RESUMO
Enhancers activate their cognate promoters over huge distances but how enhancer/promoter interactions become established is not completely understood. There is strong evidence that cohesin-mediated loop extrusion is involved but this does not appear to be a universal mechanism. Here, we identify an element within the mouse immunoglobulin lambda (Igλ) light chain locus, HSCλ1, that has characteristics of active regulatory elements but lacks intrinsic enhancer or promoter activity. Remarkably, knock-out of the YY1 binding site from HSCλ1 reduces Igλ transcription significantly and disrupts enhancer/promoter interactions, even though these elements are >10 kb from HSCλ1. Genome-wide analyses of mouse embryonic stem cells identified 2671 similar YY1-bound, putative genome organizing elements that lie within CTCF/cohesin loop boundaries but that lack intrinsic enhancer activity. We suggest that such elements play a fundamental role in locus folding and in facilitating enhancer/promoter interactions.
Assuntos
Regiões Promotoras Genéticas , Ativação Transcricional , Fator de Transcrição YY1 , Animais , Camundongos , Sítios de Ligação/genética , Cromatina/genética , Elementos Facilitadores Genéticos/genética , Estudo de Associação Genômica Ampla , Regiões Promotoras Genéticas/genética , Fator de Transcrição YY1/química , Fator de Transcrição YY1/genética , Células-Tronco EmbrionáriasRESUMO
Yin and Yang 1 gene (YY1; MIM#600,013) is recognized as a dual transcriptional activating and repressing factor, RNA-binding protein, and 3D chromatin regulator, with multi roles in neurodevelopmental and maintenance pathways. YY1 haploinsufficiency caused either by heterozygous sequence variants or deletions involving the whole gene has been recently associated with Gabriele-de Vries syndrome (GADEVS), a rare congenital autosomal dominant condition, leading to intellectual disability (ID) and multiple physical/behavioural abnormalities. Herein, we describe clinical and molecular findings from a Brazilian female harbouring a de novo missense pathogenic variant in YY1 gene (NM_003403.5:c.1106A > G; p.Asn369Ser) found by whole exome sequencing with potential implications for protein structure and function. Undescribed or uncommon clinical features in this patient included non-febrile seizures, severe scoliosis, hearing impairment, and chorioretinitis. Further bioinformatics analyses using YY1-other protein interaction networks reinforced the involvement of YY1 interactors in such phenotypes, in exception of chorioretinitis. Moreover, X-chromosome inactivation (XCI) skewing was evidenced in the patient and attributed to the haploinsufficiency of YY1, which direct and indirectly interacts with numerous XCI key regulators. Besides expanding the mutational and phenotype spectrum of GADEVS, our results highlight the role of YY1 as an essential autosomal regulator of XCI epigenetic process.
Assuntos
Coriorretinite , Deficiência Intelectual , Feminino , Humanos , Fenótipo , Mutação de Sentido Incorreto , Deficiência Intelectual/genética , Síndrome , Cromatina , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/química , Fator de Transcrição YY1/metabolismoRESUMO
As an oncogenic transcription factor, Yin Yang 1 (YY1) regulates enhancer and promoter connection. However, gaps still exist in understanding how YY1 coordinates coactivators and chromatin enhancer elements to assemble enhancers and super-enhancers. Here, we demonstrate that a histidine cluster in YY1's transactivation domain is essential for its formation of phase separation condensates, which can be extended to additional proteins. The histidine cluster is also required for YY1-promoted cell proliferation, migration, clonogenicity and tumor growth. YY1-rich nuclear puncta contain coactivators EP300, BRD4, MED1 and active RNA polymerase II, and colocalize with histone markers of gene activation, but not that of repression. Furthermore, YY1 binds to the consensus motifs in the FOXM1 promoter to activate its expression. Wild-type YY1, but not its phase separation defective mutant, connects multiple enhancer elements and the FOXM1 promoter to form an enhancer cluster. Consistently, fluorescent in situ hybridization (FISH) assays reveal the colocalization of YY1 puncta with both the FOXM1 gene locus and its nascent RNA transcript. Overall, this study demonstrates that YY1 activates target gene expression through forming liquid-liquid phase separation condensates to compartmentalize both coactivators and enhancer elements, and the histidine cluster of YY1 plays a determinant role in this regulatory mechanism.
