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1.
Cells ; 13(14)2024 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-39056784

RESUMO

SOX proteins are a family of transcription factors (TFs) that play critical functions in sex determination, neurogenesis, and chondrocyte differentiation, as well as cardiac, vascular, and lymphatic development. There are 20 SOX family members in humans, each sharing a 79-residue L-shaped high mobility group (HMG)-box domain that is responsible for DNA binding. SOX2 was recently shown to interact with long non-coding RNA and large-intergenic non-coding RNA to regulate embryonic stem cell and neuronal differentiation. The RNA binding region was shown to reside within the HMG-box domain; however, the structural details of this binding remain unclear. Here, we show that all SOX family members, except group H, interact with RNA. Our mutational experiments demonstrate that the disordered C-terminal region of the HMG-box domain plays an important role in RNA binding. Further, by determining a high-resolution structure of the HMG-box domain of the group H family member SOX30, we show that despite differences in RNA binding ability, SOX30 shares a very similar secondary structure with other SOX protein HMG-box domains. Together, our study provides insight into the interaction of SOX TFs with RNA.


Assuntos
Ligação Proteica , Fatores de Transcrição SOX , Humanos , Fatores de Transcrição SOX/metabolismo , Fatores de Transcrição SOX/genética , RNA/metabolismo , Domínios HMG-Box , Sequência de Aminoácidos
2.
Science ; 383(6689): eadk5466, 2024 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-38513029

RESUMO

In many eukaryotes, genetic sex determination is not governed by XX/XY or ZW/ZZ systems but by a specialized region on the poorly studied U (female) or V (male) sex chromosomes. Previous studies have hinted at the existence of a dominant male-sex factor on the V chromosome in brown algae, a group of multicellular eukaryotes distantly related to animals and plants. The nature of this factor has remained elusive. Here, we demonstrate that an HMG-box gene acts as the male-determining factor in brown algae, mirroring the role HMG-box genes play in sex determination in animals. Over a billion-year evolutionary timeline, these lineages have independently co-opted the HMG box for male determination, representing a paradigm for evolution's ability to recurrently use the same genetic "toolkit" to accomplish similar tasks.


Assuntos
Algas Comestíveis , Proteínas HMGB , Laminaria , Phaeophyceae , Cromossomos Sexuais , Processos de Determinação Sexual , Animais , Evolução Biológica , Phaeophyceae/genética , Cromossomos Sexuais/genética , Processos de Determinação Sexual/genética , Cromossomo Y , Proteínas HMGB/genética , Cromossomos de Plantas/genética , Domínios HMG-Box , Algas Comestíveis/genética , Laminaria/genética , Pólen/genética
3.
Int J Mol Sci ; 23(14)2022 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-35887213

RESUMO

Energy metabolism reprogramming was recently listed as a hallmark of cancer. In this process, the switch from pyruvate kinase isoenzyme type M1 to pyruvate kinase isoenzyme type M2 (PKM2) is believed to play a crucial role. Interestingly, the activity of the active form of PKM2 can efficiently be inhibited by the high-mobility group box 1 (HMGB1) protein, leading to a rapid blockage of glucose-dependent aerobic respiration and cancer cell death. HMGB1 is a member of the HMG protein family. It contains two DNA-binding HMG-box domains and an acidic C-terminal tail capable of positively or negatively modulating its biological properties. In this work, we report that the deletion of the C-terminal tail of HMGB1 increases its activity towards a large panel of cancer cells without affecting the viability of normal immortalized fibroblasts. Moreover, in silico analysis suggests that the truncated form of HMGB1 retains the capacity of the full-length protein to interact with PKM2. However, based on the capacity of the cells to circumvent oxidative phosphorylation inhibition, we were able to identify either a cytotoxic or cytostatic effect of the proteins. Together, our study provides new insights in the characterization of the anticancer activity of HMGB1.


