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1.
PLoS Pathog ; 16(7): e1008644, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32678836

RESUMO

The foamy viruses (FV) or spumaviruses are an ancient subfamily of retroviruses that infect a variety of vertebrates. FVs are endemic, but apparently apathogenic, in modern non-human primates. Like other retroviruses, FV replication is inhibited by type-I interferon (IFN). In a previously described screen of IFN-stimulated genes (ISGs), we identified the macaque PHD finger domain protein-11 (PHF11) as an inhibitor of prototype foamy virus (PFV) replication. Here, we show that human and macaque PHF11 inhibit the replication of multiple spumaviruses, but are inactive against several orthoretroviruses. Analysis of other mammalian PHF11 proteins revealed that antiviral activity is host species dependent. Using multiple reporter viruses and cell lines, we determined that PHF11 specifically inhibits a step in the replication cycle that is unique to FVs, namely basal transcription from the FV internal promoter (IP). In so doing, PHF11 prevents expression of the viral transactivator Tas and subsequent activation of the viral LTR promoter. These studies reveal a previously unreported inhibitory mechanism in mammalian cells, that targets a family of ancient viruses and may promote viral latency.


Assuntos
Proteínas de Ligação a DNA/fisiologia , Infecções por Retroviridae/virologia , Spumavirus/fisiologia , Fatores de Transcrição/fisiologia , Latência Viral/fisiologia , Replicação Viral/fisiologia , Animais , Humanos , Macaca
2.
PLoS Genet ; 16(6): e1008814, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32555650

RESUMO

The circadian clocks in chlorophyte algae have been studied in two model organisms, Chlamydomonas reinhardtii and Ostreococcus tauri. These studies revealed that the chlorophyte clocks include some genes that are homologous to those of the angiosperm circadian clock. However, the genetic network architectures of the chlorophyte clocks are largely unknown, especially in C. reinhardtii. In this study, using C. reinhardtii as a model, we characterized RHYTHM OF CHLOROPLAST (ROC) 75, a clock gene encoding a putative GARP DNA-binding transcription factor similar to the clock proteins LUX ARRHYTHMO (LUX, also called PHYTOCLOCK 1 [PCL1]) and BROTHER OF LUX ARRHYTHMO (BOA, also called NOX) of the angiosperm Arabidopsis thaliana. We observed that ROC75 is a day/subjective day-phase-expressed nuclear-localized protein that associates with some night-phased clock genes and represses their expression. This repression may be essential for the gating of reaccumulation of the other clock-related GARP protein, ROC15, after its light-dependent degradation. The restoration of ROC75 function in an arrhythmic roc75 mutant under constant darkness leads to the resumption of circadian oscillation from the subjective dawn, suggesting that the ROC75 restoration acts as a morning cue for the C. reinhardtii clock. Our study reveals a part of the genetic network of C. reinhardtii clock that could be considerably different from that of A. thaliana.


Assuntos
Chlamydomonas reinhardtii/fisiologia , Relógios Circadianos/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/fisiologia , Fatores de Transcrição/fisiologia , Cloroplastos/fisiologia , Ritmo Circadiano/genética , Redes Reguladoras de Genes/fisiologia , Mutação , Fotoperíodo , Plantas Geneticamente Modificadas
3.
Nucleic Acids Res ; 48(14): 7609-7622, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32476018

RESUMO

The splicing of tRNA introns is a critical step in pre-tRNA maturation. In archaea and eukaryotes, tRNA intron removal is catalyzed by the tRNA splicing endonuclease (TSEN) complex. Eukaryotic TSEN is comprised of four core subunits (TSEN54, TSEN2, TSEN34 and TSEN15). The human TSEN complex additionally co-purifies with the polynucleotide kinase CLP1; however, CLP1's role in tRNA splicing remains unclear. Mutations in genes encoding all four TSEN subunits, as well as CLP1, are known to cause neurodegenerative disorders, yet the mechanisms underlying the pathogenesis of these disorders are unknown. Here, we developed a recombinant system that produces active TSEN complex. Co-expression of all four TSEN subunits is required for efficient formation and function of the complex. We show that human CLP1 associates with the active TSEN complex, but is not required for tRNA intron cleavage in vitro. Moreover, RNAi knockdown of the Drosophila CLP1 orthologue, cbc, promotes biogenesis of mature tRNAs and circularized tRNA introns (tricRNAs) in vivo. Collectively, these and other findings suggest that CLP1/cbc plays a regulatory role in tRNA splicing by serving as a negative modulator of the direct tRNA ligation pathway in animal cells.