Assuntos
Cromatina , Elementos Facilitadores Genéticos , Fator de Transcrição YY1 , Regulação da Expressão Gênica , Histidina/química , Hibridização in Situ Fluorescente , Proteínas Nucleares/metabolismo , Fator de Transcrição YY1/química , Fator de Transcrição YY1/metabolismoRESUMO
RATIONALE: Disturbed flow occurring in arterial branches and curvatures induces vascular endothelial cell (EC) dysfunction and atherosclerosis. We postulated that disturbed flow plays important role in modulating phosphoprotein expression profiles to regulate endothelial functions and atherogenesis. OBJECTIVE: The goal of this study is to discover novel site-specific phosphorylation alterations induced by disturbed flow in ECs to contribute to atherosclerosis. METHODS AND RESULTS: Quantitative phosphoproteomics analysis of ECs exposed to disturbed flow with low and oscillatory shear stress (0.5±4 dynes/cm2) versus pulsatile shear stress (12±4 dynes/cm2) revealed that oscillatory shear stress induces phospho-YY1S118 (serine [S]118 phosphorylation of Yin Yang 1) in ECs. Elevated phospho-YY1S118 level in ECs was further confirmed to be present in the disturbed flow regions in experimental animals and human atherosclerotic arteries. This disturbed flow-induced EC phospho-YY1S118 is mediated by CK2α (casein kinase 2α) through its direct interaction with YY1. Yeast 2-hybrid library screening and in situ proximity ligation assays demonstrate that phospho-YY1S118 directly binds ZKSCAN4 (zinc finger with KRAB [krüppel-associated box] and SCAN [SRE-ZBP, CTfin51, AW-1 and Number 18 cDNA] domains 4) to induce promoter activity and gene expression of HDM2 (human double minute 2), which consequently induces EC proliferation through downregulation of p53 and p21CIP1. Administration of apoE-deficient (ApoE-/-) mice with CK2-specific inhibitor tetrabromocinnamic acid or atorvastatin inhibits atherosclerosis formation through downregulations of EC phospho-YY1S118 and HDM2. Generation of novel transgenic mice bearing EC-specific overexpression of S118-nonphosphorylatable mutant of YY1 in ApoE-/- mice confirms the critical role of phospho-YY1S118 in promoting atherosclerosis through EC HDM2. CONCLUSIONS: Our findings provide new insights into the mechanisms by which disturbed flow induces endothelial phospho-YY1S118 to promote atherosclerosis, thus indicating phospho-YY1S118 as a potential molecular target for atherosclerosis treatment.
Assuntos
Aterosclerose/metabolismo , Células Endoteliais/metabolismo , Fator de Transcrição YY1/metabolismo , Animais , Aterosclerose/fisiopatologia , Sítios de Ligação , Circulação Sanguínea , Caseína Quinase II/metabolismo , Linhagem Celular , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Ratos , Ratos Sprague-Dawley , Fator de Transcrição YY1/química , Fator de Transcrição YY1/genética , Dedos de ZincoRESUMO
BACKGROUND: The purpose of this study was to explore the clinical value of miR-378c and its target gene YY1 in gastric cancer. METHODS: The TCGA database was employed to analyse miR-378c expression in gastric cancer. qRT-PCR was applied to identify miR-378c and YY1 in tissues and serum of patients suffering from gastric cancer. The association of miR-378c with the clinical data of patients with gastric cancer was analysed. Receiver operating characteristics (ROC) curve analysis was used to determine the diagnostic value of miR-378c and YY1 in gastric cancer, and analyse the relationship between miR-378c and YY1 and patients' survival. Pearson's test was applied to determine the association between miR-378c and YY1 in tissue and serum of patients. Dual-Luciferase Reporter assay was employed to examine the targeting association between miR-378c and YY1. Finally, independent prognostic factors was determined in patients with gastric cancer using Cox regression analysis. RESULTS: In the TCGA database, miR-378c was weakly expressed in gastric cancer. Overall, patients with low expression had a lower survival rate. The expression of miR-378c decreased and the expression of YY1 increased in cancer tissues and serum of tumour patients. In patients with low expression of miR-378c the tumour size was ≥ 5 cm. Low differentiation, high TNM staging and lymph node invasion rate increased significantly, but the 5-year survival rate decreased in the patients. miR-378c and YY1 had better diagnostic value in gastric cancer. TargetScan, miRDB, starBase and miRTarBase predicted that YY1 was a potential gene of miR-378c, and the Dual-Luciferase Reporter assay revealed that there was a targeting relationship between the two, which was proved by correlation analysis. Multivariate Cox analysis revealed that differentiation, TNM staging and miR-378c were independent prognostic factors for patients. CONCLUSIONS: MiR-378c is weakly expressed in gastric cancer patients and may be considered as a promising diagnostic and prognostic indicator for gastric cancer.
