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1.
Cell Signal ; 27(2): 204-14, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25446257

RESUMEN

CaMKs link transient increases in intracellular Ca(2+) with biological processes. In myeloid leukemia cells, CaMKII, activated by the bcr-abl oncogene, promotes cell proliferation. Inhibition of CaMKII activity restricts cell proliferation, and correlates with growth arrest and differentiation. The mechanism by which the inhibition of CaMKII results in growth arrest and differentiation in myeloid leukemia cells is still unknown. We report that inhibition of CaMKII activity results in an upregulation of CaMKIV mRNA and protein in leukemia cell lines. Conversely, expression of CaMKIV inhibits autophosphorylation and activation of CaMKII, and elicits G0/G1cell cycle arrest,impairing cell proliferation. Furthermore, U937 cells expressing CaMKIV show elevated levels of Cdk inhibitors p27(kip1) and p16(ink4a) and reduced levels of cyclins A, B1 and D1. These findings were also confirmed in the K562 leukemic cell line. The relationship between CaMKII and CaMKIV is also observed in primary acute myeloid leukemia (AML) cells, and it correlates with their immunophenotypic profile. Indeed, immature MO/M1 AML showed increased CaMKIV expression and decreased pCaMKII, whereas highly differentiated M4/M5 AML showed decreased CaMKIV expression and increased pCaMKII levels. Our data reveal a novel cross-talk between CaMKII and CaMKIV and suggest that CaMKII suppresses the expression of CaMKIV to promote leukemia cell proliferation.


Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina/metabolismo , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina/genética , Línea Celular Tumoral , Proliferación Celular , Ciclina A/metabolismo , Ciclina B1/metabolismo , Ciclina D1/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación hacia Abajo , Puntos de Control de la Fase G1 del Ciclo Celular , Humanos , Inmunofenotipificación , Células K562 , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patología , Fosforilación , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Células U937
2.
Am J Physiol Endocrinol Metab ; 303(8): E1061-8, 2012 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-22932781

RESUMEN

Emerging research has shown that subtle factors during pregnancy and gestation can influence long-term health in offspring. In an attempt to be proactive, we set out to explore whether a nonpharmacological intervention, perinatal exercise, might improve offspring health. Female mice were separated into sedentary or exercise cohorts, with the exercise cohort having voluntary access to a running wheel prior to mating and during pregnancy and nursing. Offspring were weaned, and analyses were performed on the mature offspring that did not have access to running wheels during any portion of their lives. Perinatal exercise caused improved glucose disposal following an oral glucose challenge in both female and male adult offspring (P < 0.05 for both). Blood glucose concentrations were reduced to lower values in response to an intraperitoneal insulin tolerance test for both female and male adult offspring of parents with access to running wheels (P < 0.05 and P < 0.01, respectively). Male offspring from exercised dams showed increased percent lean mass and decreased fat mass percent compared with male offspring from sedentary dams (P < 0.01 for both), but these parameters were unchanged in female offspring. These data suggest that short-term maternal voluntary exercise prior to and during healthy pregnancy and nursing can enhance long-term glucose homeostasis in offspring.


Asunto(s)
Glucosa/metabolismo , Homeostasis/fisiología , Condicionamiento Físico Animal/fisiología , Tejido Adiposo/metabolismo , Animales , Peso al Nacer/fisiología , Glucemia/metabolismo , Composición Corporal/fisiología , Peso Corporal/fisiología , Desoxiglucosa/metabolismo , Ingestión de Alimentos/fisiología , Femenino , Prueba de Tolerancia a la Glucosa , Insulina/metabolismo , Lactancia/fisiología , Tamaño de la Camada/fisiología , Ratones , Ratones Endogámicos ICR , Músculo Esquelético/metabolismo , Embarazo , Carrera/fisiología
3.
Int J Biochem Cell Biol ; 43(9): 1252-6, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21605695

RESUMEN

Mitochondria have long been recognized as cellular energy power houses that also regulate cellular redox signaling to arbitrate cell survival. Recent studies of mitochondria in stem cells (SCs) demonstrate that they have critical roles beyond this traditional view. Embryonic (E) SCs, termed pluripotent for their ability to differentiate into all cell types within an organism, maintain a limited number of morphologically undifferentiated (electron translucent and poorly formed cristae) mitochondria. As these cells differentiate, their mitochondria undergo a tightly choreographed gain of number, mass and morphological complexity. Therefore, mechanisms that regulate mitochondrial growth, localization, division and partition must play active roles in the maintenance of pluripotency and execution of differentiation. Aberrant mitochondrial dynamics are associated with a plethora of human disorders, for which SCs hold curative potential. Hence, a comprehensive understanding of the mechanisms that regulate mitochondrial dynamics and function in SCs and their overall relationship to the maintenance of pluripotency is pivotal for the progression of therapeutic regenerative medicine.