Assuntos
Endorribonucleases/metabolismo , Precursores de RNA/metabolismo , RNA de Transferência/metabolismo , Proteínas de Drosophila/fisiologia , Éxons , Humanos , Íntrons , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Fosfotransferases/metabolismo , Fosfotransferases/fisiologia , Clivagem do RNA , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia
4.
PLoS One ; 15(5): e0233375, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32421756

RESUMO

MYB-related transcription factors play important roles in plant development and response to various environmental stresses. In the present study, a novel MYB gene, designated as BnMYB2 (GenBank accession number: MF741319.1), was isolated from Boehmeria nivea using rapid amplification of cDNA ends (RACE) and RT-PCR on a sequence fragment from a ramie transcriptome. BnMYB2 has a 945 bp open reading frame encoding a 314 amino acid protein that contains a DNA-binding domain and shares high sequence identity with MYB proteins from other plant species. The BnMYB2 promoter contains several putative cis-acting elements involved in stress or phytohormone responses. A translational fusion of BnMYB2 with enhanced green fluorescent protein (eGFP) showed nuclear and cytosolic subcellular localization. Real-time PCR results indicated that BnMYB2 expression was induced by Cadmium (Cd) stress. Overexpression of BnMYB2 in Arabidopsis thaliana resulted in a significant increase of Cd tolerance and accumulation. Thus, BnMYB2 positively regulated Cd tolerance and accumulation in Arabidopsis, and could be used to enhance the efficiency of Cd removal with plants.


Assuntos
Boehmeria/genética , Cádmio/metabolismo , Fatores de Transcrição/fisiologia , Arabidopsis/metabolismo , Cádmio/farmacologia , Tolerância a Medicamentos/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/fisiologia , Proteínas Proto-Oncogênicas c-myb/metabolismo , Proteínas Proto-Oncogênicas c-myb/fisiologia , Estresse Fisiológico/efeitos dos fármacos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
5.
Mol Cell ; 78(6): 1114-1132.e10, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32446320

RESUMO

Bromodomain-containing protein 4 (BRD4) is a cancer therapeutic target in ongoing clinical trials disrupting primarily BRD4-regulated transcription programs. The role of BRD4 in cancer has been attributed mainly to the abundant long isoform (BRD4-L). Here we show, by isoform-specific knockdown and endogenous protein detection, along with transgene expression, the less abundant BRD4 short isoform (BRD4-S) is oncogenic while BRD4-L is tumor-suppressive in breast cancer cell proliferation and migration, as well as mammary tumor formation and metastasis. Through integrated RNA-seq, genome-wide ChIP-seq, and CUT&RUN association profiling, we identify the Engrailed-1 (EN1) homeobox transcription factor as a key BRD4-S coregulator, particularly in triple-negative breast cancer. BRD4-S and EN1 comodulate the extracellular matrix (ECM)-associated matrisome network, including type II cystatin gene cluster, mucin 5, and cathepsin loci, via enhancer regulation of cancer-associated genes and pathways. Our work highlights the importance of targeted therapies for the oncogenic, but not tumor-suppressive, activity of BRD4.


Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Genes Homeobox , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos , Invasividade Neoplásica , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas/antagonistas & inibidores , Proteínas/metabolismo , Transcrição Genética/genética , Neoplasias de Mama Triplo Negativas/genética
6.
Nat Rev Endocrinol ; 16(7): 363-377, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32303708

