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1.
J Biol Chem ; 299(9): 105062, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37468105

RESUMO

SUMOylation is a post-translational modification frequently found on nuclear proteins, including transcription factors (TFs) and coactivators. By controlling the activity of several TFs, SUMOylation may have far-reaching effects. MYB is an example of a developmental TF subjected to SUMO-mediated regulation, through both SUMO conjugation and SUMO binding. How SUMO affects MYB target genes is unknown. Here, we explored the global effect of reduced SUMOylation of MYB on its downstream gene programs. RNA-Seq in K562 cells after MYB knockdown and rescue with mutants having an altered SUMO status revealed a number of differentially regulated genes and distinct gene ontology term enrichments. Clearly, the SUMO status of MYB both quantitatively and qualitatively affects its regulome. The transcriptome data further revealed that MYB upregulates the SUMO protease SENP1, a key enzyme that removes SUMO conjugation from SUMOylated proteins. Given this role of SENP1 in the MYB regulome, we expanded the analysis, mapped interaction partners of SENP1, and identified UXT as a novel player affecting the SUMO system by acting as a repressor of SENP1. MYB inhibits the expression of UXT suggesting that MYB is able not only to control a specific gene program directly but also indirectly by affecting the SUMO landscape through SENP1 and UXT. These findings suggest an autoactivation loop whereby MYB, through enhancing SENP1 and reducing UXT, is itself being activated by a reduced level of repressive SUMOylation. We propose that overexpressed MYB, seen in multiple cancers, may drive this autoactivation loop and contribute to oncogenic activation of MYB.


Assuntos
Proteínas de Ciclo Celular , Regulação da Expressão Gênica , Genes myb , Peptídeo Hidrolases , Humanos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Regulação da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Células K562 , Neoplasias/fisiopatologia , Peptídeo Hidrolases/metabolismo , Ligação Proteica , Sumoilação , Ativação Transcricional
2.
Epigenetics Chromatin ; 15(1): 13, 2022 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-35440061

RESUMO

Methylation of cytosines on DNA is a prominent modification associated with gene expression regulation. Aberrant DNA methylation patterns have recurrently been linked to dysregulation of the regulatory program in cancer cells. To shed light on the underlying molecular mechanism driving this process, we hypothesised that aberrant methylation patterns could be controlled by the binding of specific transcription factors (TFs) across cancer types. By combining DNA methylation arrays and gene expression data with TF binding sites (TFBSs), we explored the interplay between TF binding and DNA methylation in 19 cancer types. We performed emQTL (expression-methylation quantitative trait loci) analyses independently in each cancer type and identified 13 TFs whose expression levels are correlated with local DNA methylation patterns around their binding sites in at least 2 cancer types. The 13 TFs are mainly associated with local demethylation and are enriched for pioneer function, suggesting a specific role for these TFs in modulating chromatin structure and transcription in cancer patients. Furthermore, we confirmed that de novo methylation is precluded across cancers at CpGs lying in genomic regions enriched for TF binding signatures associated with SP1, CTCF, NRF1, GABPA, KLF9, and/or YY1. The modulation of DNA methylation associated with TF binding was observed at cis-regulatory regions controlling immune- and cancer-associated pathways, corroborating that the emQTL signals were derived from both cancer and tumor-infiltrating cells. As a case example, we experimentally confirmed that FOXA1 knock-down is associated with higher methylation in regions bound by FOXA1 in breast cancer MCF-7 cells. Finally, we reported physical interactions between FOXA1 with TET1 and TET2 both in an in vitro setup and in vivo at physiological levels in MCF-7 cells, adding further support for FOXA1 attracting TET1 and TET2 to induce local demethylation in cancer cells.