Asunto(s)
Células Madre Embrionarias/enzimología , GTP Fosfohidrolasas/metabolismo , Mitocondrias/enzimología , Oxidorreductasas/metabolismo , Animales , Diferenciación Celular , Reductasas del Citocromo/metabolismo , Células Madre Embrionarias/fisiología , Células Madre Embrionarias/ultraestructura , Humanos , Mitocondrias/metabolismo , Enfermedades Mitocondriales/metabolismo , Enfermedades Mitocondriales/patología , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro
4.
Am J Physiol Heart Circ Physiol ; 300(4): H1133-40, 2011 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-21217061

RESUMEN

Hyperglycemia in diabetes mellitus promotes oxidative stress in endothelial cells, which contributes to development of cardiovascular diseases. Nuclear factor erythroid 2-related factor-2 (Nrf2) is a transcription factor activated by oxidative stress that regulates expression of numerous reactive oxygen species (ROS) detoxifying and antioxidant genes. This study was designed to elucidate the homeostatic role of adaptive induction of Nrf2-driven free radical detoxification mechanisms in endothelial protection under diabetic conditions. Using a Nrf2/antioxidant response element (ARE)-driven luciferase reporter gene assay we found that in a cultured coronary arterial endothelial cell model hyperglycemia (10-30 mmol/l glucose) significantly increases transcriptional activity of Nrf2 and upregulates the expression of the Nrf2 target genes NQO1, GCLC, and HMOX1. These effects of high glucose were significantly attenuated by small interfering RNA (siRNA) downregulation of Nrf2 or overexpression of Keap-1, which inactivates Nrf2. High-glucose-induced upregulation of NQO1, GCLC, and HMOX1 was also prevented by pretreatment with polyethylene glycol (PEG)-catalase or N-acetylcysteine, whereas administration of H(2)O(2) mimicked the effect of high glucose. To test the effects of metabolic stress in vivo, Nrf2(+/+) and Nrf2(-/-) mice were fed a high-fat diet (HFD). HFD elicited significant increases in mRNA expression of Gclc and Hmox1 in aortas of Nrf2(+/+) mice, but not Nrf2(-/-) mice, compared with respective standard diet-fed control mice. Additionally, HFD-induced increases in vascular ROS levels were significantly greater in Nrf2(-/-) than Nrf2(+/+) mice. HFD-induced endothelial dysfunction was more severe in Nrf2(-/-) mice, as shown by the significantly diminished acetylcholine-induced relaxation of aorta of these animals compared with HFD-fed Nrf2(+/+) mice. Our results suggest that adaptive activation of the Nrf2/ARE pathway confers endothelial protection under diabetic conditions.


Asunto(s)
Adaptación Fisiológica , Antioxidantes/metabolismo , Vasos Coronarios/metabolismo , Hiperglucemia/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Acetilcisteína/farmacología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Aorta/química , Aorta/metabolismo , Catalasa/farmacología , Células Cultivadas , Proteínas del Citoesqueleto/genética , Grasas de la Dieta/metabolismo , Grasas de la Dieta/farmacología , Endotelio Vascular/metabolismo , Regulación de la Expresión Génica , Glutamato-Cisteína Ligasa/genética , Hemo-Oxigenasa 1/genética , Peróxido de Hidrógeno/farmacología , Proteína 1 Asociada A ECH Tipo Kelch , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos ICR , NAD(P)H Deshidrogenasa (Quinona)/genética , Factor 2 Relacionado con NF-E2/genética , Polietilenglicoles/farmacología , Especies Reactivas de Oxígeno/metabolismo
5.
Reproduction ; 137(6): 923-9, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19336471

