNA that plays a role in large-scale heterochromatin reorganization and cell differentiation.
Assuntos
Proteínas de Transporte/genética , Heterocromatina/química , Histonas/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Desenvolvimento Muscular/genética , Proteínas Nucleares/genética , RNA Longo não Codificante/genética , Proteína Nuclear Ligada ao X/genética , Animais , Sistemas CRISPR-Cas , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Correpressoras , Feminino , Edição de Genes , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Masculino , Proteína 2 de Ligação a Metil-CpG/genética , Proteína 2 de Ligação a Metil-CpG/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Chaperonas Moleculares , Músculo Esquelético/citologia , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/metabolismo , Células NIH 3T3 , Proteínas Nucleares/metabolismo , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transcrição Gênica , Proteína Nuclear Ligada ao X/metabolismo , CoesinasRESUMO
Germ cell tumours (GCTs) are one of the most threatening malignancies in young men and women. Although several reports have suggested the importance of OCT4 in human GCTs, its role has not been clearly investigated on a molecular level. In this study, we revealed GCT-specific direct transcriptional target genes of OCT4. Conditional knockdown of OCT4 in GCT cell lines reduced cell proliferation by affecting both cell cycle and death. Knockdown of OCT4 also reduced stemness of GCTs, as assessed by the expression of other stemness factors, alkaline phosphatase staining, and tumour sphere formation ability. Analysis of whole mRNA expression patterns among GCT cells harbouring endogenous, depleted, and rescued OCT4 revealed 1133 OCT4 target genes in GCT. Combined analysis of both the chromatin binding signature of OCT4 and the genes whose expression levels were changed by OCT4 revealed 258 direct target genes of OCT4 in GCTs. In a similar way, 594 direct target genes in normal embryonic stem cells (ESCs) were identified. Among these two sets of OCT4 direct target genes, 38 genes were common between GCTs and ESCs, most of which were related to regulation of pluripotency, and 220 genes were specific to GCTs, most of which were related to focal adhesion and extracellular matrix organisation. These results provide a molecular basis for how OCT4 regulates GCT stemness and will aid our understanding of the role of OCT4 in other cancers.
Assuntos
Matriz Extracelular/genética , Neoplasias Embrionárias de Células Germinativas/genética , Neoplasias Embrionárias de Células Germinativas/patologia , Células-Tronco Neoplásicas/patologia , Fator 3 de Transcrição de Octâmero/metabolismo , Fosfatase Alcalina/análise , Fosfatase Alcalina/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Doxiciclina/farmacologia , Citometria de Fluxo , Redes Reguladoras de Genes/genética , Humanos , Células-Tronco Neoplásicas/metabolismo , Fator 3 de Transcrição de Octâmero/antagonistas & inibidores , Fator 3 de Transcrição de Octâmero/genética , Transcrição Gênica/genéticaRESUMO
Cyclin-dependent kinase 1 (Cdk1) is indispensable for embryonic stem cell (ESC) maintenance and embryo development. Even though some reports have described a connection between Cdk1 and Oct4, there is no evidence that Cdk1 activity is directly linked to the ESC pluripotency transcription program. We recently reported that Aurkb/PP1-mediated Oct4 resetting is important to cell cycle maintenance and pluripotency in mouse ESCs (mESCs). In this study, we show that Cdk1 is an upstream regulator of the Oct4 phosphorylation state during cell cycle progression, and it coordinates the chromatin associated state of Oct4 for pluripotency-related gene expression within the cell cycle. Upon entry into mitosis, Aurkb in the chromosome passenger complex becomes fully activated and PP1 activity is inhibited downstream of Cdk1 activation, leading to sustaining Oct4(S229) phosphorylation and dissociation of Oct4 from chromatin during the mitotic phase. Cdk1 inhibition at the mitotic phase abnormally results in Oct4 dephosphorylation, chromosome decondensation and chromatin association of Oct4, even in replicated chromosome. Our study results suggest a molecular mechanism by which Cdk1 directly links the cell cycle to the pluripotency transcription program in mESCs.
