Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 62
Filtrar
1.
RNA ; 30(8): 1089-1105, 2024 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-38760076

RESUMO

Many transcription factors (TFs) have been shown to bind RNA, leading to open questions regarding the mechanism(s) of this RNA binding and its role in regulating TF activities. Here, we use biophysical assays to interrogate the k on, k off, and K d for DNA and RNA binding of two model human TFs, ERα and Sox2. Unexpectedly, we found that both proteins exhibit multiphasic nucleic acid-binding kinetics. We propose that Sox2 RNA and DNA multiphasic binding kinetics can be explained by a conventional model for sequential Sox2 monomer association and dissociation. In contrast, ERα nucleic acid binding exhibited biphasic dissociation paired with novel triphasic association behavior, in which two apparent binding transitions are separated by a 10-20 min "lag" phase depending on protein concentration. We considered several conventional models for the observed kinetic behavior, none of which adequately explained all the ERα nucleic acid-binding data. Instead, simulations with a model incorporating sequential ERα monomer association, ERα nucleic acid complex isomerization, and product "feedback" on isomerization rate recapitulated the general kinetic trends for both ERα DNA and RNA binding. Collectively, our findings reveal that Sox2 and ERα bind RNA and DNA with previously unappreciated multiphasic binding kinetics, and that their reaction mechanisms differ with ERα binding nucleic acids via a novel reaction mechanism.


Assuntos
DNA , Receptor alfa de Estrogênio , Ligação Proteica , RNA , Fatores de Transcrição SOXB1 , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/química , Humanos , Receptor alfa de Estrogênio/metabolismo , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/química , DNA/metabolismo , DNA/química , RNA/metabolismo , RNA/química , RNA/genética , Cinética , Sítios de Ligação
2.
Nat Commun ; 15(1): 1445, 2024 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-38365983

RESUMO

More than 1600 human transcription factors orchestrate the transcriptional machinery to control gene expression and cell fate. Their function is conveyed through intrinsically disordered regions (IDRs) containing activation or repression domains but lacking quantitative structural ensemble models prevents their mechanistic decoding. Here we integrate single-molecule FRET and NMR spectroscopy with molecular simulations showing that DNA binding can lead to complex changes in the IDR ensemble and accessibility. The C-terminal IDR of pioneer factor Sox2 is highly disordered but its conformational dynamics are guided by weak and dynamic charge interactions with the folded DNA binding domain. Both DNA and nucleosome binding induce major rearrangements in the IDR ensemble without affecting DNA binding affinity. Remarkably, interdomain interactions are redistributed in complex with DNA leading to variable exposure of two activation domains critical for transcription. Charged intramolecular interactions allowing for dynamic redistributions may be common in transcription factors and necessary for sensitive tuning of structural ensembles.


Assuntos
Proteínas Intrinsicamente Desordenadas , Fatores de Transcrição SOXB1 , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Espectroscopia de Ressonância Magnética , Conformação Proteica , Domínios Proteicos , Fatores de Transcrição/genética , Fatores de Transcrição/química , Fatores de Transcrição SOXB1/química , Fatores de Transcrição SOXB1/genética
3.
J Mol Biol ; 435(2): 167916, 2023 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-36495920

RESUMO

Pioneer transcription factors (pTFs) can bind directly to silent chromatin and promote vital transcriptional programs. Here, by integrating high-resolution nuclear magnetic resonance (NMR) spectroscopy with biochemistry, we reveal new structural and mechanistic insights into the interaction of pluripotency pTFs and functional partners Sox2 and Oct4 with nucleosomes. We find that the affinity and conformation of Sox2 for solvent-exposed nucleosome sites depend strongly on their position and DNA sequence. Sox2, which is partially disordered but becomes structured upon DNA binding and bending, forms a super-stable nucleosome complex at superhelical location +5 (SHL+5) with similar affinity and conformation to that with naked DNA. However, at suboptimal internal and end-positioned sites where DNA may be harder to deform, Sox2 favors partially unfolded and more dynamic states that are encoded in its intrinsic flexibility. Importantly, Sox2 structure and DNA bending can be stabilized by synergistic Oct4 binding, but only on adjacent motifs near the nucleosome edge and with the full Oct4 DNA-binding domain. Further mutational studies reveal that strategically impaired Sox2 folding is coupled to reduced DNA bending and inhibits nucleosome binding and Sox2-Oct4 cooperation, while increased nucleosomal DNA flexibility enhances Sox2 association. Together, our findings fit a model where the site-specific DNA bending propensity and structural plasticity of Sox2 govern distinct modes of nucleosome engagement and modulate Sox2-Oct4 synergism. The principles outlined here can potentially guide pTF site selection in the genome and facilitate interaction with other chromatin factors or chromatin opening in vivo.