RESUMEN

The Hspa1b (Hsp70.1) gene is one of the first genes expressed after fertilization, with expression occurring during the minor zygotic genome activation (ZGA) in the absence of stress. This expression can take place in the male pronucleus as early as the one-cell stage of embryogenesis. The importance of HSPA1B for embryonic viability during times of stress is supported by studies showing that depletion of this protein results in a significant reduction in embryos developing to the blastocyte stage. Recently, we have begun addressing the mechanism responsible for allowing expression of Hspa1b during the minor ZGA and found that heat shock transcription factor (HSF) 1 and 2 bind the Hspa1b promoter during late spermatogenesis. In this report, we have extended those studies using western blots and chromatin immunoprecipitation assays and found that RNA polymerase II (Pol II) is present in epididymal spermatozoa and bound to the Hspa1b promoter. These present results, in addition to our previous results, support a model in which the binding of HSF1, HSF2, SP1, and Pol II to the promoter of Hspa1b would allow the rapid formation of a transcription-competent state during the minor ZGA, thereby allowing Hspa1b expression.


Asunto(s)
Epidídimo/enzimología , Proteínas HSP70 de Choque Térmico/metabolismo , Regiones Promotoras Genéticas , ARN Polimerasa II/metabolismo , Espermatozoides/enzimología , Animales , Sitios de Unión , Western Blotting , Núcleo Celular/enzimología , Inmunoprecipitación de Cromatina , Regulación del Desarrollo de la Expresión Génica , Proteínas HSP70 de Choque Térmico/genética , Masculino , Ratones
6.
Exp Cell Res ; 314(11-12): 2224-30, 2008 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-18570919

RESUMEN

Mitosis is a series of events leading to division of a cell by the process known as cytokinesis. Protein regulating cytokinesis 1 (PRC1) is a CDK substrate that associates with the mitotic spindle and functions in microtubule bundling. Previous studies revealed that loss of PRC1 is associated with chromosomal mis-segregation and atypical chromosome alignment. HSF2 is a DNA binding protein that we previously showed bookmarks the hsp70i gene during mitosis, an epigenetic mechanism which allows the hsp70i gene to re-establish transcriptional competence early in G1. Another study demonstrated that HSF2-/- mouse embryonic fibroblasts (MEFs) exhibit increased numbers of multinucleated cells vs. wild-type MEFs. This suggests that HSF2 is important for proper cytokinesis, but the mechanism was unknown. Here we report the existence of a direct interaction between HSF2 and PRC1. HSF2 and PRC1 associate during mitosis and co-localize during this phase of the cell cycle. PRC1 does not interact with the related protein HSF1, indicating the specificity of the HSF2-PRC1 interaction. Intriguingly, PRC1 is associated with the hsp70i promoter during mitosis. These results provide a potential mechanistic basis for the defective cytokinesis phenotype exhibited by HSF2-/- cells, as well as suggest a potential role for PRC1 in HSF2-mediated gene bookmarking.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Mitosis/fisiología , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Proteínas HSP70 de Choque Térmico/genética , Células HeLa , Proteínas de Choque Térmico/genética , Humanos , Células Jurkat , Ratones , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/genética , Técnicas del Sistema de Dos Híbridos
7.
Biol Reprod ; 79(2): 283-8, 2008 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-18434628

RESUMEN

The Hspa1b gene is one of the first genes expressed after fertilization, with expression observed in the male pronucleus as early as the one-cell stage of embryogenesis. This expression can occur in the absence of stress and is initiated during the minor zygotic genome activation. There is a significant reduction in the number of embryos developing to the blastocyte stage when HSPA1B levels are depleted, which supports the importance of this protein for embryonic viability. However, the mechanism responsible for allowing expression of Hspa1b during the minor zygotic genome activation (ZGA) is unknown. In this report, we investigated the role of HSF1 and HSF2 in bookmarking Hspa1b during late spermatogenesis. Western blot results show that both HSF1 and HSF2 are present in epididymal spermatozoa, and immunofluorescence analysis revealed that some of the HSF1 and HSF2 proteins in these cells overlap the 4',6'-diamidino-2-phenylindole-stained DNA region. Results from chromatin immunoprecipitation assays showed that HSF1, HSF2, and SP1 are bound to the Hspa1b promoter in epididymal spermatozoa. Furthermore, we observed an increase in HSF2 binding to the Hspa1b promoter in late spermatids versus early spermatids, suggesting a likely period during spermatogenesis when transcription factor binding could occur. These results support a model in which the binding of HSF1, HSF2, and SP1 to the promoter of Hspa1b would allow the rapid formation of a transcription-competent state during the minor ZGA, thereby allowing Hspa1b expression.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Epidídimo/metabolismo , Proteínas HSP70 de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Regiones Promotoras Genéticas , Espermatozoides/metabolismo , Factores de Transcripción/metabolismo , Animales , Núcleo Celular/metabolismo , Inmunoprecipitación de Cromatina , Factores de Transcripción del Choque Térmico , Masculino , Ratones , Unión Proteica , Factor de Transcripción Sp1/metabolismo , Espermatogénesis/genética , Distribución Tisular , Transcripción Genética/fisiología , Cigoto/metabolismo
8.
Cell Stress Chaperones ; 12(3): 283-90, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17915561

