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1.
Blood Adv ; 3(16): 2499-2511, 2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31455666

RESUMO

Erythroid maturation requires the concerted action of a core set of transcription factors. We previously identified the Krüppel-type zinc finger transcription factor Zfp148 (also called ZBP-89) as an interacting partner of the master erythroid transcription factor GATA1. Here we report the conditional knockout of Zfp148 in mice. Global loss of Zfp148 results in perinatal lethality from nonhematologic causes. Selective Zfp148 loss within the hematopoietic system results in a mild microcytic and hypochromic anemia, mildly impaired erythroid maturation, and delayed recovery from phenylhydrazine-induced hemolysis. Based on the mild erythroid phenotype of these mice compared with GATA1-deficient mice, we hypothesized that additional factor(s) may complement Zfp148 function during erythropoiesis. We show that Zfp281 (also called ZBP-99), another member of the Zfp148 transcription factor family, is highly expressed in murine and human erythroid cells. Zfp281 knockdown by itself results in partial erythroid defects. However, combined deficiency of Zfp148 and Zfp281 causes a marked erythroid maturation block. Zfp281 physically associates with GATA1, occupies many common chromatin sites with GATA1 and Zfp148, and regulates a common set of genes required for erythroid cell differentiation. These findings uncover a previously unknown role for Zfp281 in erythroid development and suggest that it functionally overlaps with that of Zfp148 during erythropoiesis.

3.
Am J Hum Genet ; 2018 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-30503522

RESUMO

Diamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder that affects 7 out of 1,000,000 live births and has been associated with mutations in components of the ribosome. In order to characterize the genetic landscape of this heterogeneous disorder, we recruited a cohort of 472 individuals with a clinical diagnosis of DBA and performed whole-exome sequencing (WES). We identified relevant rare and predicted damaging mutations for 78% of individuals. The majority of mutations were singletons, absent from population databases, predicted to cause loss of function, and located in 1 of 19 previously reported ribosomal protein (RP)-encoding genes. Using exon coverage estimates, we identified and validated 31 deletions in RP genes. We also observed an enrichment for extended splice site mutations and validated their diverse effects using RNA sequencing in cell lines obtained from individuals with DBA. Leveraging the size of our cohort, we observed robust genotype-phenotype associations with congenital abnormalities and treatment outcomes. We further identified rare mutations in seven previously unreported RP genes that may cause DBA, as well as several distinct disorders that appear to phenocopy DBA, including nine individuals with biallelic CECR1 mutations that result in deficiency of ADA2. However, no new genes were identified at exome-wide significance, suggesting that there are no unidentified genes containing mutations readily identified by WES that explain >5% of DBA-affected case subjects. Overall, this report should inform not only clinical practice for DBA-affected individuals, but also the design and analysis of rare variant studies for heterogeneous Mendelian disorders.

4.
Artigo em Inglês | MEDLINE | ID: mdl-30346647

RESUMO

Gestational diabetes mellitus (GDM) is defined as glucose intolerance with onset or first diagnosis during pregnancy, but not to the level of being diagnostic for diabetes in a nonpregnant adult. In GDM, whole-body insulin-dependent glucose disposal decreases by 40%-60% which necessitates a 200%-250% increase in insulin secretion to maintain normoglycaemia. GDM develops when a pregnant woman does not produce sufficient insulin to compensate for the reduced glucose disposal. Fibroblast growth factor 21 (FGF21) is a hormone that is expressed predominantly in the liver, but also in other metabolically active tissues such as pancreas, skeletal muscle and adipose tissue. In animals, FGF21 lowers blood glucose levels and inhibits glucagon secretion. In humans, circulating FGF21 levels are increased in insulin-resistant morbidities such as obesity and type 2 diabetes mellitus (T2DM). An elevated FGF21 level is also an independent predictor of T2DM. GDM and T2DM are proposed to have similar underlying pathophysiologies, raising the question of whether a similar relationship exists between FGF21 and GDM as it does with T2DM. There are a limited number of studies investigating FGF21 levels in patients with GDM. Moreover, recent clinical trials investigating the therapeutic potential of FGF21 have highlighted a major gap in our understanding of the biology of FGF21. This review evaluates what is currently known about FGF21 and GDM and highlights important gaps that warrant further research.