RESUMO

Nuclear receptor crosstalk can be defined as the interplay between different nuclear receptors or between their overlapping signalling pathways. A subset of nuclear receptors (such as PPARs and RARs) engage in the formation of well-characterized 'typical' heterodimers with RXR. 'Atypical' heterodimers (such as GR with PPARs, or PPAR with ERR) might form a novel class of physical complexes that might be more transient in nature. These heterodimers might harbour strong transcriptional flexibility, with no strict need for DNA binding of both partners. Direct crosstalk could stem from a pairwise physical association between atypical nuclear receptor heterodimers, either via pre-existing interaction pairs or via interactions that are newly induced with small molecules; such crosstalk might constitute an uncharted space to target nuclear receptor physiological and/or pathophysiological actions. In this Review, we discuss the emerging aspects of crosstalk in the nuclear receptor field and present various mechanistic crosstalk modes with examples that support applicability of the atypical heterodimer concept. Stabilization or disruption, in a context-dependent or cell type-dependent manner, of these more transient heterodimers is expected to fuel unprecedented translational approaches to yield novel therapeutic agents to treat major human diseases with higher precision.


Assuntos
Receptor Cross-Talk/fisiologia , Receptores Citoplasmáticos e Nucleares/fisiologia , Terapias em Estudo/métodos , Animais , Humanos , Ligação Proteica/fisiologia , Multimerização Proteica/fisiologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais/fisiologia , Terapias em Estudo/tendências , Fatores de Transcrição/metabolismo , Fatores de Transcrição/fisiologia
7.
PLoS One ; 15(4): e0231015, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32251429

RESUMO

Hallux valgus is a serious medical concern for classical ballet dancers. Although it is well-known that progression of hallux valgus is related to inappropriate movement techniques in classical ballet, the kinematic relationship between the degree of hallux valgus and ballet techniques has not been substantiated. To develop proper training methods that prevent progression of hallux valgus, this study aimed to investigate the relationship between the degree of hallux valgus and movement techniques in classical ballet. Seventeen female classical ballet dancers at the advanced college-level participated in this study. Kinematic analysis of standing and plié in the first position was conducted via video capture technique. The Pearson product-moment correlation analysis was performed to examine the degree of hallux valgus and the following three kinematic variables: (1) the extent to which turnout is forced by other joints in the lower extremity than the hip joint, (2) the direction difference between the knee and toe in the transverse plane, and (3) the pelvis obliquity angle. Among these kinematic variables, we found a significant correlation between the hallux valgus angle and the pelvis obliquity angle during plié (P = .045). The greater the hallux valgus angle, the greater the retroversion of the pelvis, a result which was contrary to our prediction. We present the first evidence that the degree of hallux valgus correlates with kinematics in a very basic technique of classical ballet.


Assuntos
Dança , Hallux Valgus/fisiopatologia , Fenômenos Biomecânicos/fisiologia , Dança/fisiologia , Feminino , Humanos , Pelve/fisiologia , Projetos Piloto , Índice de Gravidade de Doença , Fatores de Transcrição/fisiologia , Gravação em Vídeo , Adulto Jovem
8.
Nucleic Acids Res ; 48(8): 4100-4114, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32182360

RESUMO

The evolution of regulatory networks in Bacteria has largely been explained at macroevolutionary scales through lateral gene transfer and gene duplication. Transcription factors (TF) have been found to be less conserved across species than their target genes (TG). This would be expected if TFs accumulate mutations faster than TGs. This hypothesis is supported by several lab evolution studies which found TFs, especially global regulators, to be frequently mutated. Despite these studies, the contribution of point mutations in TFs to the evolution of regulatory network is poorly understood. We tested if TFs show greater genetic variation than their TGs using whole-genome sequencing data from a large collection of Escherichia coli isolates. TFs were less diverse than their TGs across natural isolates, with TFs of large regulons being more conserved. In contrast, TFs showed higher mutation frequency in adaptive laboratory evolution experiments. However, over long-term laboratory evolution spanning 60 000 generations, mutation frequency in TFs gradually declined after a rapid initial burst. Extrapolating the dynamics of genetic variation from long-term laboratory evolution to natural populations, we propose that point mutations, conferring large-scale gene expression changes, may drive the early stages of adaptation but gene regulation is subjected to stronger purifying selection post adaptation.