Assuntos
Proteína Quinase CDC2/metabolismo , Ciclo Celular/genética , Cromatina/metabolismo , Células-Tronco Embrionárias/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Transcrição Gênica , Animais , Aurora Quinase B/metabolismo , Proteína Quinase CDC2/antagonistas & inibidores , Divisão Celular/genética , Células Cultivadas , Fase G2/genética , Humanos , Camundongos , Fosforilação , Proteína Fosfatase 1/metabolismoRESUMO
Unlike lower organisms, mammals have 2 C-terminal binding protein (Ctbp) isoforms, Ctbp1 and Ctbp2. Ctbp2 is revealed as a key factor involved in determining cell fate decisions by regulating the epigenetic state in active embryonic stem cell (ESC) genes. However, the molecular mechanism underlying how Ctbp1 and Ctbp2 have different roles remains elusive. Here we demonstrate that Ctbp isoform abundance is important for mouse embryonic ESCs (mESCs) to exit from pluripotency. Temporal expression patterns of Ctbp isoforms were quite different; Ctbp2 is more highly expressed in mESCs and decreases during differentiation, while Ctbp1 is constantly expressed at a lower level. Ctbp2 knockdown, but not Ctbp1 knockdown, in mESCs resulted in impaired exit from pluripotency. Interestingly, Ctbp1 and Ctbp2 overexpression in Ctbp2-knockdown mESCs leads to exiting from pluripotency in a manner similar to that of wild-type mESCs. Quantification of Ctbp1 and Ctbp2 revealed that differentiation ability correlates with abundance of Ctbp isoform in undifferentiated mESCs, suggesting that a sufficient amount of Ctbp isoform is a prerequisite for exiting from pluripotency. The results support the contention that 2 redundant Ctbp isoforms regulate elaborate differentiation via temporally distinctive regulatory patterns in mESCs.-Suh, M. Y., Kim, T. W., Lee, H.-T., Shin, J., Kim, J.-H., Jang, H., Kim, H. J., Kim, S.-T., Cho, E.-J., Youn, H.-D. Abundance of C-terminal binding protein isoform is a prerequisite for exit from pluripotency in mouse embryonic stem cells.
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Transcription factors and chromatin remodeling proteins control the transcriptional variability for ESC lineage commitment. During ESC differentiation, chromatin modifiers are recruited to the regulatory regions by transcription factors, thereby activating the lineage-specific genes or silencing the transcription of active ESC genes. However, the underlying mechanisms that link transcription factors to exit from pluripotency are yet to be identified. In this study, we show that the Ctbp2-interacting zinc finger proteins, Zfp217 and Zfp516, function as linkers for the chromatin regulators during ESC differentiation. CRISPR-Cas9-mediated knock-outs of both Zfp217 and Zfp516 in ESCs prevent the exit from pluripotency. Both zinc finger proteins regulate the Ctbp2-mediated recruitment of the NuRD complex and polycomb repressive complex 2 (PRC2) to active ESC genes, subsequently switching the H3K27ac to H3K27me3 during ESC differentiation for active gene silencing. We therefore suggest that some zinc finger proteins orchestrate to control the concise epigenetic states on active ESC genes during differentiation, resulting in natural lineage commitment.
Assuntos
Diferenciação Celular/genética , Células-Tronco Embrionárias/metabolismo , Inativação Gênica , Transativadores/fisiologia , Oxirredutases do Álcool/metabolismo , Animais , Células Cultivadas , Proteínas Correpressoras , Células-Tronco Embrionárias/citologia , Humanos , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Transcrição GênicaRESUMO
The canonical Wnt pathway is critical for embryonic stem cell (ESC) pluripotency and aberrant control of ß-catenin leads to failure of exit from pluripotency and lineage commitments. Hence, maintaining the appropriate level of ß-catenin is important for the decision to commit to the appropriate lineage. However, how ß-catenin links to core transcription factors in ESCs remains elusive. C-terminal-binding protein (CtBP) in Drosophila is essential for Wnt-mediated target gene expression. In addition, Ctbp acts as an antagonist of ß-catenin/TCF activation in mammals. Recently, Ctbp2, a core Oct4-binding protein in ESCs, has been reported to play a key role in ESC pluripotency. However, the significance of the connection between Ctbp2 and ß-catenin with regard to ESC pluripotency remains elusive. Here, we demonstrate that C-terminal-binding protein 2 (Ctbp2) associates with major components of the ß-catenin destruction complex and limits the accessibility of ß-catenin to core transcription factors in undifferentiated ESCs. Ctbp2 knockdown leads to stabilization of ß-catenin, which then interacts with core pluripotency-maintaining factors that are occupied by Ctbp2, leading to incomplete exit from pluripotency. These findings suggest a suppressive function for Ctbp2 in reducing the protein level of ß-catenin, along with priming its position on core pluripotency genes to hinder ß-catenin deposition, which is central to commitment to the appropriate lineage.