5.
Sci Rep ; 8(1): 5043, 2018 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-29568010

RESUMO

Tissue engineering scaffolds are used in conjunction with stem cells for the treatment of various diseases. A number of factors provided by the scaffolds affect the differentiation of stem cells. Mechanical cues that are part of the natural cellular microenvironment can both accelerate the differentiation toward particular cell lineages or induce differentiation to an alternative cell fate. Among such factors, there are externally applied strains and mechanical (stiffness and relaxation time) properties of the extracellular matrix. Here, the mechanics of a fibrous-porous scaffold is studied by applying a coordinated modeling and experimental approach. A force relaxation experiment is used, and a poroelastic model associates the relaxation process with the fluid diffusion through the fibrous matrix. The model parameters, including the stiffness moduli in the directions along and across the fibers as well as fluid diffusion time, are estimated by fitting the experimental data. The time course of the applied force is then predicted for different rates of loading and scaffold porosities. The proposed approach can help in a reduction of the technological and experimental efforts to produce 3-D scaffolds for regenerative medicine as well as in a higher accuracy of the estimation of the local factors sensed by stem cells.

6.
Cell ; 168(6): 1053-1064.e15, 2017 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-28283061

RESUMO

Cytokines are classically thought to stimulate downstream signaling pathways through monotonic activation of receptors. We describe a severe anemia resulting from a homozygous mutation (R150Q) in the cytokine erythropoietin (EPO). Surprisingly, the EPO R150Q mutant shows only a mild reduction in affinity for its receptor but has altered binding kinetics. The EPO mutant is less effective at stimulating erythroid cell proliferation and differentiation, even at maximally potent concentrations. While the EPO mutant can stimulate effectors such as STAT5 to a similar extent as the wild-type ligand, there is reduced JAK2-mediated phosphorylation of select downstream targets. This impairment in downstream signaling mechanistically arises from altered receptor dimerization dynamics due to extracellular binding changes. These results demonstrate how variation in a single cytokine can lead to biased downstream signaling and can thereby cause human disease. Moreover, we have defined a distinct treatable form of anemia through mutation identification and functional studies.


Assuntos
Anemia de Diamond-Blackfan/genética , Anemia de Diamond-Blackfan/patologia , Eritropoetina/genética , Mutação de Sentido Incorreto , Transdução de Sinais , Anemia de Diamond-Blackfan/terapia , Criança , Consanguinidade , Ativação Enzimática , Eritropoese , Eritropoetina/química , Feminino , Humanos , Janus Quinase 2/metabolismo , Cinética , Masculino , Receptores da Eritropoetina/química , Receptores da Eritropoetina/genética , Receptores da Eritropoetina/metabolismo
8.
IEEE/ACM Trans Comput Biol Bioinform ; 13(2): 350-64, 2016 Mar-Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27070978

RESUMO

This study considers the problem of describing and predicting cleft formation during the early stages of branching morphogenesis in mouse submandibular salivary glands (SMG) under the influence of varied concentrations of epidermal growth factors (EGF). Given a time-lapse video of a growing SMG, first we build a descriptive model that captures the underlying biological process and quantifies the ground truth. Tissue-scale (global) and morphological features related to regions of interest (local features) are used to characterize the biological ground truth. Second, we devise a predictive growth model that simulates EGF-modulated branching morphogenesis using a dynamic graph algorithm, which is driven by biological parameters such as EGF concentration, mitosis rate, and cleft progression rate. Given the initial configuration of the SMG, the evolution of the dynamic graph predicts the cleft formation, while maintaining the local structural characteristics of the SMG. We determined that higher EGF concentrations cause the formation of higher number of buds and comparatively shallow cleft depths. Third, we compared the prediction accuracy of our model to the Glazier-Graner-Hogeweg (GGH) model, an on-lattice Monte-Carlo simulation model, under a specific energy function parameter set that allows new rounds of de novo cleft formation. The results demonstrate that the dynamic graph model yields comparable simulations of gland growth to that of the GGH model with a significantly lower computational complexity. Fourth, we enhanced this model to predict the SMG morphology for an EGF concentration without the assistance of a ground truth time-lapse biological video data; this is a substantial benefit of our model over other similar models that are guided and terminated by information regarding the final SMG morphology. Hence, our model is suitable for testing the impact of different biological parameters involved with the process of branching morphogenesis in silico, while reducing the requirement of in vivo experiments.