Assuntos
Metilação de DNA , Neoplasias , Fatores de Transcrição/metabolismo , Sítios de Ligação , DNA/metabolismo , Genoma , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Oxigenases de Função Mista/metabolismo , Neoplasias/genética , Ligação Proteica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo
3.
Sci Rep ; 11(1): 9008, 2021 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-33903675

RESUMO

The transcription factor MYB is a master regulator in haematopoietic progenitor cells and a pioneer factor affecting differentiation and proliferation of these cells. Leukaemic transformation may be promoted by high MYB levels. Despite much accumulated molecular knowledge of MYB, we still lack a comprehensive understanding of its target genes and its chromatin action. In the present work, we performed a ChIP-seq analysis of MYB in K562 cells accompanied by detailed bioinformatics analyses. We found that MYB occupies both promoters and enhancers. Five clusters (C1-C5) were found when we classified MYB peaks according to epigenetic profiles. C1 was enriched for promoters and C2 dominated by enhancers. C2-linked genes were connected to hematopoietic specific functions and had GATA factor motifs as second in frequency. C1 had in addition to MYB-motifs a significant frequency of ETS-related motifs. Combining ChIP-seq data with RNA-seq data allowed us to identify direct MYB target genes. We also compared ChIP-seq data with digital genomic footprinting. MYB is occupying nearly a third of the super-enhancers in K562. Finally, we concluded that MYB cooperates with a subset of the other highly expressed TFs in this cell line, as expected for a master regulator.


Assuntos
Sítios de Ligação , Cromatina/genética , Cromatina/metabolismo , Regulação da Expressão Gênica , Hematopoese/genética , Proteínas Proto-Oncogênicas c-myb/metabolismo , Diferenciação Celular/genética , Montagem e Desmontagem da Cromatina , Imunoprecipitação da Cromatina , Biologia Computacional/métodos , Bases de Dados Genéticas , Epigênese Genética , Epigenômica/métodos , Perfilação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Células K562 , Modelos Biológicos , Motivos de Nucleotídeos , Regiões Promotoras Genéticas , Ligação Proteica , Transcriptoma
4.
FEBS Open Bio ; 10(11): 2329-2342, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32937031

RESUMO

Transcription factors use a DNA-binding domain to localize their action and a transactivation domain (tAD) to stimulate activation of the associated gene. Recent work has renewed interest in how tADs activate genes, which remains poorly understood. Key features in the new models are exposure of short linear motifs (SLMs) and liquid-liquid phase separation (LLPS). Inspired by the new models for tAD function, we decided to revisit the tAD of the haematopoietic transcription factor c-Myb by performing a mutational analysis to see how these new models fit and potentially explain the tAD behaviour of this master regulator. We know that c-Myb has an acidic tAD, which contains a well-characterized SLM in the form of a LxxLL motif. By testing 12 alanine-scanning mutants and three mutants with major reorganization of its tAD in two mammalian reporter systems, we found a pattern of effects very close to what would be expected from the SLM-exposure model, with strong effects exerted by both acidic replacements and SLM mutation. When the same mutants were tested in a yeast system, the pattern of effects was dramatically different, with the SLM mutation exerting no effect, and tAD behaviour was much less affected by small alterations, as would be expected from a LLPS model. These observations are discussed in the light of the two new tAD models, and a two-step hypothesis for transactivation, combining both models, is proposed.


Assuntos
Modelos Biológicos , Proteínas Proto-Oncogênicas c-myb/química , Proteínas Proto-Oncogênicas c-myb/metabolismo , Ativação Transcricional , Sequência de Aminoácidos , Animais , Cromatina/metabolismo , Análise Mutacional de DNA , Células HEK293 , Humanos , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Domínios Proteicos , Proteínas Proto-Oncogênicas c-myb/genética , Saccharomyces cerevisiae/metabolismo
5.
J Biol Chem ; 293(40): 15439-15454, 2018 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-30082317