Assuntos
Proteína HMGB1 , Domínios HMG-Box , Proteína HMGB1/metabolismo , Isoenzimas/metabolismo , Estrutura Terciária de Proteína , Piruvato Quinase/metabolismo
4.
Proc Natl Acad Sci U S A ; 118(20)2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-33975946

RESUMO

Compaction of bulky DNA is a universal issue for all DNA-based life forms. Chloroplast nucleoids (chloroplast DNA-protein complexes) are critical for chloroplast DNA maintenance and transcription, thereby supporting photosynthesis, but their detailed structure remains enigmatic. Our proteomic analysis of chloroplast nucleoids of the green alga Chlamydomonas reinhardtii identified a protein (HBD1) with a tandem repeat of two DNA-binding high mobility group box (HMG-box) domains, which is structurally similar to major mitochondrial nucleoid proteins transcription factor A, mitochondrial (TFAM), and ARS binding factor 2 protein (Abf2p). Disruption of the HBD1 gene by CRISPR-Cas9-mediated genome editing resulted in the scattering of chloroplast nucleoids. This phenotype was complemented when intact HBD1 was reintroduced, whereas a truncated HBD1 with a single HMG-box domain failed to complement the phenotype. Furthermore, ectopic expression of HBD1 in the mitochondria of yeast Δabf2 mutant successfully complemented the defects, suggesting functional similarity between HBD1 and Abf2p. Furthermore, in vitro assays of HBD1, including the electrophoretic mobility shift assay and DNA origami/atomic force microscopy, showed that HBD1 is capable of introducing U-turns and cross-strand bridges, indicating that proteins with two HMG-box domains would function as DNA clips to compact DNA in both chloroplast and mitochondrial nucleoids.


Assuntos
Chlamydomonas reinhardtii/genética , Proteínas de Cloroplastos/genética , DNA de Cloroplastos/genética , Genoma de Cloroplastos/genética , Domínios HMG-Box/genética , Sequências de Repetição em Tandem/genética , Chlamydomonas reinhardtii/metabolismo , Proteínas de Cloroplastos/classificação , Proteínas de Cloroplastos/metabolismo , DNA de Cloroplastos/metabolismo , Regulação da Expressão Gênica , Espectrometria de Massas/métodos , Mutação , Filogenia , Ligação Proteica , Proteômica/métodos
5.
Proc Natl Acad Sci U S A ; 118(2)2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33443157

RESUMO

The sex-determining region on the Y chromosome (SRY) is thought to be the central genetic element of male sex development in mammals. Pathogenic modifications within the SRY gene are associated with a male-to-female sex reversal syndrome in humans and other mammalian species, including rabbits and mice. However, the underlying mechanisms are largely unknown. To understand the biological function of the SRY gene, a site-directed mutational analysis is required to investigate associated phenotypic changes at the molecular, cellular, and morphological level. Here, we successfully generated a knockout of the porcine SRY gene by microinjection of two CRISPR-Cas ribonucleoproteins, targeting the centrally located "high mobility group" (HMG), followed by a frameshift mutation of the downstream SRY sequence. This resulted in the development of genetically male (XY) pigs with complete external and internal female genitalia, which, however, were significantly smaller than in 9-mo-old age-matched control females. Quantitative digital PCR analysis revealed a duplication of the SRY locus in Landrace pigs similar to the known palindromic duplication in Duroc breeds. Our study demonstrates the central role of the HMG domain in the SRY gene in male porcine sex determination. This proof-of-principle study could assist in solving the problem of sex preference in agriculture to improve animal welfare. Moreover, it establishes a large animal model that is more comparable to humans with regard to genetics, physiology, and anatomy, which is pivotal for longitudinal studies to unravel mammalian sex determination and relevant for the development of new interventions for human sex development disorders.


Assuntos
Processos de Determinação Sexual/genética , Proteína da Região Y Determinante do Sexo/genética , Proteína da Região Y Determinante do Sexo/metabolismo , Sequência de Aminoácidos/genética , Animais , Proteínas de Ligação a DNA/genética , Transtornos do Desenvolvimento Sexual/genética , Mutação da Fase de Leitura/genética , Genes sry/genética , Domínios HMG-Box/genética , Masculino , Mutação/genética , Proteínas Nucleares/genética , Estudo de Prova de Conceito , Domínios Proteicos/genética , Suínos/genética , Fatores de Transcrição/genética , Cromossomo Y/genética
6.
Biochem Biophys Res Commun ; 533(4): 919-924, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33010889