Assuntos
Modelos Biológicos , Modelos Estatísticos , Morfogênese/fisiologia , Biologia de Sistemas/métodos , Aprendizado de Máquina não Supervisionado , Animais , Feminino , Camundongos , Método de Monte Carlo , Glândulas Salivares/crescimento & desenvolvimento
9.
J Cancer ; 7(1): 32-6, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26722357

RESUMO

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure syndrome associated with ribosomal protein (RP) gene mutations. Recent studies have also demonstrated an increased risk of cancer predisposition among DBA patients. In this study, we report the formation of soft tissue sarcoma in the Rpl5 and Rps24 heterozygous mice. Our observation suggests that even though one wild-type allele of the Rpl5 or Rps24 gene prevents anemia in these mice, it still predisposes them to cancer development.

10.
Int J Nanomedicine ; 10: 2065-77, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25844037

RESUMO

Cancer ranks among the leading causes of human mortality. Cancer becomes intractable when it spreads from the primary tumor site to various organs (such as bone, lung, liver, and then brain). Unlike solid tumor cells, cancer stem cells and metastatic cancer cells grow in a non-attached (suspension) form when moving from their source to other locations in the body. Due to the non-attached growth nature, metastasis is often first detected in the circulatory systems, for instance in a lymph node near the primary tumor. Cancer research over the past several decades has primarily focused on treating solid tumors, but targeted therapy to treat cancer stem cells and cancer metastasis has yet to be developed. Because cancers undergo faster metabolism and consume more glucose than normal cells, glucose was chosen in this study as a reagent to target cancer cells. In particular, by covalently binding gold nanoparticles (GNPs) with thio-PEG (polyethylene glycol) and thio-glucose, the resulting functionalized GNPs (Glu-GNPs) were created for targeted treatment of cancer metastasis and cancer stem cells. Suspension cancer cell THP-1 (human monocytic cell line derived from acute monocytic leukemia patients) was selected because it has properties similar to cancer stem cells and has been used as a metastatic cancer cell model for in vitro studies. To take advantage of cancer cells' elevated glucose consumption over normal cells, different starvation periods were screened in order to achieve optimal treatment effects. Cancer cells were then fed using Glu-GNPs followed by X-ray irradiation treatment. For comparison, solid tumor MCF-7 cells (breast cancer cell line) were studied as well. Our irradiation experimental results show that Glu-GNPs are better irradiation sensitizers to treat THP-1 cells than MCF-7 cells, or Glu-GNPs enhance the cancer killing of THP-1 cells 20% more than X-ray irradiation alone and GNP treatment alone. This finding can help oncologists to design therapeutic strategies to target cancer stem cells and cancer metastasis.


Assuntos
Antineoplásicos/farmacologia , Glucose/química , Ouro/química , Nanopartículas Metálicas , Células-Tronco Neoplásicas/efeitos dos fármacos , Antineoplásicos/química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos da radiação , Feminino , Ouro/farmacologia , Humanos , Células MCF-7/efeitos dos fármacos , Células MCF-7/efeitos da radiação , Nanopartículas Metálicas/química , Células-Tronco Neoplásicas/metabolismo , Polietilenoglicóis/farmacologia , Raios X
11.
Blood ; 124(3): 437-40, 2014 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-24735966