Assuntos
DNA , Conformação de Ácido Nucleico , Nucleossomos , Fatores de Transcrição SOXB1 , Sequência de Bases , Cromatina , DNA/química , DNA/metabolismo , Nucleossomos/metabolismo , Domínios Proteicos , Fatores de Transcrição SOXB1/química , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Ressonância Magnética Nuclear Biomolecular , Fator 3 de Transcrição de Octâmero/química , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Humanos
4.
Lupus ; 31(1): 89-96, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-35019799

RESUMO

BACKGROUND: The skin and the kidney are commonly affected in systemic lupus erythematosus (SLE) with similar molecular mechanisms. Although clinical indicators of renal injury in SLE are fairly uncontroversial, few biomarkers are reliable. The role of micro-RNAs (mi-RNAs) in lupus nephritis (LN) pathogenesis has been investigated to help in early diagnosis. PURPOSE: The aim of work is to evaluate miRNA132 and SOX2 expressions in SLE Egyptian patients; with and without nephritis, and the relation between miRNA132 and its long non-coding gene SOX2 in both patients groups. RESEARCH DESIGN: This is a case-control study involving 100 SLE patients with and without LN (LN and non-LN groups), and 50 age-and sex-matched healthy controls. The study was carried out to detect miRNA132 and SOX2 expression by quantitative Real-Time Polymerase chain reaction methods. The SLE disease activity index (SLEDAI) was assessed. RESULTS: SLEDAI increased in LN compared to non-LN. Micro-RNA132 expression was significantly increased in patient groups compared to controls (p<0.01) and increased in LN more than non-LN group (p<0.001). SOX2 significantly decreased in patient groups compared to controls (p<0.001), and was more in LN compared to non-LN group (p<0.001). There was a negative correlation between miRNA132 and SOX2 expression in both patient groups (p<0.001). CONCLUSION: miRNA132 and SOX2 may play a role in SLE activity and help in the early non-invasive diagnosis of LN.


Assuntos
Lúpus Eritematoso Sistêmico , Nefrite Lúpica , MicroRNAs , Biomarcadores , Estudos de Casos e Controles , Humanos , Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/genética , Nefrite Lúpica/diagnóstico , Nefrite Lúpica/genética , MicroRNAs/genética , Fatores de Transcrição SOXB1/química , Fatores de Transcrição SOXB1/genética
5.
Int J Mol Sci ; 22(17)2021 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-34502261

RESUMO

SOX2 is an oncogenic transcription factor overexpressed in nearly half of the basal-like triple-negative breast cancers associated with very poor outcomes. Targeting and inhibiting SOX2 is clinically relevant as high SOX2 mRNA levels are positively correlated with decreased overall survival and progression-free survival in patients affected with breast cancer. Given its key role as a master regulator of cell proliferation, SOX2 represents an important scaffold for the engineering of dominant-negative synthetic DNA-binding domains (DBDs) that act by blocking or interfering with the oncogenic activity of the endogenous transcription factor in cancer cells. We have synthesized an interference peptide (iPep) encompassing a truncated 24 amino acid long C-terminus of SOX2 containing a potential SOX-specific nuclear localization sequence, and the determinants of the binding of SOX2 to the DNA and to its transcription factor binding partners. We found that the resulting peptide (SOX2-iPep) possessed intrinsic cell penetration and promising nuclear localization into breast cancer cells, and decreased cellular proliferation of SOX2 overexpressing cell lines. The novel SOX2-iPep was found to exhibit a random coil conformation predominantly in solution. Molecular dynamics simulations were used to characterize the interactions of both the SOX2 transcription factor and the SOX2-iPep with FGF4-enhancer DNA in the presence of the POU domain of the partner transcription factor OCT4. Predictions of the free energy of binding revealed that the iPep largely retained the binding affinity for DNA of parental SOX2. This work will enable the future engineering of novel dominant interference peptides to transport different therapeutic cargo molecules such as anti-cancer drugs into cells.