RESUMO

The SWI/SNF chromatin remodeling complex plays important roles in gene regulation and it is classified as the SWI/SNF complex in yeast and BAF complex in vertebrates. BAF57, one of the subunits that forms the chromatin remodeling complex core, is well conserved in the BAF complex of vertebrates, which is replaced by bap111 in the Drosophila BAP complex and does not have a counterpart in the yeast SWI/SNF complex. This suggests that BAF57 is a key component of the chromatin remodeling complex in higher eukaryotes. BAF57 contains a HMG domain, which is widely distributed among various proteins and functions as a DNA binding motif. Most proteins with HMG domain bind to four-way junction (4WJ) DNA. Here, we report the crystal structure of the HMG domain of BAF57 (BAF57HMG) at a resolution of 2.55 Å. The structure consists of three α-helices and adopts an L-shaped form. The overall structure is stabilized by a hydrophobic core, which is formed by hydrophobic residues. The binding affinity between BAF57HMG and 4WJ DNA is determined as a 295.83 ± 1.05 nM using a fluorescence quenching assay, and the structure revealed 4WJ DNA binding site of BAF57HMG. Our data will serve structural basis in understanding the roles of BAF57 during chromatin remodeling process.


Assuntos
Proteínas Cromossômicas não Histona/química , Proteínas de Ligação a DNA/química , DNA/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , DNA Cruciforme/química , DNA Cruciforme/genética , DNA Cruciforme/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Domínios HMG-Box , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , Espectrometria de Fluorescência , Eletricidade Estática
7.
Mol Carcinog ; 59(10): 1159-1173, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32794610

RESUMO

Sex-determining region Y box (SOXs) are expressed in various cells and control cell fate and differentiation in a multitude of physiologic processes. SOX6, a main representative of SOXs, is involved in the regulation of carcinogenesis in various human malignancies. However, the role of SOX6 in clear cell renal cell carcinoma (ccRCC) remains unclear. In this study, SOX6 expression in ccRCC and its clinical significance were investigated. In vitro and in vivo assays were used to explore the tumor-related function and the underlying molecular mechanism of SOX6 in ccRCC. We confirmed that SOX6 was frequently downregulated in ccRCC tissues and cell lines. Besides, downregulation of SOX6 was significantly associated with larger tumor sizes, advanced tumor stage, higher Fuhrman grades, and its expression could act as an independent prognostic factor for ccRCC (hazards ratio = 0.590, P = .026). Gain/loss-of-function experiments demonstrated that SOX6 could remarkably inhibit tumor cell growth and foci formation in vitro and xenograft tumorigenesis in vivo, respectively. Mechanistically, SOX6 could influence cell cycle by regulating the G1/the S phase transition and had an inhibitory effect on Wnt/ß-catenin signaling as well as its target genes, c-Myc and cyclin D1. Interesting, the tumor-suppressive function of SOX6 was proved to be dependent on its specific high-mobility-group (HMG) domain. In general, our findings indicated that SOX6 was a novel tumor suppressor and prognostic biomarker in ccRCC. SOX6 could inhibit tumor growth by negatively regulating the Wnt/ß-catenin signaling pathway in an HMG domain-dependent manner in ccRCC, which might provide a novel therapeutic approach for ccRCC.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/patologia , Domínios HMG-Box , Neoplasias Renais/patologia , Fatores de Transcrição SOXD/metabolismo , Proteína Wnt1/metabolismo , beta Catenina/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Proliferação de Células , Transformação Celular Neoplásica , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Prognóstico , Fatores de Transcrição SOXD/genética , Taxa de Sobrevida , Células Tumorais Cultivadas , Proteína Wnt1/genética , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/genética
8.
Hepatol Int ; 14(5): 828-841, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32607732

RESUMO

BACKGROUND AND AIM: Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease worldwide, but its pathogenesis remains imprecisely understood and requires further clarification. Recently, the tumor suppressor p53 has received growing attention for its role in metabolic diseases. In this study, we performed in vivo and in vitro experiments to identify the contribution of p53-autophagy regulation to NAFLD. METHODS: Livers from wild-type and p53 knockout mice as well as p53-functional HepG2 cells and p53-dysfunctional Huh7 cells were examined for autophagy status and HMGB1 translocation. In vivo and in vitro NAFLD models were established, and steatosis was detected. In the cell models, autophagy status and steatosis were examined by p53 and/or HMGB1 silencing. RESULTS: First, the silencing of p53 could induce autophagy both in vivo and in vitro. In addition, p53 knockout attenuated high-fat diet-induced NAFLD in mice. Similarly, knockdown of p53 could alleviate palmitate-induced lipid accumulation in cell models. Furthermore, high mobility group box 1 (HMGB1) was proven to contribute to the effect of silencing p53 on alleviating NAFLD in vitro as an autophagy regulator. CONCLUSION: The anti-NAFLD effect of functional p53 silencing is associated with the HMGB1-mediated induction of autophagy.