Assuntos
Evolução Molecular , Regulação Bacteriana da Expressão Gênica , Redes Reguladoras de Genes , Mutação Puntual , Fatores de Transcrição/genética , Escherichia coli/genética , Genes Bacterianos , Mutação , Fatores de Transcrição/fisiologia
9.
Artigo em Inglês | MEDLINE | ID: mdl-32222679

RESUMO

Brassinazole-resistant (BZR) transcription factors have important roles in the brassinosteroid (BR) signalling pathway and are widely involved in plant growth and abiotic stress processes. However, there are few studies on the functions and regulatory mechanisms of BZR TFs in birch. In this study, 5 BZR genes were identified from birch. The qRT-PCR results showed that the expression levels of most BpBZRs were significantly downregulated and/or upregulated in at least one organ following NaCl and PEG stress or ABA, GA3 and JA treatments. In particular, BpBZR1 expression was changed in all three organs after exposure to NaCl stress at all time points, indicating that this gene may be involved in salt stress. The BpBZR1 transcription factor was shown to have transcriptional activation activity in a yeast two-hybrid assay. Through a transient transformation system, we found that overexpression of BpBZR1 in birch resulted in lower H2O2 and MDA accumulation, higher SOD and POD activities and maintained a higher photosynthetic intensity and a lower chlorophyll degradation rate than those of the control plants under salt stress. These results preliminarily showed that overexpression of the BpBZR1 gene increased the tolerance of birch to salt stress.


Assuntos
Betula/fisiologia , Proteínas de Ligação a DNA/fisiologia , Proteínas de Plantas/fisiologia , Estresse Fisiológico , Fatores de Transcrição/fisiologia , Betula/genética , Regulação da Expressão Gênica de Plantas
10.
J Vasc Res ; 57(2): 86-96, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32045906

RESUMO

BACKGROUND: Aberrant proliferation, migration, and apoptosis of vascular smooth muscle cells (VSMCs) are major pathological phenomenon in hypertension. MicroRNAs (miRNAs/miRs) serve crucial roles in the progression of hypertension. We aimed to determine the role of miR-96-5p in the proliferation, migration, and apoptosis of VSMCs and its underlying mechanisms. METHODS: Angiotensin II (Ang II) was employed to treat VSMCs, and the expression of miR-96-5p was detected by RT-qPCR. Then, miR-96-5p mimic was transfected into VSMCs. Cell Counting Kit-8 assay, flow cytometry, transwell assay, and wound healing assay were applied to measure proliferation, cell cycle, and migration of VSMCs. The expression of proteins associated with proliferation, migration, and apoptosis was assessed. A luciferase reporter assay was applied to confirm the target binding between miR-96-5p and nuclear factors of activated T-cells 5 (NFAT5). Subsequently, siRNA was used to silence NFAT5, and cell proliferation, migration, and apoptosis were assessed. RESULTS: The results revealed that the expression of miR-96-5p was downregulated in Ang II-induced VSMCs. MiR-96-5p overexpression inhibited cell proliferation and migration but promoted cell apoptosis, enhanced the percentages of cells in the G1 and G2 phases, and reduced those in the S phase, accompanied by changes in the expression associated proteins. NFAT5 was confirmed as a direct target of miR-96-5p. NFAT5 silencing had the same results with miR-96-5p overexpression on VSMC proliferation, migration, and apoptosis, whereas miR-96-5p inhibitor reversed these effects. CONCLUSIONS: Our findings concluded that miR-96-5p could regulate proliferation, migration, and apoptosis of VSMCs induced by Ang II via targeting NFAT5.


Assuntos
Angiotensina II/farmacologia , Apoptose/efeitos dos fármacos , MicroRNAs/fisiologia , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/fisiologia , Fatores de Transcrição/genética , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Humanos , Fatores de Transcrição/fisiologia
11.
Biochim Biophys Acta Gene Regul Mech ; 1863(3): 194492, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32006714