Assuntos
MicroRNAs/metabolismo , Neoplasias Gástricas/diagnóstico , Fator de Transcrição YY1/metabolismo , Área Sob a Curva , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Bases de Dados Factuais , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Linfática , Masculino , MicroRNAs/sangue , MicroRNAs/química , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Modelos de Riscos Proporcionais , Curva ROC , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/mortalidade , Taxa de Sobrevida , Fator de Transcrição YY1/química , Fator de Transcrição YY1/genéticaRESUMO
Sirtuin 2 (Sirt2) belongs to the NAD+-dependent deacetylase family, is more highly expressed than other family members in adipocytes, and plays crucial roles in a wide range of biological processes. However, the mechanisms underlying Sirt2 expression during adipogenesis are poorly studied. In this study, the transcriptional start site (TSS) of Sirt2 was identified and two alternative transcript variants were spliced from Sirt2. The 5'-regulatory region of Sirt2 was also characterized; no TATA-box or CCAAT-box was presented in the 5'-flanking region. Two cytosine-phosphate diester-guanine (CpG) islands were also identified between nucleotides -563 and +4. A dual-luciferase reporter assay revealed that a 178 base pair sequence upstream from the TSS (+1) was the core promoter of Sirt2. Results from a site-directed mutagenesis experiment, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay indicated Yin Yang 1 (YY1) to be a positive regulator of bovine Sirt2 in preadipocytes. YY1 is likely to suppress adipogenesis in two different ways by regulating peroxisome proliferator-activated receptor gamma expression. Our results expand the information on the regulatory network of adipogenesis, which is an important basis for improving beef quality, treating obesity, and other related diseases.
Assuntos
Adipócitos/metabolismo , Sirtuína 2/genética , Ativação Transcricional , Fator de Transcrição YY1/metabolismo , Células 3T3-L1 , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Humanos , Camundongos , Regiões Promotoras Genéticas/genética , Fator de Transcrição YY1/químicaRESUMO
DNA sequences are often recognized by multi-domain proteins that may have higher affinity and specificity than single-domain proteins. However, the higher affinity to DNA might be coupled with slower recognition kinetics. In this study, we address this balance between stability and kinetics for multi-domain Cys2His2- (C2H2-) type zinc-finger (ZF) proteins. These proteins are the most prevalent DNA-binding domain in eukaryotes and C2H2 type zinc-finger proteins (C2H2-ZFPs) constitute nearly one-half of all known and predicted transcription factors in human. Extensive contact with DNA via tandem ZF domains confers high stability on the sequence-specific complexes. However, this can limit target search efficiency, especially for low abundance ZFPs. Earlier, we found that asymmetrical distribution of electrostatic charge among the three ZF domains of the low abundance transcription factor Egr-1 facilitates its DNA search process. Here, on a diverse set of 273 human C2H2-ZFP comprised of 3-15 tandem ZF domains, we find that, in many cases, electrostatic charge and binding specificity are asymmetrically distributed among the ZF domains so that neighbouring domains have different DNA-binding properties. For proteins containing 3-6 ZF domains, we show that the low abundance proteins possess a higher degree of non-specific asymmetry and vice versa. Our findings suggest that where the electrostatics of tandem ZF domains are similar (i.e., symmetrical), the ZFPs are more abundant to optimize their DNA search efficiency. This study reveals new insights into the fundamental determinants of recognition by C2H2-ZFPs of their DNA binding sites in the cellular landscape. The importance of electrostatic asymmetry with respect to binding site recognition by C2H2-ZFPs suggests the possibility that it may also be important in other ZFP systems and reveals a new design feature for zinc finger engineering.