Assuntos
Autofagia/fisiologia , Proteína HMGB1 , Fígado , Hepatopatia Gordurosa não Alcoólica , Proteína Supressora de Tumor p53 , Animais , Dieta Hiperlipídica , Inativação Gênica , Domínios HMG-Box/fisiologia , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Células Hep G2 , Humanos , Fígado/metabolismo , Fígado/patologia , Camundongos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
9.
Gene Expr Patterns ; 36: 119112, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32259660

RESUMO

Sox protein family is characterized by the presence of the conserved high-mobility group (HMG) box. Sox transcription factors are involved in diverse developmental process in animals, including sex-determination, organogenesis, embryogenesis, neurogenesis, and cell fate decision. In this study, 23 Sox genes were identified based on the Culter alburnus whole-genome sequence and categorized into six subfamilies according to the conserved HMG-box domain. The duplicates of four members revealed that Sox genes in the teleost fishes underwent significant expansion. Moreover, their expression pattern in gonad tissues were analyzed by RNA-seq and qRT-PCR, and Sox9b was determined as a key gene that was essential for testis development. This current study will provide new insight into the role of Sox gene family in fish sex determination and differentiation.


Assuntos
Cyprinidae/genética , Cyprinidae/metabolismo , Domínios HMG-Box/genética , Fatores de Transcrição SOX/genética , Fatores de Transcrição SOX/metabolismo , Sequência de Aminoácidos , Animais , Desenvolvimento Embrionário , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Estudo de Associação Genômica Ampla , RNA-Seq , Processos de Determinação Sexual , Transcriptoma , Sequenciamento Completo do Genoma
10.
Brain Res Bull ; 154: 68-80, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31715313

RESUMO

Traumatic brain injury (TBI) is one of the important reason of morbidity and mortality. While the primary injury due to mechanical impact is unavoidable, the secondary injury which is formed as a result of primary injury and thought to occur due to neuroinflammation in the forefront can be prevented and by this way mortality and morbidity can be reduced. High mobility group box-1 (HMGB1) is a protein that triggers the neuroinflammatory process by being released from the nucleus of necrotic tissues after primary injury. The aim of this study is to investigate the effects of HMGB1 on its receptors TLR4 and RAGE, cerebral edema, blood-brain barrier, oxidative stress and apoptosis causing secondary damage in an experimental traumatic brain injury model. Weighing between 280-320 g, 10 to 12 weeks-old, a total of 30 adult male Sprague-Dawley rats were used for the experiments. The rats were randomly assigned to 3 groups: 1) Control, 2) TBI and 3) TBI + ethyl pyruvate group (n = 10 per group). Right parietal cortical contusion was made by using a weight-dropping TBI method. Brain samples were harvested from pericontusional area at 24 h after TBI. HMGB1, TLR4, RAGE, occludin, claudin-5, ZO-1 levels are investigated by western blot analyses and immunohistochemistry examinations. HMGB-1, TLR4 and RAGE expressions increased after TBI. Major tight junction proteins in the blood-brain barrier: occludin, claudin-5 and ZO-1 expressions decreased after TBI. Brain edema increased after TBI. Also, proapoptotic bax and active caspase 3 expressions increased, antiapoptotic bcl-2 levels decreased after TBI. Total oxidant status and oxidative stress increased, total antioxidant status decreased after TBI. HMGB-1 protein plays a key role in the pathophysiology of traumatic brain injury.


Assuntos
Lesões Encefálicas Traumáticas/metabolismo , Proteína HMGB1/metabolismo , Animais , Apoptose/fisiologia , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Edema Encefálico/etiologia , Edema Encefálico/metabolismo , Lesões Encefálicas/complicações , Lesões Encefálicas Traumáticas/fisiopatologia , Claudina-5/metabolismo , Modelos Animais de Doenças , Domínios HMG-Box/fisiologia , Proteína HMGB1/fisiologia , Proteínas de Grupo de Alta Mobilidade/metabolismo , Masculino , Ocludina/metabolismo , Estresse Oxidativo/fisiologia , Piruvatos/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Receptor 4 Toll-Like/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo
11.
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
12.
FEBS J ; 286(24): 4951-4963, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31323153