RESUMO

In Drosophila, the Heterochromatin Protein 1c (HP1c) forms a transcriptional complex with the zinc-finger proteins WOC and ROW, and the extraproteasomal ubiquitin receptor Dsk2. This complex localizes at promoters of active genes and it is required for transcription. The functions played by the different components of the HP1c complex are not fully understood. In this study we show that WOC and ROW are required for chromatin binding of both Dsk2 and HP1c. However, while impairing chromatin binding strongly destabilizes HP1c, it does not affect Dsk2 stability. We also show that WOC, but not ROW, is required for nuclear localization of Dsk2. Moreover, WOC and Dsk2 co-immunoprecitate upon ROW depletion. These results suggest that WOC and Dsk2 interact to form a subcomplex that mediates nuclear translocation of Dsk2. We also show that ROW mediates chromatin binding of the WOC/Dsk2 subcomplex, as well as of HP1c. Altogether these observations favor a model by which the interaction with WOC recruits Dsk2 to the HP1c complex that, in its turn, binds chromatin in a ROW-dependent manner.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/fisiologia , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Fatores de Transcrição/fisiologia , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Drosophila/genética , Drosophila/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética
12.
PLoS One ; 15(2): e0228643, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32017793

RESUMO

Filamentous fungi reproduce asexually or sexually, and the processes of asexual and sexual development are tightly regulated by a variety of transcription factors. In this study, we characterized a Zn2Cys6 transcription factor in two Aspergillus species, A. nidulans (AN5859) and A. flavus (AFLA_046870). AN5859 encodes a Zn2Cys6 transcription factor, called ZcfA. In A. nidulans, ΔzcfA mutants exhibit decreased fungal growth, a reduction in cleistothecia production, and increased asexual reproduction. Overexpression of zcfA results in increased conidial production, suggesting that ZcfA is required for proper asexual and sexual development in A. nidulans. In conidia, deletion of zcfA causes decreased trehalose levels and decreased spore viability but increased thermal sensitivity. In A. flavus, the deletion of the zcfA homolog AFLA_046870 causes increased conidial production but decreased sclerotia production; these effects are similar to those of zcfA deletion in A. nidulans development. Overall, these results demonstrate that ZcfA is essential for maintaining a balance between asexual and sexual development and that some roles of ZcfA are conserved in Aspergillus spp.


Assuntos
Aspergillus flavus/fisiologia , Aspergillus nidulans/fisiologia , Fatores de Transcrição/fisiologia , Aspergillus flavus/crescimento & desenvolvimento , Aspergillus nidulans/crescimento & desenvolvimento , Proteínas Fúngicas/fisiologia , Metaloproteínas/fisiologia , Reprodução , Reprodução Assexuada , Especificidade da Espécie , Zinco
13.
Science ; 367(6482)2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32054698

RESUMO

Sex determination of germ cells is vital to creating the sexual dichotomy of germ cell development, thereby ensuring sexual reproduction. However, the underlying mechanisms remain unclear. Here, we show that ZGLP1, a conserved transcriptional regulator with GATA-like zinc fingers, determines the oogenic fate in mice. ZGLP1 acts downstream of bone morphogenetic protein, but not retinoic acid (RA), and is essential for the oogenic program and meiotic entry. ZGLP1 overexpression induces differentiation of in vitro primordial germ cell-like cells (PGCLCs) into fetal oocytes by activating the oogenic programs repressed by Polycomb activities, whereas RA signaling contributes to oogenic program maturation and PGC program repression. Our findings elucidate the mechanism for mammalian oogenic fate determination, providing a foundation for promoting in vitro gametogenesis and reproductive medicine.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Oócitos/fisiologia , Oogênese/genética , Proteínas Repressoras/fisiologia , Diferenciação Sexual/genética , Fatores de Transcrição/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Feminino , Feto/citologia , Masculino , Meiose/genética , Camundongos , Camundongos Knockout , Oócitos/citologia , Proteínas do Grupo Polycomb/metabolismo , Proteínas Repressoras/genética , Processos de Determinação Sexual , Transdução de Sinais , Fatores de Transcrição/genética , Transcriptoma , Tretinoína/fisiologia
14.
Dev Cell ; 52(4): 429-445.e10, 2020 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-32032549