RESUMO

Pearson marrow pancreas syndrome (PS) is a multisystem disorder caused by mitochondrial DNA (mtDNA) deletions. Diamond-Blackfan anemia (DBA) is a congenital hypoproliferative anemia in which mutations in ribosomal protein genes and GATA1 have been implicated. Both syndromes share several features including early onset of severe anemia, variable nonhematologic manifestations, sporadic genetic occurrence, and occasional spontaneous hematologic improvement. Because of the overlapping features and relative rarity of PS, we hypothesized that some patients in whom the leading clinical diagnosis is DBA actually have PS. Here, we evaluated patient DNA samples submitted for DBA genetic studies and found that 8 (4.6%) of 173 genetically uncharacterized patients contained large mtDNA deletions. Only 2 (25%) of the patients had been diagnosed with PS on clinical grounds subsequent to sample submission. We conclude that PS can be overlooked, and that mtDNA deletion testing should be performed in the diagnostic evaluation of patients with congenital anemia.


Assuntos
Acil-CoA Desidrogenase de Cadeia Longa/deficiência , Anemia de Diamond-Blackfan/diagnóstico , Anemia de Diamond-Blackfan/genética , DNA Mitocondrial/genética , Erros Inatos do Metabolismo Lipídico/diagnóstico , Erros Inatos do Metabolismo Lipídico/genética , Doenças Mitocondriais/diagnóstico , Doenças Mitocondriais/genética , Doenças Musculares/diagnóstico , Doenças Musculares/genética , Acil-CoA Desidrogenase de Cadeia Longa/genética , Criança , Pré-Escolar , Análise Mutacional de DNA , Diagnóstico Diferencial , Humanos , Lactente , Mutação , Deleção de Sequência
12.
PLoS Comput Biol ; 9(11): e1003319, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24277996

RESUMO

Cleft formation during submandibular salivary gland branching morphogenesis is the critical step initiating the growth and development of the complex adult organ. Previous experimental studies indicated requirements for several epithelial cellular processes, such as proliferation, migration, cell-cell adhesion, cell-extracellular matrix (matrix) adhesion, and cellular contraction in cleft formation; however, the relative contribution of each of these processes is not fully understood since it is not possible to experimentally manipulate each factor independently. We present here a comprehensive analysis of several cellular parameters regulating cleft progression during branching morphogenesis in the epithelial tissue of an early embryonic salivary gland at a local scale using an on lattice Monte-Carlo simulation model, the Glazier-Graner-Hogeweg model. We utilized measurements from time-lapse images of mouse submandibular gland organ explants to construct a temporally and spatially relevant cell-based 2D model. Our model simulates the effect of cellular proliferation, actomyosin contractility, cell-cell and cell-matrix adhesions on cleft progression, and it was used to test specific hypotheses regarding the function of these parameters in branching morphogenesis. We use innovative features capturing several aspects of cleft morphology and quantitatively analyze clefts formed during functional modification of the cellular parameters. Our simulations predict that a low epithelial mitosis rate and moderate level of actomyosin contractility in the cleft cells promote cleft progression. Raising or lowering levels of contractility and mitosis rate resulted in non-progressive clefts. We also show that lowered cell-cell adhesion in the cleft region and increased cleft cell-matrix adhesions are required for cleft progression. Using a classifier-based analysis, the relative importance of these four contributing cellular factors for effective cleft progression was determined as follows: cleft cell contractility, cleft region cell-cell adhesion strength, epithelial cell mitosis rate, and cell-matrix adhesion strength.