RESUMO

Capicua (CIC) is a transcriptional repressor and functions downstream of the receptor tyrosine kinase (RTK) signaling pathway. Somatic mutations found in the HMG-box DNA binding domain in CIC have been implicated in several cancers such as oligodendroglioma, oligoastrocytoma, and adenocarcinoma. However, the molecular basis of the DNA binding of CIC and the effect of the somatic mutations found in cancers on DNA binding have not been investigated. Here, we report the crystal structure of the HMG-box domain of CIC complexed with its target DNA, the promoter of Ets Translocation Variant 5 (ETV5). The structure shows that the HMG-box domain has an L-shaped structure and recognizes the minor groove leading to DNA bending. Our structure combined with an electrophoretic mobility shift assay (EMSA) revealed that cancer-associated mutations in the HMG-box domain abrogate the interaction with DNA. These results provide the molecular insight into the DNA binding of CIC and reveal the effects of carcinogenic mutations on DNA binding.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/química , DNA/metabolismo , Domínios HMG-Box/fisiologia , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Domínios HMG-Box/genética , Humanos , Mutação/genética , Neoplasias/química , Neoplasias/genética , Neoplasias/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética
13.
Am J Hum Genet ; 104(2): 246-259, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30661772

RESUMO

SOX4, together with SOX11 and SOX12, forms group C of SRY-related (SOX) transcription factors. They play key roles, often in redundancy, in multiple developmental pathways, including neurogenesis and skeletogenesis. De novo SOX11 heterozygous mutations have been shown to cause intellectual disability, growth deficiency, and dysmorphic features compatible with mild Coffin-Siris syndrome. Using trio-based exome sequencing, we here identify de novo SOX4 heterozygous missense variants in four children who share developmental delay, intellectual disability, and mild facial and digital morphological abnormalities. SOX4 is highly expressed in areas of active neurogenesis in human fetuses, and sox4 knockdown in Xenopus embryos diminishes brain and whole-body size. The SOX4 variants cluster in the highly conserved, SOX family-specific HMG domain, but each alters a different residue. In silico tools predict that each variant affects a distinct structural feature of this DNA-binding domain, and functional assays demonstrate that these SOX4 proteins carrying these variants are unable to bind DNA in vitro and transactivate SOX reporter genes in cultured cells. These variants are not found in the gnomAD database of individuals with presumably normal development, but 12 other SOX4 HMG-domain missense variants are recorded and all demonstrate partial to full activity in the reporter assay. Taken together, these findings point to specific SOX4 HMG-domain missense variants as the cause of a characteristic human neurodevelopmental disorder associated with mild facial and digital dysmorphism.


Assuntos
Anormalidades Múltiplas/genética , Mutação de Sentido Incorreto/genética , Transtornos do Neurodesenvolvimento/genética , Fatores de Transcrição SOXC/genética , Sequência de Aminoácidos , Animais , Criança , Pré-Escolar , Síndrome de Coffin-Lowry/genética , Estudos de Coortes , Sequência Conservada , DNA/genética , DNA/metabolismo , Feminino , Domínios HMG-Box/genética , Heterozigoto , Humanos , Masculino , Fatores de Transcrição SOX/química , Fatores de Transcrição SOX/genética , Fatores de Transcrição SOXC/química , Fatores de Transcrição SOXC/metabolismo , Ativação Transcricional , Xenopus/anatomia & histologia , Xenopus/embriologia , Xenopus/genética , Proteínas de Xenopus/química , Proteínas de Xenopus/genética
14.
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
15.
J Mol Biol ; 430(17): 2747-2759, 2018 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-29966609