Assuntos
Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/farmacologia , Fatores de Transcrição SOXB1/química , Fatores de Transcrição SOXB1/metabolismo , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , DNA/metabolismo , Feminino , Fator 4 de Crescimento de Fibroblastos/química , Humanos , Estimativa de Kaplan-Meier , Camundongos , Simulação de Dinâmica Molecular , Fator 3 de Transcrição de Octâmero/química , Ligação Proteica , Fatores de Transcrição SOXB1/genética , Água/química
6.
Nat Commun ; 12(1): 1085, 2021 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-33597515

RESUMO

Liquid-liquid phase separation of proteins underpins the formation of membraneless compartments in living cells. Elucidating the molecular driving forces underlying protein phase transitions is therefore a key objective for understanding biological function and malfunction. Here we show that cellular proteins, which form condensates at low salt concentrations, including FUS, TDP-43, Brd4, Sox2, and Annexin A11, can reenter a phase-separated regime at high salt concentrations. By bringing together experiments and simulations, we demonstrate that this reentrant phase transition in the high-salt regime is driven by hydrophobic and non-ionic interactions, and is mechanistically distinct from the low-salt regime, where condensates are additionally stabilized by electrostatic forces. Our work thus sheds light on the cooperation of hydrophobic and non-ionic interactions as general driving forces in the condensation process, with important implications for aberrant function, druggability, and material properties of biomolecular condensates.


Assuntos
Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Transição de Fase , Proteínas/química , Eletricidade Estática , Animais , Anexinas/química , Proteínas de Ciclo Celular/química , Proteínas de Ligação a DNA/química , Humanos , Proteína FUS de Ligação a RNA/química , Fatores de Transcrição SOXB1/química , Células Sf9 , Spodoptera , Fatores de Transcrição/química
7.
Proc Natl Acad Sci U S A ; 118(7)2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33574062

RESUMO

P27, a cell cycle inhibitor, is also able to drive repression of Sox2 This interaction plays a crucial role during development of p27-/- pituitary tumors because loss of one copy of Sox2 impairs tumorigenesis [H. Li et al., Cell Stem Cell 11, 845-852 (2012)]. However, SOX2 is expressed in both endocrine and stem cells (SCs), and its contribution to tumorigenesis in either cell type is unknown. We have thus explored the cellular origin and mechanisms underlying endocrine tumorigenesis in p27-/- pituitaries. We found that pituitary hyperplasia is associated with reduced cellular differentiation, in parallel with increased levels of SOX2 in stem and endocrine cells. Using conditional loss-of-function and lineage tracing approaches, we show that SOX2 is required cell autonomously in p27-/- endocrine cells for these to give rise to tumors, and in SCs for promotion of tumorigenesis. This is supported by studies deleting the Sox2 regulatory region 2 (Srr2), the target of P27 repressive action. Single cell transcriptomic analysis further reveals that activation of a SOX2-dependent MAPK pathway in SCs is important for tumorigenesis. Altogether, our data highlight different aspects of the role of SOX2 following loss of p27, according to cellular context, and uncover an unexpected SOX2-dependent tumor-promoting role for SCs. Our results imply that targeting SCs, in addition to tumor cells, may represent an efficient antitumoral strategy in certain contexts.


Assuntos
Carcinogênese/metabolismo , Neoplasias Hipofisárias/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Animais , Carcinogênese/genética , Linhagem da Célula , Inibidor de Quinase Dependente de Ciclina p27/deficiência , Inibidor de Quinase Dependente de Ciclina p27/genética , Células Endócrinas/metabolismo , Mutação com Perda de Função , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neoplásicas/metabolismo , Neoplasias Hipofisárias/genética , Neoplasias Hipofisárias/patologia , Domínios Proteicos , Fatores de Transcrição SOXB1/química , Fatores de Transcrição SOXB1/genética
8.
Nat Commun ; 12(1): 28, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397924