RESUMEN

Although the vast majority of genomic DNA is tightly compacted during mitosis, the promoter regions of a number of genes remain in a less compacted state throughout this stage of the cell cycle. The decreased compaction of these promoter regions, which is referred to as gene bookmarking, is thought to be important for the ability of cells to express these genes during the following interphase. Previously, we reported a role for the DNA-binding protein heat shock factor (HSF2) in bookmarking the stress-inducible 70,000-Da heat shock protein (hsp70) gene. In this report, we have extended those studies and found that during mitosis, HSF2 is bound to the HSE promoter elements of other heat shock genes, including hsp90 and hsp27, as well as the proto-oncogene c-fos. The presence of HSF2 is important for expression of these genes because blocking HSF2 levels by RNA interference techniques leads to decreased levels of these proteins. These results suggest that HSF2 is important for constitutive as well as stress-inducible expression of HSE-containing genes.


Asunto(s)
Regulación de la Expresión Génica , Proteínas HSP90 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Mitosis/genética , Proteínas de Neoplasias/metabolismo , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-fos/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Choque Térmico HSP27 , Proteínas HSP90 de Choque Térmico/genética , Células HeLa , Proteínas de Choque Térmico/genética , Humanos , Células Jurkat , Chaperonas Moleculares , Proteínas de Neoplasias/genética , Conformación de Ácido Nucleico , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-fos/genética , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Factores de Transcripción/genética
9.
J Biol Chem ; 282(47): 33902-7, 2007 Nov 23.
Artículo en Inglés | MEDLINE | ID: mdl-17897941

RESUMEN

Stress conditions inhibit mRNA export, but mRNAs encoding heat shock proteins continue to be efficiently exported from the nucleus during stress. How HSP mRNAs bypass this stress-associated export inhibition was not known. Here, we show that HSF1, the transcription factor that binds HSP promoters after stress to induce their transcription, interacts with the nuclear pore-associating TPR protein in a stress-responsive manner. TPR is brought into proximity of the HSP70 promoter after stress and preferentially associates with mRNAs transcribed from this promoter. Disruption of the HSF1-TPR interaction inhibits the export of mRNAs expressed from the HSP70 promoter, both endogenous HSP70 mRNA and a luciferase reporter mRNA. These results suggest that HSP mRNA export escapes stress inhibition via HSF1-mediated recruitment of the nuclear pore-associating protein TPR to HSP genes, thereby functionally connecting the first and last nuclear steps of the gene expression pathway, transcription and mRNA export.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Respuesta al Choque Térmico/fisiología , Proteínas de Complejo Poro Nuclear/metabolismo , Poro Nuclear/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , ARN Mensajero/metabolismo , Factores de Transcripción/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/fisiología , Proteínas HSP70 de Choque Térmico/genética , Células HeLa , Factores de Transcripción del Choque Térmico , Humanos , Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/genética , Regiones Promotoras Genéticas/fisiología , Unión Proteica/genética , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/genética , Factores de Transcripción/genética , Transcripción Genética/fisiología
10.
Science ; 307(5708): 421-3, 2005 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-15662014

RESUMEN

In contrast to most genomic DNA in mitotic cells, the promoter regions of some genes, such as the stress-inducible hsp70i gene that codes for a heat shock protein, remain uncompacted, a phenomenon called bookmarking. Here we show that hsp70i bookmarking is mediated by a transcription factor called HSF2, which binds this promoter in mitotic cells, recruits protein phosphatase 2A, and interacts with the CAP-G subunit of the condensin enzyme to promote efficient dephosphorylation and inactivation of condensin complexes in the vicinity, thereby preventing compaction at this site. Blocking HSF2-mediated bookmarking by HSF2 RNA interference decreases hsp70i induction and survival of stressed cells in the G1 phase, which demonstrates the biological importance of gene bookmarking.


