RESUMO
Yin Yang 1 (YY1) is a multifunctional transcription factor that can activate or repress transcription depending on the promotor and/or the co-factors recruited. YY1 is phosphorylated in various signaling pathways and is critical for different biological functions including embryogenesis, apoptosis, proliferation, cell-cycle regulation and tumorigenesis. Here we report that YY1 is a substrate for c-Abl kinase phosphorylation at conserved residue Y254 in the spacer region. Pharmacological inhibition of c-Abl kinase by imatinib, nilotinib and GZD824, knock-down of c-Abl using siRNA, and the use of c-Abl kinase-dead drastically reduces tyrosine phosphorylation of YY1. Both radioactive and non-radioactive in vitro kinase assays, as well as co-immunoprecipitation in different cell lines, show that the target of c-Abl phosphorylation is tyrosine residue 254. c-Abl phosphorylation has little effect on YY1 DNA binding ability or cellular localization in asynchronous cells. However, functional studies reveal that c-Abl mediated phosphorylation of YY1 regulates YY1's transcriptional ability in vivo. In conclusion, we demonstrate the novel role of c-Abl kinase in regulation of YY1's transcriptional activity, linking YY1 regulation with c-Abl tyrosine kinase signaling pathways.
Assuntos
Proteínas Oncogênicas v-abl/metabolismo , Transcrição Gênica , Fator de Transcrição YY1/química , Fator de Transcrição YY1/metabolismo , Benzamidas/farmacologia , Sequência Conservada , Técnicas de Inativação de Genes , Inativação Gênica , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Mesilato de Imatinib/farmacologia , Células MCF-7 , Proteínas Oncogênicas v-abl/genética , Fosforilação , Pirazóis/farmacologia , Pirimidinas/farmacologia , Tirosina/químicaRESUMO
The multifunctional protein Yin Yang 1 (YY1) plays critical roles in tumorigenesis. YY1 has been shown to be involved in the development, progression, resistance, and invasiveness of many types of cancers. Today, the value of YY1 as a prognostic marker and as a potential target in cancer therapy is being explored by multiple research groups around the world. Over the past 25 years, we have accumulated a wealth of information about the wide-ranging biological functions of YY1 at the molecular, cellular, and organismal levels. However, our knowledge of how YY1 is regulated and what regulates it has lagged behind. In the past few years, there has been a significant increase in the research addressing this issue. In this review, we summarize and analyze recent findings about the regulation of YY1 at multiple levels. We emphasize the necessity for deeper insights into these regulatory mechanisms if YY1 is to find its way to the clinical setting.
Assuntos
Carcinogênese/genética , Resistencia a Medicamentos Antineoplásicos , Neoplasias/genética , Fator de Transcrição YY1/genética , Progressão da Doença , Humanos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Neoplasias/patologiaRESUMO
Yin Yang 1 (YY1) is a ubiquitously expressed and highly conserved multifunctional transcription factor that is involved in a variety of cellular processes. Many YY1-regulated genes have crucial roles in cell proliferation, differentiation, apoptosis, and cell cycle regulation. Numerous mechanisms have been shown to regulate the function of YY1, such as DNA binding affinity, subcellular localization, and posttranslational modification including phosphorylation. Polo-like kinase 1(Plk1) and Casein kinase 2α (CK2 α) were the first two kinases identified to phosphorylate YY1. In this study, we identify a third kinase. We report that YY1 is a novel substrate of the Aurora B kinase both in vitro and in vivo. Serine 184 phosphorylation of YY1 by Aurora B is cell cycle regulated and peaks at G2/M and is rapidly dephosphorylated, likely by protein phosphatase 1 (PP1) as the cells enter G1. Aurora A and Aurora C can also phosphorylate YY1 in vitro, but at serine/threonine residues other than serine 184. We present evidence that phosphorylation of YY1 in the central glycine/alanine (G/A)-rich region is important for DNA binding activity, with a potential phosphorylation/acetylation interplay regulating YY1 function. Given their importance in mitosis and overexpression in human cancers, Aurora kinases have been identified as promising therapeutic targets. Increasing our understanding of Aurora substrates will add to the understanding of their signaling pathways.