RESUMO

Histone chaperones play critical roles in regulated structural transitions of chromatin in eukaryotic cells that involve nucleosome disassembly and reassembly. The histone chaperone FACT is a heterodimeric complex consisting in plants and metazoa of SSRP1/SPT16 and is involved in dynamic nucleosome reorganization during various DNA-dependent processes including transcription, replication and repair. The C-terminal HMG-box domain of the SSRP1 subunit mediates interactions with DNA and nucleosomes in vitro, but its relevance in vivo is unclear. Here, we demonstrate that Arabidopsis ssrp1-2 mutant plants express a C-terminally truncated SSRP1 protein. Although the structure of the truncated HMG-box domain is distinctly disturbed, it still exhibits residual DNA-binding activity, but has lost DNA-bending activity. Since ssrp1-2 plants are phenotypically affected but viable, the HMG-box domain may be functionally non-essential. To examine this possibility, SSRP1∆HMG completely lacking the HMG-box domain was studied. SSRP1∆HMG in vitro did not bind to DNA and its interactions with nucleosomes were severely reduced. Nevertheless, the protein showed a nuclear mobility and protein interactions similar to SSRP1. Interestingly, expression of SSRP1∆HMG is almost as efficient as that of full-length SSRP1 in supporting normal growth and development of the otherwise non-viable Arabidopsis ssrp1-1 mutant. SSRP1∆HMG is structurally similar to the fungal ortholog termed Pob3 that shares clear similarity with SSRP1, but it lacks the C-terminal HMG-box. Therefore, our findings indicate that the HMG-box domain conserved among SSRP1 proteins is not critical in Arabidopsis, and thus, the functionality of SSRP1/SPT16 in plants/metazoa and Pob3/Spt16 in fungi is perhaps more similar than anticipated.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cromatina/química , Proteínas Cromossômicas não Histona/metabolismo , Domínios HMG-Box , Chaperonas de Histonas/metabolismo , Nucleossomos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Replicação do DNA , DNA de Plantas/química , DNA de Plantas/genética , DNA de Plantas/metabolismo , Chaperonas de Histonas/química , Chaperonas de Histonas/genética , Nucleossomos/química , Nucleossomos/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
16.
FEMS Microbiol Lett ; 365(12)2018 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-29617942

RESUMO

Ustilago esculenta, an obligate parasite of Zizania latifolia, is a typical dimorphic fungus which induces host stem swelling and inhibits host inflorescence development, but is not found in host leaves. Previous studies have shown that dimorphic switching is essential for fungal pathogenicity and is regulated by protein kinase A and mitogen-activated protein kinase (MAPK) signaling pathways that are integrated by Prf1 in Ustilago maydis. In this study we identified a Prf1 homolog in U. esculenta, designated UePrf1, encoding 830 amino acids with a conserved high mobility group domain located between amino acids 124 and 195. UePrf1 was upregulated during the mating process, which induces dimorphism in U. esculenta. In vitro, UePrf1 mutants showed defects in the mating process, including cell fusion and hyphal growth. UePrf1 mutants also show reduced expression of a genes, even during the cell fusion process. Additionally, the defect in hyphal growth of the UeKpp2 and UeKpp6 mutants (MAPK signaling pathway mutants) was partially counteracted by UePrf1 overexpression, along with induced b gene expression. These results provide evidence that UePrf1 is a key factor coordinating dimorphism in U. esculenta and suggest a conserved role for UePrf1 in the regulation of the a and b genes.


Assuntos
Proteínas Fúngicas/genética , Ustilago/genética , Clonagem Molecular , Proteínas Fúngicas/isolamento & purificação , Genes Fúngicos Tipo Acasalamento/genética , Domínios HMG-Box/genética , Interações Hospedeiro-Patógeno/genética , Hifas/genética , Hifas/crescimento & desenvolvimento , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação , Doenças das Plantas/microbiologia , Fatores de Transcrição/genética , Ustilago/crescimento & desenvolvimento , Ustilago/patogenicidade
17.
Sci Rep ; 8(1): 5156, 2018 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-29581456

RESUMO

For decades, outbred guinea pigs (GP) have been used as research models. Various past research studies using guinea pigs used measures that, unknown at the time, may be sex-dependent, but from which today, archival tissues may be all that remain. We aimed to provide a protocol for sex-typing archival guinea pig tissue, whereby past experiments could be re-evaluated for sex effects. No PCR sex-genotyping protocols existed for GP. We found that published sequence of the GP Sry gene differed from that in two separate GP stocks. We used sequences from other species to deduce PCR primers for Sry. After developing a genomic DNA extraction for archival, fixed, decalcified, immunolabeled, guinea pig cochlear half-turns, we used a multiplex assay (Y-specific Sry; X-specific Dystrophin) to assign sex to tissue as old as 3 years. This procedure should allow reevaluation of prior guinea pig studies in various research areas for the effects of sex on experimental outcomes.


