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1.
Methods Mol Biol ; 2351: 275-288, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382195

RESUMO

Functionalization of the genome is carried out by proteins that bind to DNA to regulate gene expression. Since this process is highly dynamic, context-dependent, and rarely performed by single proteins alone, we here describe ChIP-SICAP to identify proteins that co-localize with a protein of interest on the genome. Benefiting from its nature as a dual purification approach via ChIP and DNA biotinylation, ChIP-SICAP distinguishes genuine chromatin-binders and is uniquely placed to identify novel players in genome regulation.


Assuntos
Imunoprecipitação da Cromatina/métodos , Cromatina/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Sítios de Ligação , Biotinilação , DNA/genética , DNA/metabolismo , Histonas/metabolismo , Espectrometria de Massas , Peptídeo Hidrolases , Ligação Proteica , Proteômica/métodos
2.
Leukemia ; 2021 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-33911178

RESUMO

Deregulation of the EVI1 proto-oncogene by the GATA2 distal hematopoietic enhancer (G2DHE) is a key event in high-risk acute myeloid leukemia carrying 3q21q26 aberrations (3q-AML). Upon chromosomal rearrangement, G2DHE acquires characteristics of a super-enhancer and causes overexpression of EVI1 at 3q26.2. However, the transcription factor (TF) complex of G2DHE remains poorly characterized. The aim of this study was to unravel key components of G2DHE-bound TFs involved in the deregulation of EVI1. We have identified several CEBPA and RUNX1 binding sites to be enriched and critical for G2DHE function in 3q-AML cells. Using ChIP-SICAP (ChIP followed by selective isolation of chromatin-associated proteins), a panel of chromatin interactors of RUNX1 and CEBPA were detected in 3q-AML, including PARP1 and IKZF1. PARP1 inhibition (PARPi) caused a reduction of EVI1 expression and a decrease in EVI1-G2DHE interaction frequency, highlighting the involvement of PARP1 in oncogenic super-enhancer formation. Furthermore, 3q-AML cells were highly sensitive to PARPi and displayed morphological changes with higher rates of differentiation and apoptosis as well as depletion of CD34 + cells. In summary, integrative analysis of the 3q-AML super-enhancer complex identified CEBPA and RUNX1 associated proteins and nominated PARP1 as a potential new therapeutic target in EVI1 + 3q-AML.

3.
Mol Syst Biol ; 16(5): e9370, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32400114

RESUMO

Streptavidin-mediated enrichment is a powerful strategy to identify biotinylated biomolecules and their interaction partners; however, intense streptavidin-derived peptides impede protein identification by mass spectrometry. Here, we present an approach to chemically modify streptavidin, thus rendering it resistant to proteolysis by trypsin and LysC. This modification results in over 100-fold reduction of streptavidin contamination and in better coverage of proteins interacting with various biotinylated bait molecules (DNA, protein, and lipid) in an overall simplified workflow.


Assuntos
Espectrometria de Massas/métodos , Metaloendopeptidases/química , Proteínas/análise , Proteômica/métodos , Estreptavidina/química , Tripsina/química , Arginina/análogos & derivados , Arginina/química , Biotinilação/métodos , Imunoprecipitação da Cromatina/métodos , Células HeLa , Humanos , Lisina/análogos & derivados , Lisina/química , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Complexo Repressor Polycomb 2/metabolismo , Proteólise , Fatores de Transcrição/metabolismo
4.
iScience ; 23(5): 101127, 2020 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-32422593

RESUMO

Regulatory T cells are important regulators of the immune system and have versatile functions for the homeostasis and repair of tissues. They express the forkhead box transcription factor Foxp3 as a lineage-defining protein. Negative regulators of Foxp3 expression are not well understood. Here, we generated double-stranded DNA probes complementary to the Foxp3 promoter sequence and performed a pull-down with nuclear protein in vitro, followed by elution of bound proteins and quantitative mass spectrometry. Of the Foxp3-promoter-binding transcription factors identified with this approach, one was T cell factor 1 (TCF1). Using viral over-expression, we identified TCF1 as a repressor of Foxp3 expression. In TCF1-deficient animals, increased levels of Foxp3intermediateCD25negative T cells were identified. CRISPR-Cas9 knockout studies in primary human and mouse conventional CD4 T (Tconv) cells revealed that TCF1 protects Tconv cells from inadvertent Foxp3 expression. Our data implicate a role of TCF1 in suppressing Foxp3 expression in activated T cells.

5.
Nat Commun ; 11(1): 1388, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32170121

RESUMO

Transcription factors (TFs) control cell fates by precisely orchestrating gene expression. However, how individual TFs promote transcriptional diversity remains unclear. Here, we use the Hox TF Ultrabithorax (Ubx) as a model to explore how a single TF specifies multiple cell types. Using proximity-dependent Biotin IDentification in Drosophila, we identify Ubx interactomes in three embryonic tissues. We find that Ubx interacts with largely non-overlapping sets of proteins with few having tissue-specific RNA expression. Instead most interactors are active in many cell types, controlling gene expression from chromatin regulation to the initiation of translation. Genetic interaction assays in vivo confirm that they act strictly lineage- and process-specific. Thus, functional specificity of Ubx seems to play out at several regulatory levels and to result from the controlled restriction of the interaction potential by the cellular environment. Thereby, it challenges long-standing assumptions such as differential RNA expression as determinant for protein complexes.