Asunto(s)
Regulación de la Expresión Génica , Proteínas HSP70 de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Mitosis , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Adenosina Trifosfatasas/metabolismo , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/metabolismo , Células HeLa , Proteínas de Choque Térmico/genética , Calor , Humanos , Inmunoprecipitación , Interfase , Complejos Multiproteicos , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Unión Proteica , Proteína Fosfatasa 2 , Subunidades de Proteína/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/farmacología , Factores de Transcripción/genética , Técnicas del Sistema de Dos Híbridos
12.
J Cell Biochem ; 91(2): 375-83, 2004 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-14743396

RESUMEN

Transcription of the mammalian testis-specific linker histone H1t gene occurs only in pachytene primary spermatocytes during spermatogenesis. Studies of the wild type (Wt) and mutant H1t promoters in transgenic mice show that transcription of the H1t gene is dependent upon the TE promoter element. We purified an 85 kDa protein from rat testis nuclear extracts using the TE1 subelement as an affinity chromatography probe and analysis revealed that the protein was RFX2. The TE1 element is essentially an X-box DNA consensus element and regulatory factor X (RFX) binds specifically to this element. Polyclonal antibodies directed against RFX2 supershift the low mobility testis nuclear protein complex formed in electrophoretic mobility shift assays (EMSA). RFX2 derived from primary spermatocytes, where the transcription factor is relatively abundant, binds with high affinity to the TE1 element. Coexpression of RFX2 together with an H1t promoter/reporter vector activates the H1t promoter in a cultured GC-2spd germinal cell line, but mutation of either the TE1 subelement or the TE2 subelements represses activity. These observations lead us to conclude that the TE1 and TE2 subelements of the testis-specific histone H1t promoter are targets of the transcription factor RFX2 and that this factor plays a key role in activating transcription of the H1t gene in primary spermatocytes. Published 2003 Wiley-Liss, Inc.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Histonas/genética , Regiones Promotoras Genéticas , Testículo/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Animales , Secuencia de Bases , Regulación de la Expresión Génica , Vectores Genéticos , Masculino , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Ratas , Ratas Sprague-Dawley , Espermatocitos/metabolismo
13.
J Cell Biochem ; 88(6): 1177-87, 2003 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-12647300

RESUMEN

The testis-specific linker histone H1t gene is transcribed exclusively in pachytene primary spermatocytes. Tissue specific expression of the gene is mediated in part by transcriptional factors that bind elements located within the proximal and distal promoter. A 40 bp promoter element, designated H1t/TE, that is located within the proximal promoter between the CCAAT-box and AC-box, is known to be essential for H1t gene transcription in transgenic animals. In the present study, we show by SDS-PAGE analysis of UV crosslinked protein and DNA and by electrophoretic mobility shift assays (EMSA) of testis nuclear proteins separated on a non-denaturing glycerol gradient that the TE1 sub-element is bound by a protein complex. Mutation of TE1 leads to a drop in H1t promoter activity in germinal GC-2spd cells as well as in nongerminal Leydig, NIH3T3, and C127I cell lines. Although TE1 and TE2 sub-elements have similar sequences, mutation of the TE2 sub-element causes an increase in promoter activity in C127I and Leydig cells. The rat TE1 but not TE2 contains a CpG dinucleotide and this cytosine is methylated in liver but not in primary spermatocytes. Methylation of the cytosine at this site almost eliminates nuclear protein binding. Thus, there are significant functional differences in the TE2 and TE1 sub-elements of the H1t promoter with TE1 serving as a transcriptional activator binding site and TE2 serving as a repressor binding site in some cell lines.


Asunto(s)
Regulación de la Expresión Génica/fisiología , Histonas/fisiología , Regiones Promotoras Genéticas/fisiología , Testículo/metabolismo , Animales , Línea Celular , Regulación de la Expresión Génica/genética , Genes Reguladores , Histonas/química , Histonas/genética , Masculino , Metilación/efectos de los fármacos , Ratones , Proteínas Nucleares/análisis , Proteínas Nucleares/biosíntesis , Proteínas Nucleares/metabolismo , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Análisis de Secuencia de ADN , Espermatozoides/metabolismo
14.
Biol Reprod ; 67(4): 1157-64, 2002 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-12297531