Assuntos
Modelos Biológicos , Morfogênese/fisiologia , Glândula Submandibular/embriologia , Algoritmos , Animais , Adesão Celular , Embrião de Mamíferos , Feminino , Camundongos , Método de Monte Carlo
13.
PLoS One ; 6(10): e25830, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21998704

RESUMO

Auxotrophic marker genes such as URA3, LEU2, and HIS3 in Saccharomyces cerevisiae have long been used to select cells that have been successfully transformed with recombinant DNA. A longstanding challenge in working with these genes is that counterselection procedures are often lacking. This paper describes the unexpected discovery of a simple plate assay that imparts a bright red stain to cells experiencing nutritional stress from the lack of a marker gene. The procedure specifically stains a zinc-rich vesicular compartment analogous to the zinc-rich secretory vesicles found in insulin-secreting pancreatic islet cells and glutamate-secreting neurons. Staining was greatly diminished in zap1 mutants, which lack a homeostatic activator of zinc uptake, and in cot1 zrc1 double mutants, which lack the two yeast homologs of mammalian vesicle-specific zinc export proteins. Only one of 93 strains with temperature-sensitive alleles of essential genes exhibited an increase in dithizone staining at its non-permissive temperature, indicating that staining is not simply a sign of growth-arrested or dying cells. Remarkably, the procedure works with most commonly used marker genes, highlights subtle defects, uses no reporter constructs or expensive reagents, requires only a few hours of incubation, yields visually striking results without any instrumentation, and is not toxic to the cells. Many potential applications exist for dithizone staining, both as a versatile counterscreen for auxotrophic marker genes and as a powerful new tool for the genetic analysis of a biomedically important vesicular organelle.


Assuntos
Ditizona/metabolismo , Genes Fúngicos/genética , Marcadores Genéticos/genética , Espaço Intracelular/metabolismo , Saccharomyces cerevisiae/citologia , Coloração e Rotulagem/métodos , Zinco/metabolismo , Sobrevivência Celular , Células Clonais/citologia , Cor , Permeabilidade , Saccharomyces cerevisiae/genética , Vesículas Secretórias/metabolismo
14.
Genetics ; 186(2): 725-34, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20660648

RESUMO

The exact molecular mechanisms by which the environmental pollutant arsenic works in biological systems are not completely understood. Using an unbiased chemogenomics approach in Saccharomyces cerevisiae, we found that mutants of the chaperonin complex TRiC and the functionally related prefoldin complex are all hypersensitive to arsenic compared to a wild-type strain. In contrast, mutants with impaired ribosome functions were highly arsenic resistant. These observations led us to hypothesize that arsenic might inhibit TRiC function, required for folding of actin, tubulin, and other proteins postsynthesis. Consistent with this hypothesis, we found that arsenic treatment distorted morphology of both actin and microtubule filaments. Moreover, arsenic impaired substrate folding by both bovine and archaeal TRiC complexes in vitro. These results together indicate that TRiC is a conserved target of arsenic inhibition in various biological systems.


Assuntos
Chaperonina com TCP-1/antagonistas & inibidores , Óxidos/toxicidade , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Actinas/química , Actinas/metabolismo , Trióxido de Arsênio , Arsenicais , Western Blotting , Chaperonina com TCP-1/química , Chaperonina com TCP-1/metabolismo , Imunofluorescência , Mathanococcus/efeitos dos fármacos , Proteínas dos Microtúbulos , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Chaperonas Moleculares/antagonistas & inibidores , Chaperonas Moleculares/química , Mutação , Dobramento de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo
15.
J Bacteriol ; 191(4): 1152-61, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19060143

RESUMO

DNA double-strand breaks are particularly deleterious lesions that can lead to genomic instability and cell death. We investigated the SOS response to double-strand breaks in both Escherichia coli and Bacillus subtilis. In E. coli, double-strand breaks induced by ionizing radiation resulted in SOS induction in virtually every cell. E. coli strains incapable of SOS induction were sensitive to ionizing radiation. In striking contrast, we found that in B. subtilis both ionizing radiation and a site-specific double-strand break causes induction of prophage PBSX and SOS gene expression in only a small subpopulation of cells. These results show that double-strand breaks provoke global SOS induction in E. coli but not in B. subtilis. Remarkably, RecA-GFP focus formation was nearly identical following ionizing radiation challenge in both E. coli and B. subtilis, demonstrating that formation of RecA-GFP foci occurs in response to double-strand breaks but does not require or result in SOS induction in B. subtilis. Furthermore, we found that B. subtilis cells incapable of inducing SOS had near wild-type levels of survival in response to ionizing radiation. Moreover, B. subtilis RecN contributes to maintaining low levels of SOS induction during double-strand break repair. Thus, we found that the contribution of SOS induction to double-strand break repair differs substantially between E. coli and B. subtilis.