RESUMO

SOX (SRY-related HMG-box) transcription factors perform critical functions in development and cell differentiation. These roles depend on precise nuclear trafficking, with mutations in the nuclear targeting regions causing developmental diseases and a range of cancers. SOX protein nuclear localization is proposed to be mediated by two nuclear localization signals (NLSs) positioned within the extremities of the DNA-binding HMG-box domain and, although mutations within either cause disease, the mechanistic basis has remained unclear. Unexpectedly, we find here that these two distantly positioned NLSs of SOX2 contribute to a contiguous interface spanning 9 of the 10 ARM domains on the nuclear import adapter IMPα3. We identify key binding determinants and show this interface is critical for neural stem cell maintenance and for Drosophila development. Moreover, we identify a structural basis for the preference of SOX2 binding to IMPα3. In addition to defining the structural basis for SOX protein localization, these results provide a platform for understanding how mutations and post-translational modifications within these regions may modulate nuclear localization and result in clinical disease, and also how other proteins containing multiple NLSs may bind IMPα through an extended recognition interface.


Assuntos
Núcleo Celular/metabolismo , Fatores de Transcrição SOXB1/química , Fatores de Transcrição SOXB1/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Drosophila/metabolismo , Células HEK293 , Humanos , Camundongos , Modelos Moleculares , Proteínas Mutantes/metabolismo , Células-Tronco Neurais/metabolismo , Sinais de Localização Nuclear/metabolismo , Mutação Puntual/genética , Ligação Proteica , Domínios Proteicos , Isoformas de Proteínas/metabolismo , Fatores de Transcrição SOXB1/genética , Relação Estrutura-Atividade
9.
Cells ; 9(11)2020 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-33202879

RESUMO

The transcription factor SOX1 is a key regulator of neural stem cell development, acting to keep neural stem cells (NSCs) in an undifferentiated state. Postnatal expression of Sox1 is typically confined to the central nervous system (CNS), however, its expression in non-neural tissues has recently been implicated in tumorigenesis. The mechanism through which SOX1 may exert its function is not fully understood, and studies have mainly focused on changes in SOX1 expression at a transcriptional level, while its post-translational regulation remains undetermined. To investigate this, data were extracted from different publicly available databases and analysed to search for putative SOX1 post-translational modifications (PTMs). Results were compared to PTMs associated with SOX2 in order to identify potentially key PTM motifs common to these SOXB1 proteins, and mapped on SOX1 domain structural models. This approach identified several putative acetylation, phosphorylation, glycosylation and sumoylation sites within known functional domains of SOX1. In particular, a novel SOXB1 motif (xKSExSxxP) was identified within the SOX1 protein, which was also found in other unrelated proteins, most of which were transcription factors. These results also highlighted potential phospho-sumoyl switches within this SOXB1 motif identified in SOX1, which could regulate its transcriptional activity. This analysis indicates different types of PTMs within SOX1, which may influence its regulatory role as a transcription factor, by bringing changes to its DNA binding capacities and its interactions with partner proteins. These results provide new research avenues for future investigations on the mechanisms regulating SOX1 activity, which could inform its roles in the contexts of neural stem cell development and cancer.


Assuntos
Simulação por Computador , Processamento de Proteína Pós-Traducional , Fatores de Transcrição SOXB1/química , Fatores de Transcrição SOXB1/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência Consenso , Ontologia Genética , Humanos , Modelos Moleculares , Fosforilação , Sumoilação
10.
Nature ; 580(7805): 669-672, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32350470

RESUMO

'Pioneer' transcription factors are required for stem-cell pluripotency, cell differentiation and cell reprogramming1,2. Pioneer factors can bind nucleosomal DNA to enable gene expression from regions of the genome with closed chromatin. SOX2 is a prominent pioneer factor that is essential for pluripotency and self-renewal of embryonic stem cells3. Here we report cryo-electron microscopy structures of the DNA-binding domains of SOX2 and its close homologue SOX11 bound to nucleosomes. The structures show that SOX factors can bind and locally distort DNA at superhelical location 2. The factors also facilitate detachment of terminal nucleosomal DNA from the histone octamer, which increases DNA accessibility. SOX-factor binding to the nucleosome can also lead to a repositioning of the N-terminal tail of histone H4 that includes residue lysine 16. We speculate that this repositioning is incompatible with higher-order nucleosome stacking, which involves contacts of the H4 tail with a neighbouring nucleosome. Our results indicate that pioneer transcription factors can use binding energy to initiate chromatin opening, and thereby facilitate nucleosome remodelling and subsequent transcription.