Assuntos
Proteínas de Ligação a DNA/química , DNA/química , Regulação da Expressão Gênica , Dedos de Zinco , Sítios de Ligação , Proteína 1 de Resposta de Crescimento Precoce/química , Humanos , Cinética , Ligação Proteica , Domínios Proteicos , Eletricidade Estática , Fator de Transcrição YY1/químicaRESUMO
Long non-coding RNA (lncRNA) LINC00899 is one kind cytoplasmic lncRNA, however, there is rarely little information about its function in physiological process. Here, we demonstrated that lncRNA LINC00899 was upregulated in acute myeloid leukaemia (AML) cells and was quite correlated with poor prognosis of AML patients. High expression of LINC00899 in AML cells could promote cell proliferation and inhibit cell apoptosis, and facilitate the progression of AML consequently both in vitro and in vivo. Besides, LINC00899 acted as a molecular sponge of miR-744-3p. Furthermore, we characterized YY1 as the direct target of miR-744-3p, and LINC00899/miR-744-3p interaction modulated YY1 expression in AML cells. Finally, we verified LINC00899 modulated AML cell proliferation and apoptosis via regulating YY1. Our study revealed novel mechanism about how did lncRNA LINC00899 execute function in AML and thus provided potential therapeutic interventions for AML. SIGNIFICANCE OF THE STUDY: LncRNA LINC00899 is upregulated in AML cells and is correlated with poor prognosis of AML patients. LncRNA LINC00899 mediates cell proliferation and apoptosis of acute myeloid leukaemia cells. Knockdown of LINC00899 inhibited the growth of xenograft glioma tumour in vivo. LINC00899 acts as a molecular sponge of miR-744-3p. YY1 is the downstream target of LINC00899/miR-744-3p signalling.
Assuntos
Leucemia Mieloide Aguda/patologia , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Regiões 3' não Traduzidas , Animais , Antagomirs/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Progressão da Doença , Humanos , Estimativa de Kaplan-Meier , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/mortalidade , Camundongos , Camundongos Nus , Prognóstico , Interferência de RNA , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/genética , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/uso terapêutico , Regulação para Cima , Fator de Transcrição YY1/química , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismoRESUMO
Yin Yang 1 is a human transcription factor that controls a number of genes and takes part in the regulation of cell cycle, proliferation, differentiation, and neuronal development. Yin Yang 1 is composed of an N-terminal intrinsically disordered fragment and a C-terminal domain responsible for binding to DNA, composed of four zinc fingers. Recently, various alterations in the Yin Yang 1's DNA binding domain were linked with an unexplained intellectual disability named Gabriele-de Vries syndrome. In this paper, a repetitively occurring substitution of aspartate-380 for tyrosine was analyzed to assess its impact on Yin Yang 1's structure and DNA binding. The substitution was found to affect Yin Yang 1's secondary and tertiary structure to a limited extent and to impair the specificity of its interaction with DNA.
Assuntos
Mutação de Sentido Incorreto , Fator de Transcrição YY1/genética , Ácido Aspártico , DNA/metabolismo , Humanos , Conformação Proteica , Estrutura Secundária de Proteína/genética , Estrutura Terciária de Proteína/genética , Fatores de Transcrição/genética , Tirosina , Fator de Transcrição YY1/química , Fator de Transcrição YY1/metabolismo , Fator de Transcrição YY1/fisiologiaRESUMO
As a transcription factor, Yin Yang 1 (YY1) either activates or represses gene expression depending on its recruited cofactors. The YY1 C-terminal consists of four zinc fingers (ZF) that are responsible for its DNA binding. However, the contribution of each YY1 ZF to its functions have not been fully elucidated. In this study, we used alanines to replace YY1 cysteines that are crucial to ZFs in binding to DNA. We characterized these YY1 mutants for their DNA binding, transcriptional activity, and functional role in maintaining MDA-MB-231 cell proliferation. We demonstrated that ZFs 2 and 3 are essential to the general biological activity of YY1. ZF 1 showed relatively low importance, while ZF 4 is virtually dispensable for YY1 function.