RESUMO

The mechanisms regulating meiotic initiation in mammals are enigmatic. It is known that retinoic acid (RA) signaling plays a pivotal role during meiotic initiation. STRA8, which is expressed in response to RA, is thought to be a key factor promoting meiotic initiation. However, the specific role of STRA8 in meiotic initiation has remained elusive. Here, we identified MEIOSIN as a germ-cell-specific factor that associates with STRA8. MEIOSIN, like STRA8, is expressed in response to RA and plays an essential role in meiotic initiation in both males and females. Functional analyses revealed that MEIOSIN acts as a transcription factor together with STRA8, and that both factors are critical for driving meiotic gene activation. Furthermore, temporally restricted expression of MEIOSIN leads to meiotic entry decision during spermatogenesis. The present study demonstrates that MEIOSIN, in collaboration with STRA8, plays a central role in regulating the mitosis to meiosis germ cell fate decision in mammals.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Ciclo Celular , Regulação da Expressão Gênica , Células Germinativas/fisiologia , Meiose , Mitose , Fatores de Transcrição/fisiologia , Animais , Diferenciação Celular , Feminino , Células Germinativas/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais , Espermatogênese
15.
Arterioscler Thromb Vasc Biol ; 40(4): e105-e113, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32075417

RESUMO

OBJECTIVE: Vascular progenitor cells (VPCs), which are able to differentiate into both endothelial cells and smooth muscle cells, have the potential for treatment of ischemic diseases. Generated by pluripotent stem cells, VPCs carry the risk of tumorigenicity in clinical application. This issue could be resolved by direct lineage conversion, the induction of functional cells from another lineage by using only lineage-restricted transcription factors. Here, we show that induced VPCs (iVPCs) can be generated from fibroblasts by ETS (E-twenty six) transcription factors, Etv2 and Fli1. Approach and Results: Mouse fibroblasts were infected with lentivirus encoding Etv2 and Fli1. Cell colonies appeared in Fli1- and Etv2/Fli1-infected groups and were mechanically picked. The identity of cell colonies was confirmed by proliferation assay and reverse-transcription polymerase chain reaction with vascular markers. Etv2/Fli1- infected cell colonies were sorted by CD144 (also known as CDH5, VE-cadherin). We defined that CD144-positive iVPCs maintained its own population and expanded stably at multiple passages. iVPCs could differentiate into functional endothelial cells and smooth muscle cells by a defined medium. The functionalities of iVPC-derived endothelial cells and smooth muscle cells were confirmed by analyzing LDL (low-density lipoprotein) uptake, carbachol-induced contraction, and tube formation in vitro. Transplantation of iVPCs into the ischemic hindlimb model enhanced blood flow without tumor formation in vivo. Human iVPCs were generated by human ETS transcription factors ETV2 and FLI1. CONCLUSIONS: We demonstrate that ischemic disease curable iVPCs, which have self-renewal and bipotency, can be generated from mouse fibroblasts by enforced ETS family transcription factors, Etv2 and Fli1 expression. Our simple strategy opens insights into stem cell-based ischemic disease therapy.


Assuntos
Fibroblastos/citologia , Isquemia/fisiopatologia , Proteína Proto-Oncogênica c-fli-1/fisiologia , Células-Tronco/fisiologia , Fatores de Transcrição/fisiologia , Animais , Antígenos CD , Caderinas , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Modelos Animais de Doenças , Células Endoteliais/citologia , Membro Posterior/irrigação sanguínea , Isquemia/terapia , Miócitos de Músculo Liso/citologia , Transplante de Células-Tronco , Células-Tronco/imunologia
16.
Development ; 147(4)2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-32001439

RESUMO

Primordial germ cells (PGCs), the founder cells of the germline, are specified in pre-gastrulating embryos in mammals, and subsequently migrate towards gonads to mature into functional gametes. Here, we investigated PGC development in rats, by genetically modifying Prdm14, a unique marker and an essential PGC transcriptional regulator. We trace PGC development in rats, for the first time, from specification until the sex determination stage in fetal gonads using Prdm14 H2BVenus knock-in rats. We uncover that the crucial role of Prdm14 in PGC specification is conserved between rat and mice, by analyzing Prdm14-deficient rat embryos. Notably, loss of Prdm14 completely abrogates the PGC program, as demonstrated by failure of the maintenance and/or activation of germ cell markers and pluripotency genes. Finally, we profile the transcriptome of the post-implantation epiblast and all PGC stages in rat to reveal enrichment of distinct gene sets at each transition point, thereby providing an accurate transcriptional timeline for rat PGC development. Thus, the novel genetically modified rats and data sets obtained in this study will advance our knowledge on conserved versus species-specific features for germline development in mammals.