Assuntos
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Quebras de DNA de Cadeia Dupla , Escherichia coli/genética , Escherichia coli/metabolismo , Resposta SOS (Genética)/fisiologia , Bacillus subtilis/efeitos da radiação , Desoxirribonucleases de Sítio Específico do Tipo II , Escherichia coli/efeitos da radiação , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/fisiologia
16.
Cell ; 134(6): 1066-78, 2008 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-18805098

RESUMO

Nucleosome structural integrity underlies the regulation of DNA metabolism and transcription. Using a synthetic approach, a versatile library of 486 systematic histone H3 and H4 substitution and deletion mutants that probes the contribution of each residue to nucleosome function was generated in Saccharomyces cerevisiae. We probed fitness contributions of each residue to perturbations of chromosome integrity and transcription, mapping global patterns of chemical sensitivities and requirements for transcriptional silencing onto the nucleosome surface. Each histone mutant was tagged with unique molecular barcodes, facilitating identification of histone mutant pools through barcode amplification, labeling, and TAG microarray hybridization. Barcodes were used to score complex phenotypes such as competitive fitness in a chemostat, DNA repair proficiency, and synthetic genetic interactions, revealing new functions for distinct histone residues and new interdependencies among nucleosome components and their modifiers.


Assuntos
Histonas/genética , Histonas/metabolismo , Nucleossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Cromossomos Fúngicos/metabolismo , Dano ao DNA , Reparo do DNA , Deleção de Genes , Biblioteca Gênica , Inativação Gênica , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Plasmídeos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Especificidade da Espécie
17.
Biol Reprod ; 79(6): 1176-82, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-18753606

RESUMO

We previously discovered a germ cell-specific spermatogenesis and oogenesis basic helix-loop-helix transcription factor, Sohlh2. We generated Sohlh2-deficient mice to understand physiologic consequences of Sohlh2 deletion. We discovered that Sohlh2-knockout adult female mice are infertile due to lack of ovarian follicles. Sohlh2-deficient ovaries can form primordial follicles and, despite limited oocyte growth, do not differentiate surrounding granulosa cells into cuboidal and multilayered structures. Oocytes are rapidly lost in Sohlh2-deficient ovaries, and few are present by 14 days of postnatal life. However, the primordial oocytes are abnormal at the molecular level because they misexpress numerous germ cell- and oocyte-specific genes, including Sohlh1, Nobox, Figla, Gdf9, Pou5f1, Zp1, Zp3, Kit, Oosp1, Nlrp14, H1foo, and Stra8. Our findings show that Sohlh2 is a critical factor for maintenance and differentiation of the oocyte during early oogenesis.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Expressão Gênica/fisiologia , Células Germinativas/fisiologia , Oócitos/metabolismo , Oogênese/genética , Oogênese/fisiologia , Folículo Ovariano/fisiologia , Animais , Animais Recém-Nascidos , Diferenciação Celular/fisiologia , Feminino , Genótipo , Células da Granulosa/fisiologia , Imuno-Histoquímica , Infertilidade Feminina/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA/biossíntese , RNA/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa
18.
Genes Dev ; 22(15): 2062-74, 2008 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-18676811

RESUMO

Histone acetylation and deacetylation are among the principal mechanisms by which chromatin is regulated during transcription, DNA silencing, and DNA repair. We analyzed patterns of genetic interactions uncovered during comprehensive genome-wide analyses in yeast to probe how histone acetyltransferase (HAT) and histone deacetylase (HDAC) protein complexes interact. The genetic interaction data unveil an underappreciated role of HDACs in maintaining cellular viability, and led us to show that deacetylation of the histone variant Htz1p at Lys 14 is mediated by Hda1p. Studies of the essential nucleosome acetyltransferase of H4 (NuA4) revealed acetylation-dependent protein stabilization of Yng2p, a potential nonhistone substrate of NuA4 and Rpd3C, and led to a new functional organization model for this critical complex. We also found that DNA double-stranded breaks (DSBs) result in local recruitment of the NuA4 complex, followed by an elaborate NuA4 remodeling process concomitant with Rpd3p recruitment and histone deacetylation. These new characterizations of the HDA and NuA4 complexes demonstrate how systematic analyses of genetic interactions may help illuminate the mechanisms of intricate cellular processes.