Assuntos
Cisteína/fisiologia , Mutagênese , Fator de Transcrição YY1/fisiologia , Dedos de Zinco , Células HeLa , Humanos , Fator de Transcrição YY1/químicaRESUMO
The transcription factor YY2 is a recently discovered paralog of YY1. The two proteins exhibit substantial sequence similarity and partially similar transcriptional activity. They recognize the same DNA sequence in vitro yet bind different promoters in vivo. YY1 comprises two structurally distinct parts: an intrinsically disordered regulatory part and a compact DNA-binding domain. The structure of YY2 is yet unknown. We show that YY2 is structurally similar to YY1, although the conformational state of YY2 is more ordered, as shown by its composition, hydrodynamic properties, spectroscopic signal, and proteolytic susceptibility. As such, YY2's range of molecular partners might be distinct from that of YY1. This could explain different effects of YY1 and YY2 on gene expression patterns and the mechanism of YY proteins in transcriptional regulation.
Assuntos
Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Fator de Transcrição YY1/química , Fator de Transcrição YY1/metabolismo , Sequência de Aminoácidos , Regulação da Expressão Gênica , Humanos , Desnaturação Proteica , Estrutura Secundária de ProteínaRESUMO
BACKGROUND: The oncoprotein binding (OPB) domain of Yin Yang 1 (YY1) consists of 26 amino acids between G201 and S226, and is involved in YY1 interaction with multiple oncogene products, including MDM2, AKT, EZH2 and E1A. Through the OPB domain, YY1 promotes the oncogenic or proliferative regulation of these oncoproteins in cancer cells. We previously demonstrated that a peptide with the OPB sequence blocked YY1-AKT interaction and inhibited breast cancer cell proliferation. OBJECTIVE: In the current study, we characterized the OPB domain and determined a minimal region for peptide design to suppress cancer cells. METHODS: Using alanine-scan method, we identified that the amino acids at OPB C-terminal are essential to YY1 binding to AKT. Further studies suggested that serine and threonine residues, but not lysines, in OPB play a key role in YY1-AKT interaction. We generated GFP fusion expression vectors to express OPB peptides with serially deleted N-terminal and found that OPB1 (i.e. G201-S226) is cytoplasmic, but OPB2 (i.e. E206-S226), OPB3 (i.e. E206-S226) and control peptide were both nuclear and cytoplasmic. RESULTS: Both OPB1 and 2 inhibited breast cancer cell proliferation and migration, but OPB3 exhibited similar effects to control. OPB1 and 2 caused cell cycle arrest at G1 phase, increased p53 and p21 expression, and reduced AKT(S473) phosphorylation in MCF-7 cells, but not in MDA-MB-231 cells. CONCLUSION: Overall, the serines and threonines of OPB are essential to YY1 binding to oncoproteins, and OPB peptide can be minimized to E206-S226 that maintain inhibitory activity to YY1- promoted cell proliferation.
Assuntos
Analgésicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Transcrição YY1/química , Sequência de Aminoácidos , Apoptose/efeitos dos fármacos , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Feminino , Humanos , Fragmentos de Peptídeos/química , Fosforilação , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Homologia de Sequência , Transdução de Sinais , Células Tumorais Cultivadas , Fator de Transcrição YY1/metabolismoRESUMO
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
Moloney Murine Leukemia Virus (M-MLV) proviral DNA is transcriptionally silenced in embryonic cells by a large repressor complex tethered to the provirus by two sequence-specific DNA binding proteins, ZFP809 and YY1. A central component of the complex is Trim28, a scaffold protein that regulates many target genes involved in cell cycle progression, DNA damage responses, and viral gene expression. The silencing activity of Trim28, and its interactions with corepressors are often regulated by post-translational modifications such as sumoylation and phosphorylation. We defined the interaction domains of Trim28 and YY1, and investigated the role of sumoylation and phosphorylation of Trim28 in mediating M-MLV silencing. The RBCC domain of Trim28 was sufficient for interaction with YY1, and acidic region 1 and zinc fingers of YY1 were necessary and sufficient for its interaction with Trim28. Additionally, we found that residue K779 was critical for Trim28-mediated silencing of M-MLV in embryonic cells.