Assuntos
Proteínas de Ligação a DNA/genética , Células Germinativas/citologia , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética , Animais , Cruzamentos Genéticos , Proteínas de Ligação a DNA/fisiologia , Feminino , Gástrula/fisiologia , Deleção de Genes , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Heterozigoto , Masculino , Camundongos , Proteínas de Ligação a RNA/fisiologia , Ratos , Processos de Determinação Sexual , Fatores de Transcrição/fisiologia , Transcrição Genética
17.
Plant Mol Biol ; 102(4-5): 389-401, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31894456

RESUMO

KEY MESSAGE: This study revealed that the Arabidopsis UGT75B1 plays an important role in modulating ABA activity by glycosylation when confronting stress environments. The cellular ABA content and activity can be tightly controlled in several ways, one of which is glycosylation by family 1 UDP-glycosyltransferases (UGTs). Previous analysis has shown UGT75B1 activity towards ABA in vitro. However, the biological role of UGT75B1 remains to be elucidated. Here, we characterized the function of UGT75B1 in abiotic stress responses via ABA glycosylation. GUS assay and qRT-PCR indicated that UGT75B1 is significantly upregulated by adverse conditions, such as osmotic stress, salinity and ABA. Overexpression of UGT75B1 in Arabidopsis leads to higher seed germination rates and seedling greening rates upon exposure to salt and osmotic stresses. In contrast, the big UGT75B1 overexpression plants are more sensitive under salt and osmotic stresses. Additionally, the UGT75B1 overexpression plants showed larger stomatal aperture and more water loss under drought condition, which can be explained by lower ABA levels examined in UGT75B1 OE plants in response to water deficit conditions. Consistently, UGT75B1 ectopic expression leads to downregulation of many ABA-responsive genes under stress conditions, including ABI3, ABI5 newly germinated seedlings and RD29A, KIN1, AIL1 in big plants. In summary, our results revealed that the Arabidopsis UGT75B1 plays an important role in coping with abiotic stresses via glycosylation of ABA.


Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Regulação da Expressão Gênica de Plantas , Glucosiltransferases/fisiologia , Glicosiltransferases/metabolismo , Estresse Fisiológico , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Catálise , Secas , Genes de Plantas , Germinação , Glucosiltransferases/genética , Glicosilação , Glicosiltransferases/genética , Pressão Osmótica , Plantas Geneticamente Modificadas/genética , Salinidade , Plântula/genética , Plântula/fisiologia , Cloreto de Sódio , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia
18.
Plant Mol Biol ; 102(4-5): 447-462, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31898148

RESUMO

KEY MESSAGE: ZjICE2 works as a positive regulator in abiotic stress responses and ZjICE2 is a valuable genetic resource to improve abiotic stress tolerance in the molecular breeding program of Zoysia japonica. The basic helix-loop-helix (bHLH) family transcription factors (TFs) play an important role in response to biotic or abiotic stresses in plants. However, the functions of bHLH TFs in Zoysia japonica, one of the warm-season turfgrasses, remain poorly understood. Here, we identified ZjICE2 from Z. japonica, a novel MYC-type bHLH transcription factor that was closely related to ICE homologs in the phylogenetic tree, and its expression was regulated by various abiotic stresses. Transient expression of ZjICE2-GFP in onion epidermal cells revealed that ZjICE2 was a nuclear-localized protein. Also, ZjICE2 bound the MYC cis-element in the promoter of dehydration responsive element binding 1 of Z. japonica (ZjDREB1) using yeast one-hybrid assay. A phenotypic analysis showed that overexpression of the ZjICE2 in Arabidopsis enhanced tolerance to cold, drought, and salt stresses. The transgenic Arabidopsis and Z. japonica accumulated more transcripts of cold-responsive DREB/CBFs and their downstream genes than the wild type (WT) after cold treatment. Furthermore, the transgenic plants exhibited an enhanced Reactive oxygen species (ROS) scavenging ability, which resulted in an efficient maintenance of oxidant-antioxidant homeostasis. In addition, overexpression of the ZjICE2 in Z. japonica displayed intensive cold tolerance with increases in chlorophyll contents and photosynthetic efficiency. Our study suggests that ZjICE2 works as a positive regulator in abiotic stress responses and the ICE-DREB/CBFs response pathway involved in cold stress tolerance is also conserved in Z. japonica. These results provide a valuable genetic resource for the molecular breeding program especially for warm-season grasses as well as other leaf crop plants.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/fisiologia , Poaceae/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Temperatura Baixa , Resposta ao Choque Frio , Secas , Filogenia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/fisiologia , Poaceae/genética , Regulon , Tolerância ao Sal , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Ativação Transcricional
19.
Plant Mol Biol ; 102(4-5): 537-551, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31916084