RESUMO

The small ubiquitin-like modifier (SUMO) post-translationally modifies lysine residues of transcription factors and co-regulators and thereby contributes to an important layer of control of the activities of these transcriptional regulators. Likewise, deSUMOylation of these factors by the sentrin-specific proteases (SENPs) also plays a role in gene regulation, but whether SENPs functionally interact with other regulatory factors that control gene expression is unclear. In the present work, we focused on SENP1, specifically, on its role in activation of gene expression investigated through analysis of the SENP1 interactome, which revealed that SENP1 physically interacts with the chromatin remodeler chromodomain helicase DNA-binding protein 3 (CHD3). Using several additional methods, including GST pulldown and co-immunoprecipitation assays, we validated and mapped this interaction, and using CRISPR-Cas9-generated CHD3- and SENP1-KO cells (in the haploid HAP1 cell line), we investigated whether these two proteins are functionally linked in regulating chromatin remodeling and gene expression. Genome-wide ATAC-Seq analysis of the CHD3- and SENP1-KO cells revealed a large degree of overlap in differential chromatin openness between these two mutant cell lines. Moreover, motif analysis and comparison with ChIP-Seq profiles in K562 cells pointed to an association of CHD3 and SENP1 with CCCTC-binding factor (CTCF) and SUMOylated chromatin-associated factors. Lastly, genome-wide RNA-Seq also indicated that these two proteins co-regulate the expression of several genes. We propose that the functional link between chromatin remodeling by CHD3 and deSUMOylation by SENP1 uncovered here provides another level of control of gene expression.


Assuntos
Montagem e Desmontagem da Cromatina , Cromatina/química , Cisteína Endopeptidases/metabolismo , DNA Helicases/metabolismo , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Células COS , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Chlorocebus aethiops , Cromatina/metabolismo , Cromatina/ultraestrutura , Clonagem Molecular , Cisteína Endopeptidases/genética , DNA Helicases/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Edição de Genes/métodos , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Células K562 , Leucócitos/metabolismo , Leucócitos/patologia , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
6.
Epigenetics Chromatin ; 11(1): 35, 2018 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-29954426

RESUMO

BACKGROUND: The concept of pioneer transcription factors is emerging as an essential part of the epigenetic regulation, taking place during cell development and differentiation. However, the precise molecular mechanism underlying pioneer factor activity remains poorly understood. We recently reported that the transcription factor c-Myb acts as a pioneer factor in haematopoiesis, and a point mutation in its DNA binding domain, D152V, is able to abrogate this function. RESULTS: Here, we show that specific histone modifications, including H3K27ac, prevent binding of c-Myb to histone tails, representing a novel effect of histone modifications, namely restricting binding of a pioneer factor to chromatin. Furthermore, we have taken advantage of the pioneer-defect D152V mutant to investigate mechanisms of c-Myb's pioneer factor activity. We show that c-Myb-dependent transcriptional activation of a gene in inaccessible chromatin relies on c-Myb binding to histones, as well as on c-Myb interacting with the histone acetyltransferase p300. ChIP assays show that both wild type and the D152V mutant of c-Myb bind to a selected target gene at its promoter and enhancer, but only wild-type c-Myb causes opening and activation of the locus. Enhancement of histone acetylation restores activation of the same gene in the absence of c-Myb, suggesting that facilitating histone acetylation is a crucial part of the pioneer factor function of c-Myb. CONCLUSIONS: We suggest a pioneer factor model in which c-Myb binds to regions of closed chromatin and then recruits histone acetyltransferases. By binding to histones, c-Myb facilitates histone acetylation, acting as a cofactor for p300 at c-Myb bound sites. The resulting H3K27ac leads to chromatin opening and detachment of c-Myb from the acetylated chromatin. We propose that the latter phenomenon, acetylation-induced chromatin dissociation, represents a mechanism for controlling the dynamics of pioneer factor binding to chromatin.


Assuntos
Cromatina/genética , Proteína p300 Associada a E1A/metabolismo , Histonas/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Acetilação , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Epigênese Genética , Humanos , Células K562 , Mutação , Proteínas Proto-Oncogênicas c-myb/genética , Transcrição Gênica
7.
Biochim Biophys Acta Gene Regul Mech ; 1860(7): 751-760, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28495617