Assuntos
Vírus da Leucemia Murina de Moloney/fisiologia , Provírus/genética , Infecções por Retroviridae/veterinária , Doenças dos Roedores/metabolismo , Proteína 28 com Motivo Tripartido/metabolismo , Fator de Transcrição YY1/metabolismo , Motivos de Aminoácidos , Animais , Inativação Gênica , Camundongos , Vírus da Leucemia Murina de Moloney/genética , Ligação Proteica , Domínios Proteicos , Provírus/metabolismo , Infecções por Retroviridae/genética , Infecções por Retroviridae/metabolismo , Infecções por Retroviridae/virologia , Doenças dos Roedores/genética , Doenças dos Roedores/virologia , Proteína 28 com Motivo Tripartido/química , Proteína 28 com Motivo Tripartido/genética , Fator de Transcrição YY1/química , Fator de Transcrição YY1/genéticaRESUMO
Site-specific labeling of proteins with fluorescent dyes allows the study of protein structure and function using a wide variety of fluorescent techniques. However, specific labeling is not trivial in the case of proteins containing multiple cysteine residues. An example of such a protein is transcription factor Yin Yang 1, which comprises eight cysteine residues in four C2H2 type zinc fingers in the C-terminal region. Kinetic measurements of the labeling process allowed us to develop preparative labeling of three cysteine residues differently introduced to the N-terminal, disordered fragment of the protein. The protocol developed in the present study allows to prepare the protein with high recovery yield and high selectivity of the labeling. This was confirmed using proteolytic digestion and spectroscopic approach. The labeling process was significantly affected by the presence of zinc ions and was dependent on the localization of the engineered cysteine residue. This is the first known example of the use of cysteine metal protection and labeling (CyMPL) technology for the labeling of protein regions with no stable secondary structures.
Assuntos
Corantes Fluorescentes/química , Coloração e Rotulagem/métodos , Fator de Transcrição YY1/química , Zinco/farmacologia , Sítios de Ligação , Cisteína/química , Humanos , Mutação , Dobramento de Proteína , Proteólise , Fator de Transcrição YY1/genética , Dedos de ZincoRESUMO
BACKGROUND/AIMS: An increase in intracellular lipid droplet formation and hepatic triglyceride (TG) content usually results in nonalcoholic fatty liver disease. However, the mechanisms underlying the regulation of hepatic TG homeostasis remain unclear. METHODS: Oil red O staining and TG measurement were performed to determine the lipid content. miRNA expression was evaluated by quantitative PCR. A luciferase assay was performed to validate the regulation of Yin Yang 1 (YY1) by microRNA (miR)-122. The effects of miR-122 expression on YY1 and its mechanisms involving the farnesoid X receptor and small heterodimer partner (FXR-SHP) pathway were evaluated by quantitative PCR and Western blot analyses. RESULTS: miR-122 was downregulated in free fatty acid (FFA)-induced steatotic hepatocytes, and streptozotocin and high-fat diet (STZ-HFD) induced nonalcoholic steatohepatitis (NASH) in mice. Transfection of hepatocytes with miR-122 mimics before FFA induction inhibited lipid droplet formation and TG accumulation in vitro. These results were verified by overexpressing miR-122 in the livers of STZ-HFD-induced NASH mice. The 3'-untranslated region (3'UTR) of YY1 mRNA is predicted to contain an evolutionarily conserved miR-122 binding site. In silico searches, a luciferase reporter assay and quantitative PCR analysis confirmed that miR-122 directly bound to the YY1 3'UTR to negatively regulate YY1 mRNA in HepG2 and Huh7 cells. The (FXR-SHP) signaling axis, which is downstream of YY1, may play a key role in the mechanism of miR-122-regulated lipid homeostasis. YY1-FXR-SHP signaling, which is negatively regulated by FFA, was enhanced by miR-122 overexpression. This finding was also confirmed by overexpression of miR-122 in the livers of NASH mice. CONCLUSIONS: The present results indicate that miR-122 plays an important role in lipid (particularly TG) accumulation in the liver by reducing YY1 mRNA stability to upregulate FXR-SHP signaling.