RESUMO

KEY MESSAGE: Silencing of SlCAND1 expression resulted in dwarfish, loss of apical dominance, early flowering, suppression of seed germination, and abnormal root architecture in tomato Cullin-RING E3 ligases (CRLs)-dependent ubiquitin proteasome system mediates degradation of numerous proteins that controls a wide range of developmental and physiological processes in eukaryotes. Cullin-associated Nedd8-dissociated protein 1 (CAND1) acts as an exchange factor allowing substrate recognition part exchange and plays a vital role in reactivating CRLs. The present study reports on the identification of SlCAND1, the only one CAND gene in tomato. SlCAND1 expression is ubiquitous and positively regulated by multiple plant hormones. Silencing of SlCAND1 expression using RNAi strategy resulted in a pleiotropic and gibberellin/auxin-associated phenotypes, including dwarf plant with reduced internode length, loss of apical dominance, early flowering, low seed germination percentage, delayed seed germination speed, short primary root, and increased lateral root proliferation and elongation. Moreover, application of exogenous GA3 or IAA could partly rescue some SlCAND1-silenced phenotypes, and the expression levels of gibberellin/auxin-related genes were altered in SlCAND1-RNAi lines. These facts revealed that SlCAND1 is required for gibberellin/auxin-associated regulatory network in tomato. Although SlCAND1 is crucial for multiple developmental processes during vegetative growth stage, SlCAND1-RNAi lines didn't exhibit visible effect on fruit development and ripening. Meanwhile, we discussed that multiple physiological functions of SlCAND1 in tomato are different to previous report of its ortholog in Arabidopsis. Our study adds a new perspective on the functional roles of CAND1 in plants, and strongly supports the hypothesis that CAND1 and its regulated ubiquitin proteasome system are pivotal for plant vegetative growth but possibly have different roles in diverse plant species.


Assuntos
Flores/fisiologia , Germinação , Lycopersicon esculentum/fisiologia , Proteínas de Plantas/fisiologia , Raízes de Plantas/fisiologia , Arabidopsis/genética , Proteínas Culina , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Giberelinas/metabolismo , Lycopersicon esculentum/genética , Fenótipo , Reguladores de Crescimento de Planta/fisiologia , Proteínas de Plantas/genética , Interferência de RNA , Sementes/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia
20.
Gene ; 732: 144368, 2020 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-31954859

RESUMO

The INO80 complex, including the Ino80 protein, forms a highly conserved canonical complex that remodels chromatin in the context of multiple cellular functions. The Drosophila homologue, dIno80, is involved in homeotic gene regulation during development as a canonical Pho-dIno80 complex. Previously, we found that dIno80 regulates homeotic genes by interacting with epigenetic regulators, such as polycomb and trithorax, suggesting the occurrence of non-canonical Ino80 complexes. Here using spectroscopic methods and gel retardation assays, we identified a set of consensus DNA sequences that DNA binding domain of dIno80 (DBINO) interacts with having differential affinity and high specificity. Testing these sequences in reporter assays, showed that this interaction can positively regulate transcription. These results suggest that, dIno80 has a sequence preference for interaction with DNA leading to transcriptional changes.


Assuntos
Proteínas de Ligação a DNA/fisiologia , DNA/metabolismo , Proteínas de Drosophila/fisiologia , Fatores de Transcrição/fisiologia , Animais , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Fatores de Transcrição/metabolismo , Transcrição Genética
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