Assuntos
Montagem e Desmontagem da Cromatina , Microscopia Crioeletrônica , Nucleossomos/metabolismo , Fatores de Transcrição SOXB1/química , Fatores de Transcrição SOXB1/metabolismo , Fatores de Transcrição SOXC/química , Fatores de Transcrição SOXC/metabolismo , Sequência de Bases , DNA Super-Helicoidal/química , DNA Super-Helicoidal/genética , DNA Super-Helicoidal/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Lisina/metabolismo , Modelos Biológicos , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestrutura , Nucleossomos/química , Nucleossomos/ultraestrutura , Fatores de Transcrição SOXB1/ultraestrutura , Fatores de Transcrição SOXC/ultraestrutura
11.
Science ; 368(6498): 1460-1465, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32327602

RESUMO

Transcription factors (TFs) regulate gene expression through chromatin where nucleosomes restrict DNA access. To study how TFs bind nucleosome-occupied motifs, we focused on the reprogramming factors OCT4 and SOX2 in mouse embryonic stem cells. We determined TF engagement throughout a nucleosome at base-pair resolution in vitro, enabling structure determination by cryo-electron microscopy at two preferred positions. Depending on motif location, OCT4 and SOX2 differentially distort nucleosomal DNA. At one position, OCT4-SOX2 removes DNA from histone H2A and histone H3; however, at an inverted motif, the TFs only induce local DNA distortions. OCT4 uses one of its two DNA-binding domains to engage DNA in both structures, reading out a partial motif. These findings explain site-specific nucleosome engagement by the pluripotency factors OCT4 and SOX2, and they reveal how TFs distort nucleosomes to access chromatinized motifs.


Assuntos
Regulação da Expressão Gênica , Nucleossomos/química , Fator 3 de Transcrição de Octâmero/química , Fatores de Transcrição SOXB1/química , Animais , Microscopia Crioeletrônica , DNA/química , Histonas/química , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo
12.
Nat Commun ; 11(1): 1805, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286318

RESUMO

Certain transcription factors are proposed to form functional interactions with RNA to facilitate proper regulation of gene expression. Sox2, a transcription factor critical for maintenance of pluripotency and neurogenesis, has been found associated with several lncRNAs, although it is unknown whether these interactions are direct or via other proteins. Here we demonstrate that human Sox2 interacts directly with one of these lncRNAs with high affinity through its HMG DNA-binding domain in vitro. These interactions are primarily with double-stranded RNA in a non-sequence specific fashion, mediated by a similar but not identical interaction surface. We further determined that Sox2 directly binds RNA in mouse embryonic stem cells by UV-cross-linked immunoprecipitation of Sox2 and more than a thousand Sox2-RNA interactions in vivo were identified using fRIP-seq. Together, these data reveal that Sox2 employs a high-affinity/low-specificity paradigm for RNA binding in vitro and in vivo.


Assuntos
RNA/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Animais , Sequência de Bases , Ligação Competitiva , DNA/metabolismo , Masculino , Camundongos , Modelos Moleculares , Células-Tronco Embrionárias Murinas/metabolismo , Ligação Proteica , Domínios Proteicos , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Fatores de Transcrição SOXB1/química , Deleção de Sequência , Eletricidade Estática
13.
Nucleic Acids Res ; 48(7): 3869-3887, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32016422

RESUMO

Some transcription factors that specifically bind double-stranded DNA appear to also function as RNA-binding proteins. Here, we demonstrate that the transcription factor Sox2 is able to directly bind RNA in vitro as well as in mouse and human cells. Sox2 targets RNA via a 60-amino-acid RNA binding motif (RBM) positioned C-terminally of the DNA binding high mobility group (HMG) box. Sox2 can associate with RNA and DNA simultaneously to form ternary RNA/Sox2/DNA complexes. Deletion of the RBM does not affect selection of target genes but mitigates binding to pluripotency related transcripts, switches exon usage and impairs the reprogramming of somatic cells to a pluripotent state. Our findings designate Sox2 as a multi-functional factor that associates with RNA whilst binding to cognate DNA sequences, suggesting that it may co-transcriptionally regulate RNA metabolism during somatic cell reprogramming.