Assuntos
Gotículas Lipídicas/metabolismo , MicroRNAs/metabolismo , Triglicerídeos/metabolismo , Fator de Transcrição YY1/metabolismo , Regiões 3' não Traduzidas , Animais , Antagomirs/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patologia , Dieta Hiperlipídica , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Ácidos Graxos não Esterificados/farmacologia , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Células Hep G2 , Humanos , Gotículas Lipídicas/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Alinhamento de Sequência , Fator de Transcrição YY1/química , Fator de Transcrição YY1/genéticaRESUMO
MicroRNAs (miRNAs) have been suggested to repress transcription via binding the 3'-untranslated regions of mRNAs. However, the involvement and details of miRNA-mediated epigenetic regulation, particularly in targeting genomic DNA and mediating epigenetic regulation, remain largely uninvestigated. In the present study, transcription factor CCAAT/enhancer binding protein delta (CEBPD) was responsive to the anticancer drug bortezomib, a clinical and highly selective drug for leukemia treatment, and contributed to bortezomib-induced cell death. Interestingly, following the identification of CEBPD-induced miRNAs, we found that miR-744, miR-3154 and miR-3162 could target CpG islands in the 5'-flanking region of the CEBPD gene. We previously demonstrated that the Yin Yang 1 (YY1)/polycomb group (PcG) protein/DNA methyltransferase (DNMT) complex is important for CCAAT/enhancer binding protein delta (CEBPD) gene inactivation; we further found that Argonaute 2 (Ago2) interacts with YY1 and binds to the CEBPD promoter. The miRNA/Ago2/YY1/PcG group protein/DNMT complex linked the inactivation of CEBPD and genes adjacent to its 5'-flanking region, including protein kinase DNA-activated catalytic polypeptide (PRKDC), minichromosome maintenance-deficient 4 (MCM4) and ubiquitin-conjugating enzyme E2 variant 2 (UBE2V2), upon bortezomib treatment. Moreover, we revealed that miRNA binding is necessary for YY1/PcG group protein/DNMT complex-mediated epigenetic gene silencing and is associated with bortezomib-induced methylation on genomic DNA. The present study successfully characterized the interactions of the miRNA/Ago2/YY1/PcG group protein/DNMT complex and provided new insights for miRNA-mediated epigenetic regulation in bortezomib-induced leukemic cell arrest and cell death.
Assuntos
Apoptose/efeitos dos fármacos , Bortezomib/farmacologia , Leucemia/fisiopatologia , MicroRNAs/metabolismo , Regiões 3' não Traduzidas , Antineoplásicos/farmacologia , Proteínas Argonautas/química , Proteínas Argonautas/metabolismo , Proteína delta de Ligação ao Facilitador CCAAT/genética , Proteína delta de Ligação ao Facilitador CCAAT/metabolismo , Linhagem Celular Tumoral , Ilhas de CpG , Metilação de DNA/efeitos dos fármacos , Proteína Quinase Ativada por DNA/genética , Proteína Quinase Ativada por DNA/metabolismo , Inativação Gênica , Humanos , Leucemia/metabolismo , Ligases/genética , Ligases/metabolismo , MicroRNAs/genética , Componente 4 do Complexo de Manutenção de Minicromossomo/genética , Componente 4 do Complexo de Manutenção de Minicromossomo/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Transcrição Gênica/efeitos dos fármacos , Enzimas de Conjugação de Ubiquitina , Fator de Transcrição YY1/química , Fator de Transcrição YY1/metabolismoRESUMO
Successful execution of mitotic cell division requires the tight synchronisation of numerous biochemical pathways. The underlying mechanisms that govern chromosome segregation have been thoroughly investigated. However, the mechanisms that regulate transcription factors in coordination with mitotic progression remain poorly understood. In this report, we identify the transcription factor YY1 as a novel mitotic substrate for the Aurora A kinase, a key regulator of critical mitotic events, like centrosome maturation and spindle formation. Using in vitro kinase assays, we show that Aurora A directly phosphorylates YY1 at serine 365 in the DNA-binding domain. Using a new phospho-specific antibody, we show that YY1 phosphorylation at serine 365 occurs during mitosis, and that this phosphorylation is significantly reduced upon inhibition of Aurora A. Furthermore, we show, using electrophoretic mobility shift and chromatin immunoprecipitation assays, that phosphorylation of YY1 at this site abolishes its DNA binding activity in vitro and in vivo. In conformity with this loss of binding activity, phosphorylated YY1 also loses its transctivation ability as demonstrated by a luciferase reporter assay. These results uncover a novel mechanism that implicates Aurora A in the mitotic inactivation of transcription factors.