Assuntos
Histonas/metabolismo , Saccharomyces cerevisiae/metabolismo , Biologia de Sistemas , Acetilação , Acetiltransferases , Análise por Conglomerados , Reparo do DNA , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/genética , Cinética , Modelos Biológicos , Modelos Genéticos , Mutação , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
19.
Methods Mol Biol ; 416: 221-47, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18392971

RESUMO

Comprehensive collections of open reading frame (ORF) deletion mutant strains exist for the budding yeast Saccharomyces cerevisiae. With great prescience, these strains were designed with short molecular bar codes or TAGs that uniquely mark each deletion allele, flanked by shared priming sequences. These features have enabled researchers to handle yeast mutant collections as complex pools of approximately 6000 strains. The presence of any individual mutant within a pool can be assessed indirectly by measuring the relative abundance of its corresponding TAG(s) in genomic DNA prepared from the pool. This is readily accomplished by wholesale polymerase chain reaction (PCR) amplification of the TAGs using fluorescent oligonucleotide primers that recognize the common flanking sequences, followed by hybridization of the labeled PCR products to a TAG oligonucleotide microarray. Here we describe a method-diploid-based synthetic lethality analysis by microarray (dSLAM)-whereby such pools can be manipulated to rapidly construct and assess the fitness of 6000 double-mutant strains in a single experiment. Analysis of double-mutant strains is of growing importance in defining the spectrum of essential cellular functionalities and in understanding how these functionalities interrelate.


Assuntos
Regulação Fúngica da Expressão Gênica/fisiologia , Genes Letais , Mutação/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Saccharomyces cerevisiae/genética , Deleção de Genes , Perfilação da Expressão Gênica/métodos , Regulação Fúngica da Expressão Gênica/genética , Genoma Fúngico
20.
Mol Microbiol ; 67(2): 350-63, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18067541

RESUMO

The bacterial non-homologous end-joining (NHEJ) apparatus is a two-component system that uses Ku and LigD to repair DNA double-strand breaks. Although the reaction mechanism has been extensively studied, much less is known about the physiological role of bacterial NHEJ. Recent studies suggest that NHEJ acts under conditions where DNA replication is reduced or absent (such as in a spore or stationary phase). Interestingly, genes encoding Ku and LigD have been identified in a wide range of bacteria that can chronically infect eukaryotic hosts. Strikingly, Sinohizobium meliloti, an intracellular symbiont of legume plants, carries four genes encoding Ku homologues (sku1 to sku4). Deletion analysis of the sku genes indicated that all Ku homologues are functional. One of these genes, sku2, is strongly expressed in free-living cells, as well as in bacteroid cells residing inside of the host plant. To visualize the NHEJ apparatus in vivo, SKu2 protein was fused to yellow fluorescent protein (YFP). Ionizing radiation (IR) induced focus formation of SKu2-YFP in free-living cells in a dosage-dependent manner. Moreover, SKu2-YFP foci formed in response to IR in non-dividing bacteroids, indicating that NHEJ system is functional even during the chronic infection phase of symbiosis.


Assuntos
Proteínas de Bactérias/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Medicago sativa/microbiologia , Sinorhizobium meliloti/fisiologia , Proteínas de Bactérias/genética , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Genes Reporter , Medicago sativa/enzimologia , Medicago sativa/fisiologia , Radiação Ionizante , Deleção de Sequência , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/efeitos da radiação , Simbiose
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