Assuntos
Divisão Celular , Fase G2 , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Transcrição YY1/metabolismo , Acetilação , Sequência de Aminoácidos , Animais , Aurora Quinase A , Aurora Quinase B , Aurora Quinase C , Aurora Quinases , DNA/metabolismo , Regulação Enzimológica da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Camundongos , Mitose , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Serina/metabolismo , Transcrição Gênica , Fator de Transcrição YY1/químicaRESUMO
In this report, we describe the phosphorylation of Yin Yang 1 (YY1) in vitro and in vivo by CK2α (casein kinase II), a multifunctional serine/threonine protein kinase. YY1 is a ubiquitously expressed multifunctional zinc finger transcription factor implicated in regulation of many cellular and viral genes. The products of these genes are associated with cell growth, the cell cycle, development, and differentiation. Numerous studies have linked YY1 to tumorigenesis and apoptosis. YY1 is a target for cleavage by caspases in vitro and in vivo as well, but very little is known about the mechanisms that regulate its cleavage during apoptosis. Here, we identify serine 118 in the transactivation domain of YY1 as the site of CK2α phosphorylation, proximal to a caspase 7 cleavage site. CK2α inhibitors, as well as knockdown of CK2α by small interfering RNA, reduce S118 phosphorylation in vivo and enhance YY1 cleavage under apoptotic conditions, whereas increased CK2α activity by overexpression in vivo elevates S118 phosphorylation. A serine-to-alanine substitution at serine 118 also increases the cleavage of YY1 during apoptosis compared to wild-type YY1. Taken together, we have discovered a regulatory link between YY1 phosphorylation at serine 118 and regulation of its cleavage during programmed cell death.
Assuntos
Caspase 7/metabolismo , Fator de Transcrição YY1/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Apoptose/fisiologia , Sequência de Bases , Sítios de Ligação , Caseína Quinase II/metabolismo , Primers do DNA/genética , Células HEK293 , Células HeLa , Humanos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/química , Fator de Transcrição YY1/química , Fator de Transcrição YY1/genéticaRESUMO
DXZ4 is an X-linked macrosatellite composed of 12-100 tandemly arranged 3-kb repeat units. In females, it adopts opposite chromatin arrangements at the two alleles in response to X-chromosome inactivation. In males and on the active X chromosome, it is packaged into heterochromatin, but on the inactive X chromosome (Xi), it adopts a euchromatic conformation bound by CTCF. Here we report that the ubiquitous transcription factor YY1 associates with the euchromatic form of DXZ4 on the Xi. The binding of YY1 close to CTCF is reminiscent of that at other epigenetically regulated sequences, including sites of genomic imprinting, and at the X-inactivation centre, suggesting a common mode of action in this arrangement. As with CTCF, binding of YY1 to DXZ4 in vitro is not blocked by CpG methylation, yet in vivo both proteins are restricted to the hypomethylated form. In several male carcinoma cell lines, DXZ4 can adopt a Xi-like conformation in response to cellular transformation, characterized by CpG hypomethylation and binding of YY1 and CTCF. Analysis of a male melanoma cell line and normal skin cells from the same individual confirmed that a transition in chromatin state occurred in response to transformation.