Assuntos
Linhagem da Célula/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila/embriologia , Drosophila/metabolismo , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição/metabolismo , Animais , Cromatina/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Genes de Insetos , Proteínas de Homeodomínio/genética , Masculino , Mesoderma/citologia , Mesoderma/metabolismo , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , RNA/metabolismo , Fatores de Transcrição/genética
6.
Mol Cell ; 64(3): 624-635, 2016 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-27773674

RESUMO

Maintenance of pluripotency is regulated by a network of transcription factors coordinated by Oct4, Sox2, and Nanog (OSN), yet a systematic investigation of the composition and dynamics of the OSN protein network specifically on chromatin is still missing. Here we have developed a method combining ChIP with selective isolation of chromatin-associated proteins (SICAP) followed by mass spectrometry to identify chromatin-bound partners of a protein of interest. ChIP-SICAP in mouse embryonic stem cells (ESCs) identified over 400 proteins associating with OSN, including several whose interaction depends on the pluripotent state. Trim24, a previously unrecognized protein in the network, converges with OSN on multiple enhancers and suppresses the expression of developmental genes while activating cell cycle genes. Consistently, Trim24 significantly improved efficiency of cellular reprogramming, demonstrating its direct functionality in establishing pluripotency. Collectively, ChIP-SICAP provides a powerful tool to decode chromatin protein composition, further enhanced by its integrative capacity to perform ChIP-seq.


Assuntos
Cromatina/química , Células-Tronco Embrionárias Murinas/metabolismo , Proteína Homeobox Nanog/genética , Proteínas Nucleares/genética , Fator 3 de Transcrição de Octâmero/genética , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição/genética , Animais , Sítios de Ligação , Diferenciação Celular , Reprogramação Celular , Cromatina/metabolismo , Imunoprecipitação da Cromatina/métodos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Marcação por Isótopo , Espectrometria de Massas/métodos , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Proteína Homeobox Nanog/metabolismo , Proteínas Nucleares/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/citologia , Ligação Proteica , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição/metabolismo
7.
Cell J ; 16(4): 494-505, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25685740

RESUMO

OBJECTIVE: MiR-302-367 is a cluster of polycistronic microRNAs that are exclusively expressed in embryonic stem (ES) cells. The miR-302-367 promoter is functional during embryonic development but is turned off in later stages. Motivated by the cancer stem cell hypothesis, we explored the potential expression of miR-302 in brain tumor cell lines. MATERIALS AND METHODS: In the present experimental study, we have tried to expand our knowledge on the expression pattern and functionality of miR302 cluster by quantifying its expression in a series of glioma (A-172, 1321N1, U87MG) and medulloblastoma (DAOY) cell lines. To further assess the functionality of miR-302 in these cell lines, we cloned its promoter core region upstream of the enhanced green fluorescent protein (EGFP) or luciferase encoding genes. RESULTS: Our data demonstrated a very low expression of miR-302 in glioma cell lines, compared with that of embryonal carcinoma cell line NT2 being used as a positive control. The expression of miR-302 promoter-EGFP construct in the aforementioned cell lines demonstrated GFP expression in a rare subpopulation of the cells. Serum deprivation led to the generation of tumorospheres, enrichment of miR-302 positive cells and upregulation of a number of pluripotency genes. CONCLUSION: Taken together, our data suggest that miR-302 could potentially be used as a novel putative cancer stem cell marker to identify and target cancer stem cells within tumor tissues.

8.
Stem Cells ; 32(1): 126-34, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24105929

RESUMO

Long noncoding RNAs (lncRNAs) have emerged as new regulators of stem cell pluripotency and tumorigenesis. The SOX2 gene, a master regulator of pluripotency, is embedded within the third intron of a lncRNA known as SOX2 overlapping transcript (SOX2OT). SOX2OT has been suspected to participate in regulation of SOX2 expression and/or other related processes; nevertheless, its potential involvement in tumor initiation and/or progression is unclear. Here, we have evaluated a possible correlation between expression patterns of SOX2OT and those of master regulators of pluripotency, SOX2 and OCT4, in esophageal squamous cell carcinoma (ESCC) tissue samples. We have also examined its potential function in the human embryonic carcinoma stem cell line, NTERA2 (NT2), which highly expresses SOX2OT, SOX2, and OCT4. Our data revealed a significant coupregulation of SOX2OT along with SOX2 and OCT4 in tumor samples, compared to the non-tumor tissues obtained from the margin of same tumors. We also identified two novel splice variants of SOX2OT (SOX2OT-S1 and SOX2OT-S2) which coupregulated with SOX2 and OCT4 in ESCCs. Suppressing SOX2OT variants caused a profound alteration in cell cycle distribution, including a 5.9 and 6.9 time increase in sub-G1 phase of cell cycle for SOX2OT-S1 and SOX2OT-S2, respectively. The expression of all variants was significantly diminished, upon the induction of neural differentiation in NT2 cells, suggesting their potential functional links to the undifferentiated state of the cells. Our data suggest a part for SOX2OT spliced variants in tumor initiation and/or progression as well as regulating pluripotent state of stem cells.