Assuntos
Autorrenovação Celular/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , beta Catenina/metabolismo , Oxirredutases do Álcool , Animais , Sítios de Ligação , Linhagem Celular , Proteínas Correpressoras , Células-Tronco Embrionárias , Expressão Gênica , Técnicas de Silenciamento de Genes , Genes Reporter , Camundongos , Modelos Biológicos , Motivos de Nucleotídeos , Ligação Proteica , Estabilidade Proteica , RNA Interferente Pequeno/genéticaRESUMO
The C-terminal domain of RNA polymerase II is an unusual series of repeated residues appended to the C-terminus of the largest subunit and serves as a flexible binding scaffold for numerous nuclear factors. The binding of these factors is determined by the phosphorylation patterns on the repeats in the domain. In this study, we generated a synthetic antibody library by replacing the third heavy chain complementarity-determining region of an anti-HER2 (human epidermal growth factor receptor 2) antibody (trastuzumab) with artificial sequences of 7-18 amino-acid residues. From this library, antibodies were selected that were specific to serine phosphopeptides that represent typical phosphorylation patterns on the functional unit (YSPTSPS)2 of the RNA polymerase II C-terminal domain (CTD). Antibody clones pCTD-1stS2 and pCTD-2ndS2 showed specificity for peptides with phosphoserine at the second residues of the first or second heptamer repeat, respectively. Additional clones specifically reacted to peptides with phosphoserine at the fifth serine of the first repeat (pCTD-1stS5), the seventh residue of the first repeat and fifth residue of the second repeat (pCTD-S7S5) or the seventh residue of either the first or second repeat (pCTD-S7). All of these antibody clones successfully reacted to RNA polymerase II in immunoblot analysis. Interestingly, pCTD-2ndS2 precipitated predominately RNA polymerase II from the exonic regions of genes in genome-wide chromatin immunoprecipitation sequencing analysis, which suggests that the phosphoserine at the second residue of the second repeat of the functional unit (YSPTSPS)2 is a mediator of exon definition.
Assuntos
Anticorpos/metabolismo , Imunoprecipitação da Cromatina/métodos , Éxons , RNA Polimerase II/metabolismo , Anticorpos/imunologia , Células HEK293 , Células HeLa , Humanos , Fosforilação , Ligação Proteica , RNA Polimerase II/imunologiaRESUMO
In embryonic stem cells (ESCs), cell cycle regulation is deeply connected to pluripotency. Especially, core transcription factors (CTFs) which are essential to maintaining the pluripotency transcription programs should be reset during M/G1 transition. However, it remains unknown about how CTFs are governed during cell cycle progression. Here, we describe that the regulation of Oct4 by Aurora kinase b (Aurkb)/protein phosphatase 1 (PP1) axis during the cell cycle is important for resetting Oct4 to pluripotency and cell cycle related target genes in determining the identity of ESCs. Aurkb starts to phosphorylate Oct4(S229) at the onset of G2/M phase, inducing the dissociation of Oct4 from chromatin, whereas PP1 binds Oct4 and dephosphorylates Oct4(S229) during M/G1 transition, which resets Oct4-driven transcription for pluripotency and the cell cycle. Furthermore, Aurkb phosphormimetic and PP1 binding-deficient mutations in Oct4 disrupt the pluripotent cell cycle, lead to the loss of pluripotency in ESCs, and decrease the efficiency of somatic cell reprogramming. Based on our findings, we suggest that the cell cycle is directly linked to pluripotency programs in ESCs. [BMB Reports 2016; 49(10): 527-528].