Assuntos
Aurora Quinase A/genética , DNA/química , Mitose , Transcrição Gênica , Fator de Transcrição YY1/química , Sequência de Aminoácidos , Aurora Quinase A/metabolismo , Sítios de Ligação , Segregação de Cromossomos , DNA/genética , DNA/metabolismo , Genes Reporter , Células HEK293 , Células HeLa , Humanos , Luciferases/genética , Luciferases/metabolismo , Modelos Moleculares , Fosforilação , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Fuso Acromático/metabolismo , Fuso Acromático/ultraestrutura , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismoRESUMO
Yin and yang 1 (YY1) is a well-known zinc-finger transcription factor with crucial roles in normal development and malignancy. YY1 acts both as a repressor and as an activator of gene expression. We have identified 23 individuals with de novo mutations or deletions of YY1 and phenotypic features that define a syndrome of cognitive impairment, behavioral alterations, intrauterine growth restriction, feeding problems, and various congenital malformations. Our combined clinical and molecular data define "YY1 syndrome" as a haploinsufficiency syndrome. Through immunoprecipitation of YY1-bound chromatin from affected individuals' cells with antibodies recognizing both ends of the protein, we show that YY1 deletions and missense mutations lead to a global loss of YY1 binding with a preferential retention at high-occupancy sites. Finally, we uncover a widespread loss of H3K27 acetylation in particular on the YY1-bound enhancers, underscoring a crucial role for YY1 in enhancer regulation. Collectively, these results define a clinical syndrome caused by haploinsufficiency of YY1 through dysregulation of key transcriptional regulators.
Assuntos
Cromatina/metabolismo , Haploinsuficiência/genética , Deficiência Intelectual/genética , Transcrição Gênica , Fator de Transcrição YY1/genética , Acetilação , Adolescente , Sequência de Bases , Pré-Escolar , Imunoprecipitação da Cromatina , Estudos de Coortes , Elementos Facilitadores Genéticos/genética , Feminino , Ontologia Genética , Haplótipos/genética , Hemizigoto , Histonas/metabolismo , Humanos , Linfócitos/metabolismo , Masculino , Metilação , Modelos Moleculares , Mutação de Sentido Incorreto/genética , Ligação Proteica/genética , Domínios Proteicos , Fator de Transcrição YY1/químicaRESUMO
Yin Yang 1 (YY1)'s interaction with DNA can result in various, even contradicting, effects on transcription in the form of initiation, activation, or repression. This surprising activity can be explained in the context of the YY1-DNA's complex structure. YY1's DNA-binding domain is formed by four zinc finger motifs. However, the sequence of both the zinc fingers and the linkers is non-canonical, which impairs their docking to the DNA duplex. Short linkers between the zinc fingers impose a concerted binding mechanism. Analysis of the sequences known to be recognized by YY1 suggests different contributions of particular zinc fingers in specific recognition of activated versus repressed promoters. Thermodynamic and kinetic studies show that, although the YY1's N-terminal fragment does not itself bind to DNA, it might regulate the interaction because its presence influences the binding parameters. Meta-analysis of YY1-DNA binding allowed us to observe that YY1's avidity to multiple binding sites is crucial in providing high-affinity specific sequence recognition. Alternatively, other trans-acting factors can modulate the YY1-DNA interaction and influence its outcome. This complex mechanism causes great sensitivity for individual point mutations, an increasing number of which are found in YY1 in cancer tissues.