RESUMEN

The testis-specific linker histone H1t gene is transcribed exclusively in mid to late pachytene primary spermatocytes. Tissue-specific expression of the gene is mediated primarily through elements located within the proximal promoter. Previous work in transgenic animals identified a unique 40-base pair promoter element designated H1t/TE that is essential for spermatocyte-specific expression. The H1t/TE element contains three subelements designated TE2, GC-box, and TE1 based on in vitro footprinting and electrophoretic mobility shift assays. Because GC-box is a consensus site for binding of Sp transcription-factor family members, experiments were performed demonstrating that two Sp family members, Sp1 and Sp3, were present in testis cells from 9-day-old and adult rats and in pachytene primary spermatocytes and early spermatids. A 95- to 105-kDa form of Sp1 is most abundant in the tissues and cell lines examined, but a 60-kDa form of Sp1 is the most abundant species in spermatocytes and early spermatids. Further examination of Sp1 and Sp3 from adult testis, primary spermatocytes, and early spermatids showed that they can bind to the H1t/TE element. In order to determine the contributions of the subelements to H1t transcription, we mutated each of them in H1t promoter luciferase reporter vectors. Mutation of the GC-box and TE1 subelement reduced expression 77% and 49%, respectively, compared with the wild-type H1t promoter in transient expression assays in a testis GC-2spd cell line that was derived from germinal cells. These studies suggest that Sp transcription factors may be involved in transcription of the H1t gene and the GC-box and the TE1 subelement are required for activation of the H1t promoter.


Asunto(s)
Histonas/genética , Regiones Promotoras Genéticas , Testículo/química , Animales , Sitios de Unión , Secuencia Conservada , ADN/metabolismo , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Masculino , Mutagénesis , Ratas , Ratas Sprague-Dawley , Elementos de Respuesta , Factor de Transcripción Sp1/análisis , Factor de Transcripción Sp1/metabolismo , Factor de Transcripción Sp3 , Espermátides/química , Espermatozoides/química , Factores de Transcripción/análisis , Factores de Transcripción/metabolismo , Transcripción Genética , Transfección
15.
J Cell Biochem ; 86(4): 716-25, 2002.
Artículo en Inglés | MEDLINE | ID: mdl-12210738

RESUMEN

The testis-specific linker histone H1t gene is transcribed exclusively in mid to late pachytene primary spermatocytes. Tissue specific expression of the gene is mediated in large part through elements located within the proximal promoter. Previous work in transgenic animals showed that a unique 40 bp promoter element designated H1t/TE is essential for spermatocyte-specific expression. The H1t/TE element contains a GC-box, which is a perfect consensus binding site for members of the Sp family of transcription factors. We have shown that Sp1 and Sp3 are present in testis cells from 9-day-old and adult rats and in pachytene primary spermatocytes and early spermatids and that they can bind to the H1t/GC-box. Mutagenesis of the GC-box reduced H1t promoter activity. Furthermore, a CpG dinucleotide within the GC-box was totally unmethylated in rat testis primary spermatocytes where the gene is transcribed but it was methylated in liver where the gene is silenced. These previous studies supported the importance of the GC-box and suggested that Sp transcription factors contribute to expression of the H1t gene. In this study, we show that co-transfection of Sp1 and Sp3 expression vectors leads to an upregulation of histone H1t promoter activity in several cell lines including testis GC-2spd cells. However, very low H1t promoter activity is seen in GC-2spd cells grown at 39 degrees C, which correlates with lower levels of Sp1 and Sp3 in these cells grown at this elevated temperature. Upregulation of the H1t promoter by Sp1 and Sp3 was also seen in cotransfected NIH3T3 and C127I cell lines. On the other hand, co-transfection of the Sp1 and Sp3 expression vectors does not lead to upregulation of activity of the cell-cycle dependent histone H1d promoter.


Asunto(s)
Histonas/genética , Proteínas Gestacionales/genética , Glicoproteínas beta 1 Específicas del Embarazo/genética , Testículo/fisiología , Animales , Composición de Base , Western Blotting , Línea Celular , Metilación de ADN , Ensayo de Cambio de Movilidad Electroforética , Histonas/química , Masculino , Ratones , Sondas de Oligonucleótidos , Proteínas Gestacionales/biosíntesis , Proteínas Gestacionales/metabolismo , Glicoproteínas beta 1 Específicas del Embarazo/biosíntesis , Glicoproteínas beta 1 Específicas del Embarazo/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Ratas , Ratas Sprague-Dawley , Temperatura , Transfección
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