Assuntos
Reprogramação Celular/genética , DNA/metabolismo , RNA/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Motivos de Aminoácidos , Animais , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Ligação Proteica , Domínios Proteicos , Splicing de RNA , Fatores de Transcrição SOXB1/química
14.
Biochim Biophys Acta Biomembr ; 1862(2): 183106, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31669571

RESUMO

Gastric cancer is associated with high mortality and is preceded by an infection with Helicobacter pylori (H. pylori). H. pylori stimulates inflammation which involves the activation of Toll-like receptor 4 by lipopolysaccharide molecules from the H. pylori. This leads to chronic inflammation that can eventually lead to gastric cancer. Sox2 is a member of the high mobility group (HMG) box family of proteins, and recent studies have shown that HMG box proteins can modulate immune response by altering signaling to Toll-like receptors. Sox2 is overexpressed in most types of cancer with the exception of gastric cancer where expression of Sox2 is decreased. Here, we demonstrate that Sox2 can bind LPS and we investigated the thermodynamic drivers of the Sox2/LPS interaction.


Assuntos
Domínios HMG-Box , Lipopolissacarídeos/química , Simulação de Acoplamento Molecular , Fatores de Transcrição SOXB1/química , Helicobacter pylori/química , Humanos , Lipopolissacarídeos/metabolismo , Ligação Proteica , Fatores de Transcrição SOXB1/metabolismo
15.
Cytogenet Genome Res ; 159(3): 143-150, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31760386

RESUMO

Two species of spiny rats, Tokudaia osimensis and Tokudaia tokunoshimensis, show an X0/X0 sex chromosome constitution due to the lack of a Y chromosome. The Sry gene has been completely lost from the genome of these species. We hypothesized that Sox3, which is thought to be originally a homologue of Sry, could function in sex determination in these animals in the absence of Sry. Sox3 was localized in a region of the X chromosome in T. osimensis homologous to mouse. A similar testis- and ovary-specific pattern of expression was observed in mouse and T. osimensis. Although the sequence of the Sox3 gene and its promoter are highly conserved, a 13-bp deletion was specifically found in the promoter region of the 2 spiny rat species. Reporter gene assays were performed to examine the effect of the 13-bp deletion in the promoter region on Sox3 regulation. Although an approximately 60% decrease in activity was observed using the Tokudaia promoters with the 13-bp deletion, the activity was recovered using a mutated promoter in which the deletion was filled with mouse sequence. To evaluate whether SOX3 could regulate Sox9 expression, a reporter gene assay was carried out using testis-specific enhancer of Sox9 core (TESCO). Co-transfection with a combination of mouse SF1 and mouse SOX3 or T. osimensis SOX3 resulted in a greater than 2-fold increase in activity of mouse and T. osimensis TESCO. These results support the idea that the function of SOX3 as a transcription factor, as has been reported in mice and humans, is conserved in T. osimensis. Therefore, we conclude that the Sox3 gene has no function in sex determination in Sry-lacking Tokudaia species.


Assuntos
Murinae/genética , Fatores de Transcrição SOXB1/genética , Proteína da Região Y Determinante do Sexo/genética , Sequência de Aminoácidos , Animais , Espécies em Perigo de Extinção , Feminino , Deleção de Genes , Genes Reporter , Masculino , Regiões Promotoras Genéticas , Fatores de Transcrição SOXB1/química , Homologia de Sequência de Aminoácidos
16.
Oncogene ; 38(10): 1764-1777, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30382189