RESUMO

LIM-domain proteins, containing multiple cysteine-rich zinc finger-like motifs, have been shown to play diverse roles in several cellular processes. A common theme is that they mediate important protein-protein interactions that are key to their function. Androgen receptor-associated protein 55 (ARA55) belongs to this family of bridging proteins containing four C-terminal LIM domains. It has a dual role with functions both at focal adhesions and in the nucleus, apparently shuttling between the two compartments. In the present work, we have expanded our understanding of its nuclear functions by showing that it interacts with three nuclear regulators not previously linked to ARA55. We first identified ARA55 as a novel interaction partner of the nuclear kinase HIPK1 and found that ARA55, like HIPK1, also interacts with the transcription factor c-Myb. In search of a function for these associations, we observed that the coactivator p300 not only binds to c-Myb, but to ARA55 as well. When combined, c-Myb, p300, HIPK1 and ARA55 caused strong synergistic activation of a chromatinized reporter gene. In parallel, all partners, including p300, were efficiently recruited to chromatin at the c-Myb-bound promoter. Consistent with this cooperation, we found that c-Myb and ARA55 share a common set of target genes in an osteosarcoma cellular context. We propose that ARA55 and HIPK1 assist c-Myb in recruiting the coactivator and acetyltransferase p300 to chromatin.


Assuntos
Cromatina/metabolismo , Proteína p300 Associada a E1A/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Animais , Células COS , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Chlorocebus aethiops , Proteínas de Ligação a DNA/metabolismo , Genes Reporter/genética , Células HEK293 , Humanos , Células K562 , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética
8.
Biochim Biophys Acta ; 1859(5): 705-18, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-27032383

RESUMO

The PIAS proteins (Protein Inhibitor of Activated STATs) constitute a family of multifunctional nuclear proteins operating as SUMO E3 ligases and being involved in a multitude of interactions. They participate in a range of biological processes, also beyond their well-established role in the immune system and cytokine signalling. They act both as transcriptional corepressors and coactivators depending on the context. In the present work, we investigated mechanisms by which PIAS1 causes activation or repression of c-Myb dependent target genes. Analysis of global expression data shows that c-Myb and PIAS1 knockdowns affect a subset of common targets, but with a dual outcome consistent with a role of PIAS1 as either a corepressor or coactivator. Our mechanistic studies show that PIAS1 engages in a novel interaction with the acetyltransferase and coactivator p300. Interaction and ChIP analysis suggest a bridging function where PIAS1 enhances p300 recruitment to c-Myb-bound sites through interaction with both proteins. In addition, the E3 activity of PIAS1 enhances further its coactivation. Remarkably, the SUMO status of c-Myb had a decisive role, indicating a SUMO-dependent switch in the way PIAS1 affects c-Myb, either as a coactivator or corepressor. Removal of the two major SUMO-conjugation sites in c-Myb (2KR mutant), which enhances its activity significantly, turned PIAS1 into a corepressor. Also, p300 was less efficiently recruited to chromatin by c-Myb-2KR. We propose that PIAS1 acts as a "protein inhibitor of activated c-Myb" in the absence of SUMOylation while, in its presence, PIAS behaves as a "protein activator of repressed c-Myb".


Assuntos
Proteínas Inibidoras de STAT Ativados/genética , Proteínas Proto-Oncogênicas c-myb/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Fatores de Transcrição de p300-CBP/genética , Cromatina/genética , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Ligação Proteica/genética , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Proto-Oncogênicas c-myb/biossíntese , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Sumoilação/genética , Fatores de Transcrição de p300-CBP/metabolismo
9.
Mol Cancer ; 10: 21, 2011 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-21338522