Assuntos
Carcinoma de Células Escamosas/genética , Células-Tronco de Carcinoma Embrionário/fisiologia , Neoplasias Esofágicas/genética , Fator 3 de Transcrição de Octâmero/genética , Fatores de Transcrição SOXB1/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Técnicas de Cultura de Células , Diferenciação Celular/genética , Células-Tronco de Carcinoma Embrionário/citologia , Células-Tronco de Carcinoma Embrionário/metabolismo , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago , Humanos , Fator 3 de Transcrição de Octâmero/biossíntese , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/fisiologia , Isoformas de Proteínas , Interferência de RNA , Splicing de RNA , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Fatores de Transcrição SOXB1/biossíntese , Regulação para Cima
9.
Cell Rep ; 2(6): 1579-92, 2012 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-23260666

RESUMO

Generation of induced pluripotent stem cells (iPSCs) is a process whose mechanistic underpinnings are only beginning to emerge. Here, we applied in-depth quantitative proteomics to monitor proteome changes during the course of reprogramming of fibroblasts to iPSCs. We uncover a two-step resetting of the proteome during the first and last 3 days of reprogramming, with multiple functionally related proteins changing in expression in a highly coordinated fashion. This comprised several biological processes, including changes in the stoichiometry of electron transport-chain complexes, repressed vesicle-mediated transport during the intermediate stage, and an EMT-like process in the late phase. In addition, we demonstrate that the nucleoporin Nup210 is essential for reprogramming by its permitting of rapid cellular proliferation and subsequent progression through MET. Along with the identification of proteins expressed in a stage-specific manner, this study provides a rich resource toward an enhanced mechanistic understanding of cellular reprogramming.


Assuntos
Proliferação de Células , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteoma/metabolismo , Animais , Linhagem Celular , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo
10.
Urol J ; 9(3): 574-80, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22903480

RESUMO

PURPOSE: To investigate and compare the expression of OCT4B1 between tumor and non-tumor bladder tissues. MATERIALS AND METHODS: We investigated the expression of OCT4B1 in 30 tumor and non-tumor surgical specimens of the bladder, using the TaqMan real-time polymerase chain reaction approach and by carefully designing primers and probes specific for the amplification of the variant. RESULTS: Most tumor and non-tumor samples of the bladder showed OCT4B1 expression, but its expression level was significantly higher in the tumors (P < .002). Moreover, the up-regulation of OCT4B1 was more significant in high-grade tumors compared to the low-grade ones (P < .05). We have also employed the RNA interference strategy to evaluate the functional role of OCT4B1 in a bladder cancer cell line, 5637. Suppression of OCT4B1 caused some changes in cell cycle distribution, and significantly elevated the rate of apoptosis in the cells. CONCLUSION: Our findings suggest that OCT4B1 plays a potential role in tumor initiation and/or progression of the bladder cancer. Additionally, OCT4B1 can be regarded as a new tumor marker for detection, classification, and treatment of the bladder cancer. However, more experimental studies are needed to replicate our findings.


Assuntos
Biomarcadores Tumorais/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Apoptose , Ciclo Celular , Linhagem Celular Tumoral , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Fator 3 de Transcrição de Octâmero/genética , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima
11.
J Biosci Bioeng ; 104(3): 178-81, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17964480

RESUMO

Strain XII, a moderately halophilic bacterium, expressed a peptide in response to saline media. This peptide was designated as salt-inducible factor (Sif-A). The purpose of this study is to describe Sif-A, which might be involved in the osmoresistance mechanism of strain XII. The complete sequence of sif-A was determined using PCR. sif-A codes for a polypeptide of 20.518 kDa. The polypeptide has a putative signal peptide of 27 amino acids (2.667 kDa) preceding the mature protein (17.869 kDa). Motif analysis of the deduced amino acid sequence indicated that there is a p-loop NTPase domain on the C-terminal of the peptide, which might correlate with its function. The sequence of the 16S rRNA gene was analyzed phylogenetically to classify strain XII. This organism was found to have the closest association with Virgibacillus halodenitrificans, which was proven by its phenotypic characteristics.


Assuntos
Halobacteriales/enzimologia , Halobacteriales/genética , Peptídeos/química , Peptídeos/genética , Fosfotransferases/química , Fosfotransferases/genética , Sais/química , Sequência de Aminoácidos , Sequência de Bases , Ativação Enzimática , Dados de Sequência Molecular , Equilíbrio Hidroeletrolítico/fisiologia
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