RESUMO

Pancreatic cancer, mostly pancreatic ductal adenocarcinomas (PDAC), is one of the most lethal cancers, with a dismal median survival around 8 months. PDAC is notoriously resistant to chemotherapy. Thus far, numerous attempts using novel targeted therapies and immunotherapies yielded limited clinical benefits for pancreatic cancer patients. It is hoped that delineating the molecular mechanisms underlying drug resistance in pancreatic cancer may provide novel therapeutic options. Using acquired gemcitabine resistant pancreatic cell lines, we revealed an important role of the GLI-SOX2 signaling axis for regulation of gemcitabine sensitivity in vitro and in animal models. Down-regulation of GLI transcriptional factors (GLI1 or GLI2), but not SMO signaling inhibition, reduces tumor sphere formation, a characteristics of tumor initiating cell (TIC). Down-regulation of GLI transcription factors also decreased expression of TIC marker CD24. Similarly, high SOX2 expression is associated with gemcitabine resistance whereas down-regulation of SOX2 sensitizes pancreatic cancer cells to gemcitabine treatment. We further revealed that elevated SOX2 expression is associated with an increase in GLI1 or GLI2 expression. Our ChIP assay revealed that GLI proteins are associated with a putative Gli binding site within the SOX2 promoter, suggesting a more direct regulation of SOX2 by GLI transcription factors. The relevance of our findings to human disease was revealed in human cancer specimens. We found that high SOX2 protein expression is associated with frequent tumor relapse and poor survival in stage II PDAC patients (all of them underwent gemcitabine treatment), indicating that reduced SOX2 expression or down-regulation of GLI transcription factors may be effective in sensitizing pancreatic cancer cells to gemcitabine treatment.


Assuntos
Carcinoma Ductal Pancreático/patologia , Resistencia a Medicamentos Antineoplásicos , Proteínas Nucleares/genética , Neoplasias Pancreáticas/patologia , Fatores de Transcrição SOXB1/genética , Proteína GLI1 em Dedos de Zinco/genética , Proteína Gli2 com Dedos de Zinco/genética , Animais , Sítios de Ligação , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Desoxicitidina/análogos & derivados , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Transplante de Neoplasias , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fatores de Transcrição SOXB1/química , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Análise de Sobrevida , Regulação para Cima , Proteína GLI1 em Dedos de Zinco/metabolismo , Proteína Gli2 com Dedos de Zinco/metabolismo , Gencitabina
17.
Int J Mol Sci ; 19(12)2018 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-30518054

RESUMO

Sox2 is a pioneer transcription factor that initiates cell fate reprogramming through locus-specific differential regulation. Mechanistically, it was assumed that Sox2 achieves its regulatory diversity via heterodimerization with partner transcription factors. Here, utilizing single-molecule fluorescence spectroscopy, we show that Sox2 alone can modulate DNA structural landscape in a dosage-dependent manner. We propose that such stoichiometric tuning of regulatory DNAs is crucial to the diverse biological functions of Sox2, and represents a generic mechanism of conferring functional plasticity and multiplicity to transcription factors.


Assuntos
DNA/química , Domínios HMG-Box , Conformação de Ácido Nucleico , Fatores de Transcrição SOXB1/química , Imagem Individual de Molécula , Transferência Ressonante de Energia de Fluorescência , Modelos Moleculares , Regiões Promotoras Genéticas/genética , Ligação Proteica
18.
Mol Cell Endocrinol ; 478: 133-140, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30125608

RESUMO

SOX3, a transcription factor of the SRY-related high mobility group box family, has been implicated in the etiology of X-linked hypopituitarism. Here, we report a Chinese pedigree of X-linked hypopituitarism with variable phenotypes. Despite the complete growth hormone deficiency, the growth failure of the patients was relatively modest. A rare point variant of SOX3 (c.424C > A; p. P142T) was identified in the pedigree via target panel sequencing. An in vitro study showed that both the expression and nuclear targeting of SOX3 remained unaffected by the variant. However, increased transcriptional activation and impaired repression of ß-catenin-mediated transcription were noticed as a result of the SOX3 variant. This is the first study to report that the rare SOX3 missense variant associated with hypopituitarism possibly due to increased activation of SOX3 target genes and disregulation of ß-catenin target genes. In addition, we have expanded the phenotypic spectrum associated with SOX3 mutations.


Assuntos
Doenças Genéticas Ligadas ao Cromossomo X/genética , Crescimento e Desenvolvimento/genética , Hipopituitarismo/genética , Mutação de Sentido Incorreto/genética , Fatores de Transcrição SOXB1/genética , Transcrição Gênica , Ativação Transcricional/genética , beta Catenina/genética , Sequência de Aminoácidos , Povo Asiático/genética , Sequência de Bases , Criança , Simulação por Computador , Feminino , Teste de Tolerância a Glucose , Hormônio Liberador de Gonadotropina/farmacologia , Células HEK293 , Humanos , Imageamento por Ressonância Magnética , Masculino , Linhagem , Fatores de Transcrição SOXB1/química
19.
Evol Dev ; 20(3-4): 108-118, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29569814