Assuntos
Aurora Quinase B/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Proteína Fosfatase 1/metabolismo , Animais , Reprogramação Celular , Cromatina/metabolismo , Pontos de Checagem da Fase G1 do Ciclo Celular , Pontos de Checagem da Fase M do Ciclo Celular , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Mutação , Fator 3 de Transcrição de Octâmero/genética , Fosforilação , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismoRESUMO
Replication-independent incorporation of variant histone H3.3 has a profound impact on chromatin function and numerous cellular processes, including the differentiation of muscle cells. The histone chaperone HIRA and H3.3 have essential roles in MyoD regulation during myoblast differentiation. However, the precise mechanism that determines the onset of H3.3 deposition in response to differentiation signals is unclear. Here we show that HIRA is phosphorylated by Akt kinase, an important signaling modulator in muscle cells. By generating a phosphospecific antibody, we found that a significant amount of HIRA was phosphorylated in myoblasts. The phosphorylation level of HIRA and the occupancy of phosphorylated protein on muscle genes gradually decreased during cellular differentiation. Remarkably, the forced expression of the phosphomimic form of HIRA resulted in reduced H3.3 deposition and suppressed the activation of muscle genes in myotubes. Our data show that HIRA phosphorylation limits the expression of myogenic genes, while the dephosphorylation of HIRA is required for proficient H3.3 deposition and gene activation, demonstrating that the phosphorylation switch is exploited to modulate HIRA/H3.3-mediated muscle gene regulation during myogenesis.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Chaperonas de Histonas/metabolismo , Desenvolvimento Muscular , Mioblastos/citologia , Fatores de Transcrição/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Humanos , Camundongos , Mioblastos/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ativação TranscricionalRESUMO
Embryonic stem cells (ESCs) undergo coordinated epigenetic and metabolic changes to differentiate properly. However, the precise mechanisms by which these alterations are fine-tuned in the early stages of differentiation have not been identified. In this study, we demonstrate that phosphoserine aminotransferase 1 (Psat1), an Oct4/Sox2/Nanog (OSN) target protein, regulates changes in α-ketoglutarate (α-KG), determining the fate of mouse ESCs (mESCs). Maintaining Psat1 levels was essential for mESC self-renewal and pluripotency. Moderate knockdown (KD) of Psat1 in mESCs lowered DNA 5'-hydroxymethylcytosine (5'-hmC) and increased histone methylation levels by downregulating permissive amounts of α-KG, ultimately accelerating differentiation. We found that intracellular α-KG declined transiently during differentiation and that its dysregulation by treatment with dimethyl-α-KG impeded differentiation. Further, by in vivo teratoma formation assay, pluripotency of Psat1 KD mESCs was impaired, especially into the ectodermal lineage. Thus, we have established how Psat1 is regulated in maintaining intracellular α-KG levels and determining the fate of mESCs.
Assuntos
Diferenciação Celular , Ácidos Cetoglutáricos/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Transaminases/metabolismo , Animais , Metilação de DNA , Histonas/metabolismo , Espaço Intracelular/metabolismo , Camundongos , Fatores de Tempo , Fatores de Transcrição/metabolismoRESUMO
Since the molecular mechanism of hypoxic adaptation in cancer cells is cell-type specific, we investigated whether glycogen synthase kinase-3ß (GSK-3ß) activation is involved in hypoxia-induced gastric tumor promotion. Stable gastric cancer cell lines (SNU-638, SNU-484, MKN1, and MKN45) were cultured under hypoxic conditions. Cells overexpressing wild-type GSK-3ß (WT-GSK-3ß) or kinase-dead mutant of GSK-3ß (KD-GSK-3ß) were generated and used for cell culture and animal studies. In cell culture experiments, hypoxia decreased GSK-3ß activation in gastric cancer cells. Cell viability and the expressions of HIF-1α protein and VEGF mRNA in gastric cancer cells were higher in KD-GSK-3ß transfectants than in WT-GSK-3ß transfectants under hypoxic conditions, but not under normoxic conditions. Gastric cancer xenografts showed that tumor growth, microvessel area, HIF-1α activation, and VEGF expression were higher in KD-GSK-3ß tumors than in WT-GSK-3ß tumors in vivo. In addition, the expression of hypoxia-induced HIF-1α protein was regulated by GSK-3ß at the translational level. Our data suggest that GSK-3ß is involved in hypoxic adaptation of gastric cancer cells as an inhibitory upstream regulator of the HIF-1α/VEGF signaling pathway.