Assuntos
Carcinoma/genética , Cromossomos Humanos X/metabolismo , Proteínas Repressoras/metabolismo , Sequências de Repetição em Tandem , Fator de Transcrição YY1/metabolismo , Sequência de Bases , Fator de Ligação a CCCTC , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Células Cultivadas , Cromatina/metabolismo , Cromossomos Humanos X/química , Sequência Consenso , Ilhas de CpG , Metilação de DNA , Feminino , Histonas/metabolismo , Humanos , Masculino , Fator de Transcrição YY1/análiseRESUMO
Cessation of transcriptional activity is a hallmark of cell division. Many biochemical pathways have been shown and proposed over the past few decades to explain the silence of this phase. In particular, many individual transcription factors have been shown to be inactivated by phosphorylation. In this report, we show the simultaneous phosphorylation and mitotic redistribution of a whole class of modified transcription factors. C(2)H(2) zinc finger proteins (ZFPs) represent the largest group of gene expression regulators in the human genome. Despite their diversity, C(2)H(2) ZFPs display striking conservation of small linker peptides joining their adjacent zinc finger modules. These linkers are critical for DNA binding activity. It has been proposed that conserved phosphorylation of these linker peptides could be a common mechanism for the inactivation of the DNA binding activity of C(2)H(2) ZFPs, during mitosis. Using a novel antibody, raised against the phosphorylated form of the most conserved linker peptide sequence, we are able to visualize the massive and simultaneous mitotic phosphorylation of hundreds of these proteins. We show that this wave of phosphorylation is tightly synchronized, starting in mid-prophase right after DNA condensation and before the breakdown of the nuclear envelope. This global phosphorylation is completely reversed in telophase. In addition, the exclusion of the phospho-linker signal from condensed DNA clearly demonstrates a common mechanism for the mitotic inactivation of C(2)H(2) ZFPs.
Assuntos
Proteínas de Transporte/metabolismo , Mitose , Proteínas Nucleares/metabolismo , Sequência de Aminoácidos , Anticorpos/imunologia , Linhagem Celular Tumoral , DNA/metabolismo , Humanos , Peptídeos/química , Peptídeos/imunologia , Peptídeos/farmacologia , Fosforilação , Proteínas Repressoras , Telófase , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/imunologia , Fator de Transcrição YY1/metabolismoRESUMO
Yin-Yang 1 (YY1) is an essential multifunctional zinc-finger protein. It has been shown over the past two decades to be a critical regulator of a vast array of biological processes, including development, cell proliferation and differentiation, DNA repair, and apoptosis. YY1 exerts its functions primarily as a transcription factor that can activate or repress gene expression, dependent on its spatial and temporal context. YY1 regulates a large number of genes involved in cell cycle transitions, many of which are oncogenes and tumor-suppressor genes. YY1 itself has been classified as an oncogene and was found to be upregulated in many cancer types. Unfortunately, our knowledge of what regulates YY1 is very minimal. Although YY1 has been shown to be a phosphoprotein, no kinase has ever been identified for the phosphorylation of YY1. Polo-like kinase 1 (Plk1) has emerged in the past few years as a major cell cycle regulator, particularly for cell division. Plk1 has been shown to play important roles in the G/M transition into mitosis and for the proper execution of cytokinesis, processes that YY1 has been shown to regulate also. Here, we present evidence that Plk1 directly phosphorylates YY1 in vitro and in vivo at threonine 39 in the activation domain. We show that this phosphorylation is cell cycle regulated and peaks at G2/M. This is the first report identifying a kinase for which YY1 is a substrate.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Fase G2 , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fator de Transcrição YY1/metabolismo , Células HeLa , Humanos , Fosforilação , Especificidade por Substrato , Treonina/metabolismo , Fatores de Transcrição/metabolismo , Quinase 1 Polo-LikeRESUMO
Yin-Yang 1 (YY1) is a ubiquitously expressed zinc finger transcription factor. It regulates a vast array of genes playing critical roles in development, differentiation, and cell cycle. Very little is known about the mechanisms that regulate the functions of YY1. It has long been proposed that YY1 is a phosphoprotein; however, a direct link between phosphorylation and the function of YY1 has never been proven. Investigation of the localization of YY1 during mitosis shows that it is distributed to the cytoplasm during prophase and remains excluded from DNA until early telophase. Immunostaining studies show that YY1 is distributed equally between daughter cells and rapidly associates with decondensing chromosomes in telophase, suggesting a role for YY1 in early marking of active and repressed genes. The exclusion of YY1 from DNA in prometaphase HeLa cells correlated with an increase in the phosphorylation of YY1 and loss of DNA-binding activity that can be reversed by dephosphorylation. We have mapped three phosphorylation sites on YY1 during mitosis and show that phosphorylation of two of these sites can abolish the DNA-binding activity of YY1. These results demonstrate a novel mechanism for the inactivation of YY1 through phosphorylation of its DNA-binding domain.