RESUMO

A number of studies have established that in vertebrates, Sox3 is involved in a wide range of developmental processes, including sex differentiation and neurogenesis. However, the exact functions of the Sox3 gene have not been documented so far in teleosts. Here, we cloned the full length cDNA of Sox3 from the teleost fish, Paramisgurnus dabryanus, which we designated PdSox3. Sequence analysis revealed that PdSox3 encodes a hydrophilic protein, and shares high homology with Sox3 in other species, ranging from mammals to fishes. Quantitative real-time reverse transcription PCR, and in situ hybridization showed that PdSox3 is consistently expressed during embryogenesis, mainly localized in the developing central nervous system. Tissue distribution analyses revealed that PdSox3 is abundant in the adult brain, especially in particle cell layer. Furthermore, PdSox3 expression was higher in gonads, in primary spermatocyte cells, primary oocytes, and previtellogenic oocyte cells. All of these results suggest that PdSox3 plays an important role in early embryonic development, in particular the formation and development of the nervous system, and gonad development, similarly to other vertebrates. This is the first report describing Sox3 gene expression from this species, and the results are necessary to provide fundamental information on both the functional and evolutionary role of Sox3 across different species.


Assuntos
Cipriniformes/embriologia , Cipriniformes/genética , Proteínas de Peixes/genética , Fatores de Transcrição SOXB1/genética , Animais , Clonagem Molecular , Proteínas de Peixes/química , Filogenia , Fatores de Transcrição SOXB1/química , Homologia de Sequência de Aminoácidos , Transcriptoma
20.
J Biol Chem ; 293(10): 3663-3674, 2018 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-29358331

RESUMO

The pluripotency-controlling stem-cell protein SRY-box 2 (SOX2) plays a pivotal role in maintaining the self-renewal and pluripotency of embryonic stem cells and also of teratocarcinoma or embryonic carcinoma cells. SOX2 is monomethylated at lysine 119 (Lys-119) in mouse embryonic stem cells by the SET7 methyltransferase, and this methylation triggers ubiquitin-dependent SOX2 proteolysis. However, the molecular regulators and mechanisms controlling SET7-induced SOX2 proteolysis are unknown. Here, we report that in human ovarian teratocarcinoma PA-1 cells, methylation-dependent SOX2 proteolysis is dynamically regulated by the LSD1 lysine demethylase and a methyl-binding protein, PHD finger protein 20-like 1 (PHF20L1). We found that LSD1 not only removes the methyl group from monomethylated Lys-117 (equivalent to Lys-119 in mouse SOX2), but it also demethylates monomethylated Lys-42 in SOX2, a reaction that SET7 also regulated and that also triggered SOX2 proteolysis. Our studies further revealed that PHF20L1 binds both monomethylated Lys-42 and Lys-117 in SOX2 and thereby prevents SOX2 proteolysis. Down-regulation of either LSD1 or PHF20L1 promoted SOX2 proteolysis, which was prevented by SET7 inactivation in both PA-1 and mouse embryonic stem cells. Our studies also disclosed that LSD1 and PHF20L1 normally regulate the growth of pluripotent mouse embryonic stem cells and PA-1 cells by preventing methylation-dependent SOX2 proteolysis. In conclusion, our findings reveal an important mechanism by which the stability of the pluripotency-controlling stem-cell protein SOX2 is dynamically regulated by the activities of SET7, LSD1, and PHF20L1 in pluripotent stem cells.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Histona Desmetilases/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Ovarianas/metabolismo , Processamento de Proteína Pós-Traducional , Fatores de Transcrição SOXB1/metabolismo , Substituição de Aminoácidos , Animais , Linhagem Celular Tumoral , Células Cultivadas , Proteínas Cromossômicas não Histona/antagonistas & inibidores , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Células HEK293 , Histona Desmetilases/antagonistas & inibidores , Histona Desmetilases/química , Histona Desmetilases/genética , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/genética , Humanos , Metilação , Camundongos Endogâmicos C57BL , Mutação , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/patologia , Estabilidade Proteica , Proteólise , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição SOXB1/química , Fatores de Transcrição SOXB1/genética , Teratocarcinoma/enzimologia , Teratocarcinoma/metabolismo , Teratocarcinoma/patologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...