Assuntos
Cóclea , Genes sry/genética , Genótipo , Técnicas de Genotipagem/métodos , Cobaias/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Bancos de Tecidos , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA/isolamento & purificação , Primers do DNA , Distrofina/genética , Domínios HMG-Box/genética , Imuno-Histoquímica , Fatores Sexuais
18.
Gene ; 638: 52-59, 2018 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-28918251

RESUMO

The homeodomain-containing transcription factor Anf (also known as Rpx/Hesx1 in mammals) plays an important role during the forebrain development in vertebrates. Here we demonstrate the ability of the Xenopus laevis Anf, Xanf1/Hesx1, to directly bind SRY-related HMG-box-containing transcription factor SoxD/Sox15 and to cooperate with the latter during regulating of the expression of Xanf1/Hesx1 own gene. As we have shown by GST pull-down, EMSA and the luciferase reporter assays, Xanf1/Hesx1 and SoxD/Sox15 bind the Xanf1/Hesx1 promoter region counteracting the inhibitory effect of Xanf1/Hesx1 alone. This finding explains how Xanf1/Hesx1 could escape the repressive activity of its own protein during early patterning of the forebrain rudiment.


Assuntos
Proteínas de Homeodomínio/metabolismo , Prosencéfalo/embriologia , Fatores de Transcrição SOXD/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/crescimento & desenvolvimento , Animais , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Domínios HMG-Box , Proteínas de Homeodomínio/genética , Prosencéfalo/metabolismo , Fatores de Transcrição SOXD/química , Técnicas do Sistema de Duplo-Híbrido , Proteínas de Xenopus/genética , Xenopus laevis/metabolismo
19.
Open Biol ; 7(11)2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-29167311

RESUMO

A dynamic multi-protein assembly known as the replisome is responsible for DNA synthesis in eukaryotic cells. In yeast, the hub protein Ctf4 bridges DNA helicase and DNA polymerase and recruits factors with roles in metabolic processes coupled to DNA replication. An important question in DNA replication is the extent to which the molecular architecture of the replisome is conserved between yeast and higher eukaryotes. Here, we describe the biochemical basis for the interaction of the human CTF4-orthologue AND-1 with DNA polymerase α (Pol α)/primase, the replicative polymerase that initiates DNA synthesis. AND-1 has maintained the trimeric structure of yeast Ctf4, driven by its conserved SepB domain. However, the primary interaction of AND-1 with Pol α/primase is mediated by its C-terminal HMG box, unique to mammalian AND-1, which binds the B subunit, at the same site targeted by the SV40 T-antigen for viral replication. In addition, we report a novel DNA-binding activity in AND-1, which might promote the correct positioning of Pol α/primase on the lagging-strand template at the replication fork. Our findings provide a biochemical basis for the specific interaction between two critical components of the human replisome, and indicate that important principles of replisome architecture have changed significantly in evolution.


Assuntos
DNA Polimerase I/metabolismo , DNA Primase/metabolismo , Proteínas de Ligação a DNA/metabolismo , Domínios HMG-Box , Sítios de Ligação , Biologia Computacional , Humanos , Modelos Moleculares , Ligação Proteica
20.
Mol Cell Biol ; 37(22)2017 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-28874518

RESUMO

Upstream binding factor (UBF) is a member of the high-mobility group (HMG) box protein family, characterized by multiple HMG boxes and a C-terminal acidic region (AR). UBF is an essential transcription factor for rRNA genes and mediates the formation of transcriptionally active chromatin in the nucleolus. However, it remains unknown how UBF is specifically localized to the nucleolus. Here, we examined the molecular mechanisms that localize UBF to the nucleolus. We found that the first HMG box (HMG box 1), the linker region (LR), and the AR cooperatively regulate the nucleolar localization of UBF1. We demonstrated that the AR intramolecularly associates with and attenuates the DNA binding activity of HMG boxes and confers the structured DNA preference to HMG box 1. In contrast, the LR was found to serve as a nuclear localization signal and compete with HMG boxes to bind the AR, permitting nucleolar localization of UBF1. The LR sequence binds DNA and assists the stable chromatin binding of UBF. We also showed that the phosphorylation status of the AR does not clearly affect the localization of UBF1. Our results strongly suggest that associations of the AR with HMG boxes and the LR regulate UBF nucleolar localization.


Assuntos
Nucléolo Celular/metabolismo , Mutação , Proteínas Pol1 do Complexo de Iniciação de Transcrição/química , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , Sítios de Ligação , Linhagem Celular , Nucléolo Celular/genética , Células HEK293 , Domínios HMG-Box , Células HeLa , Humanos , Fosforilação , Proteínas Pol1 do Complexo de Iniciação de Transcrição/genética , Ligação Proteica
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