RESUMO

BACKGROUND: FLASH is a huge nuclear protein involved in various cellular functions such as apoptosis signalling, NF-κB activation, S-phase regulation, processing of histone pre-mRNAs, and co-regulation of transcription. Recently, we identified FLASH as a co-activator of the transcription factor c-Myb and found FLASH to be tightly associated with active transcription foci. As a huge multifunctional protein, FLASH is expected to have many interaction partners, some which may shed light on its function as a transcriptional regulator. RESULTS: To find additional FLASH-associated proteins, we performed a yeast two-hybrid (Y2H) screening with FLASH as bait and identified the SUMO E3 ligase PIAS1 as an interaction partner. The association appears to involve two distinct interaction surfaces in FLASH. We verified the interaction by Y2H-mating, GST pulldowns, co-IP and ChIP. FLASH and PIAS1 were found to co-localize in nuclear speckles. Functional assays revealed that PIAS1 enhances the intrinsic transcriptional activity of FLASH in a RING finger-dependent manner. Furthermore, PIAS1 also augments the specific activity of c-Myb, and cooperates with FLASH to further co-activate c-Myb. The three proteins, FLASH, PIAS1, and c-Myb, are all co-localized with active RNA polymerase II foci, resembling transcription factories. CONCLUSIONS: We conclude that PIAS1 is a common partner for two cancer-related nuclear factors, c-Myb and FLASH. Our results point to a functional cooperation between FLASH and PIAS1 in the enhancement of c-Myb activity in active nuclear foci.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Núcleo Celular/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Animais , Células COS , Chlorocebus aethiops , Cromatina/metabolismo , Regulação da Expressão Gênica , Humanos , Ligação Proteica , Transporte Proteico , RNA Polimerase II/metabolismo
10.
Biochem Biophys Res Commun ; 388(1): 150-4, 2009 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-19646965

RESUMO

The transcription factor v-Myb is a potent inducer of myeloid leukaemias, and its cellular homologue c-Myb plays a crucial role in the regulation of haematopoiesis. In a yeast two-hybrid (Y2H) screening we identified the nuclear kinase HIPK1 as an interaction partner for human c-Myb. The interaction involves a C-terminal region of HIPK1, while a bipartite interaction surface was identified in c-Myb, including regions in its N-terminal DNA-binding domain as well as in its C-terminal region. HIPK1 and c-Myb co-localize in distinct nuclear foci upon co-transfection. c-Myb appears to be phosphorylated by HIPK1 in its negative regulatory domain as supported by both in vivo and in vitro data. A functional assay revealed that HIPK1 repressed the ability of c-Myb to activate a chromatin embedded target gene, mim-1, in haematopoetic cells. Our findings point to a novel link between an important kinase and a key regulator of haematopoiesis.


Assuntos
Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Humanos , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-myb/genética , Ativação Transcricional , Transfecção , Técnicas do Sistema de Duplo-Híbrido
11.
J Biol Chem ; 282(19): 13994-4005, 2007 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-17344210

RESUMO

The c-Myb protein belongs to a group of early hematopoietic transcription factors that are important for progenitor generation and proliferation. These factors have been hypothesized to participate in establishing chromatin patterns specific for hematopoietic genes. In a two-hybrid screening we identified the chromatin remodeling factor Mi-2alpha as an interaction partner for human c-Myb. The main interacting domains were mapped to the N-terminal region of Mi-2alpha and the DNA-binding domain of c-Myb. Surprisingly, functional analysis revealed that Mi-2alpha, previously studied as a subunit in the NuRD co-repressor complex, enhanced c-Myb-dependent reporter activation. Consistently, knock-down of endogenous Mi-2alpha in c-Myb-expressing K562 cells, led to down-regulation of the c-Myb target genes NMU and ADA. When wild-type and helicase-dead Mi-2alpha were compared, the Myb-Mi-2alpha co-activation appeared to be independent of the ATPase/DNA helicase activity of Mi-2alpha. The rationale for the unexpected co-activator function seems to lie in a dual function of Mi-2alpha, by which this factor is able to repress transcription in a helicase-dependent and activate in a helicase-independent fashion, as revealed by Gal4-tethering experiments. Interestingly, desumoylation of c-Myb potentiated the Myb-Mi-2alpha transactivational co-operation, as did co-transfection with p300.


Assuntos
Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina , DNA Helicases/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Ativação Transcricional , Adenosina Trifosfatases/genética , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , DNA Helicases/genética , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Genes Reporter , Humanos , Células K562 , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myb/genética , Proteína SUMO-1/metabolismo , Transativadores , Transcrição Gênica , Transfecção , Técnicas do Sistema de Duplo-Híbrido
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