Assuntos
DNA/metabolismo , Mitose/fisiologia , Fator de Transcrição YY1/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , DNA/genética , Regulação da Expressão Gênica , Células HeLa/efeitos dos fármacos , Células HeLa/fisiologia , Humanos , Mitose/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Nocodazol/farmacologia , Fosforilação , Serina/metabolismo , Treonina/metabolismo , Moduladores de Tubulina/farmacologia , Fator de Transcrição YY1/genéticaRESUMO
The transcriptional regulator Yin Yang 1 (YY1) controls many aspects of cell behavior and is essential for development. We analyzed the fate of YY1 during apoptosis and studied the functional consequences. We observed that this factor is rapidly translocated into the cell nucleus in response to various apoptotic stimuli, including activation of Fas, stimulation by tumor necrosis factor, and staurosporine and etoposide treatment. Furthermore, YY1 is cleaved by caspases in vitro and in vivo at two distinct sites, IATD(12)G and DDSD(119)G, resulting in the deletion of the first 119 amino acids early in the apoptotic process. This activity generates an N-terminally truncated YY1 fragment (YY1Delta119) that has lost its transactivation domain but retains its DNA binding domain. Indeed, YY1Delta119 is no longer able to stimulate gene transcription but interacts with DNA. YY1Delta119 but not the wild-type protein or the caspase-resistant mutant YY1D12A/D119A enhances Fas-induced apoptosis, suggesting that YY1 is involved in a positive feedback loop during apoptosis. Our findings provide evidence for a new mode of regulation of YY1 and define a novel aspect of the involvement of YY1 in the apoptotic process.
Assuntos
Apoptose/fisiologia , Caspases/fisiologia , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Diferenciação Celular , Linhagem Celular , Núcleo Celular/química , Núcleo Celular/fisiologia , Proliferação de Células , Proteínas de Ligação a DNA/análise , Fatores de Ligação de DNA Eritroide Específicos , Humanos , Estrutura Terciária de Proteína , Fatores de Transcrição/análise , Transcrição Gênica , Fator de Transcrição YY1RESUMO
The essential Yin Yang-1 gene (YY1) encodes a ubiquitous, conserved, multifunctional zinc-finger transcription factor in animals. The YY1 protein regulates initiation, activation, or repression of transcription from a variety of genes required for cell growth, development, differentiation, or tumor suppression, as well as from genes in some retroviruses and DNA viruses. Among the specific functions attributed to YY1 is a role in cell-cycle-specific upregulation of the replication-dependent histone genes. The YY1 protein binds to the histone alpha element, a regulatory sequence found in all replication-dependent histone genes. We therefore examined the abundance, DNA-binding activity and localization of the YY1 protein throughout the cell cycle in unperturbed, shake-off-synchronized Chinese hamster ovary and HeLa cells. We found that, whereas the DNA-binding activity of YY1 increased dramatically early in S phase, the YY1 mRNA and protein levels did not. YY1 changed subcellular distribution patterns during the cell cycle, from mainly cytoplasmic at G1 to mainly nuclear at early and middle S phase, then back to primarily cytoplasmic later in S phase. Nuclear accumulation of YY1 near the G1/S boundary coincided with both an increase in YY1 DNA-binding activity and the coordinate up-regulation of the replication-dependent histone genes. The DNA synthesis inhibitor aphidicolin caused a nearly complete loss of nuclear YY1, whereas addition of caffeine or 2-aminopurine to aphidicolin-treated cells restored both DNA synthesis and YY1 localization in the nucleus. These findings reveal a mechanism by which YY1 localization is coupled to DNA synthesis and responsive to cell-cycle signaling pathways. Taken together, our results provide insight into how YY1 might participate in the cell-cycle control over a variety of nuclear events required for cell division and proliferation.