RESUMO
The SALL2 transcription factor, an evolutionarily conserved gene through vertebrates, is involved in normal development and neuronal differentiation. In disease, SALL2 is associated with eye, kidney, and brain disorders, but mainly is related to cancer. Some studies support a tumor suppressor role and others an oncogenic role for SALL2, which seems to depend on the cancer type. An additional consideration is tissue-dependent expression of different SALL2 isoforms. Human and mouse SALL2 gene loci contain two promoters, each controlling the expression of a different protein isoform (E1 and E1A). Also, several improvements on the human genome assembly and gene annotation through next-generation sequencing technologies reveal correction and annotation of additional isoforms, obscuring dissection of SALL2 isoform-specific transcriptional targets and functions. We here integrated current data of normal/tumor gene expression databases along with ChIP-seq binding profiles to analyze SALL2 isoforms expression distribution and infer isoform-specific SALL2 targets. We found that the canonical SALL2 E1 isoform is one of the lowest expressed, while the E1A isoform is highly predominant across cell types. To dissect SALL2 isoform-specific targets, we analyzed publicly available ChIP-seq data from Glioblastoma tumor-propagating cells and in-house ChIP-seq datasets performed in SALL2 wild-type and E1A isoform knockout HEK293 cells. Another available ChIP-seq data in HEK293 cells (ENCODE Consortium Phase III) overexpressing a non-canonical SALL2 isoform (short_E1A) was also analyzed. Regardless of cell type, our analysis indicates that the SALL2 long E1 and E1A isoforms, but not short_E1A, are mostly contributing to transcriptional control, and reveals a highly conserved network of brain-specific transcription factors (i.e., SALL3, POU3F2, and NPAS3). Our data integration identified a conserved molecular network in which SALL2 regulates genes associated with neural function, cell differentiation, development, and cell adhesion between others. Also, we identified PODXL as a gene that is likely regulated by SALL2 across tissues. Our study encourages the validation of publicly available ChIP-seq datasets to assess a specific gene/isoform's transcriptional targets. The knowledge of SALL2 isoforms expression and function in different tissue contexts is relevant to understanding its role in disease.
RESUMO
BACKGROUND: Recent studies demonstrated that changes in DNA methylation (DNAm) and inactivation of two imprinted genes (MKRN3 and DLK1) alter the onset of female puberty. We aimed to investigate the association of DNAm profiling with the timing of human puberty analyzing the genome-wide DNAm patterns of peripheral blood leukocytes from ten female patients with central precocious puberty (CPP) and 33 healthy girls (15 pre- and 18 post-pubertal). For this purpose, we performed comparisons between the groups: pre- versus post-pubertal, CPP versus pre-pubertal, and CPP versus post-pubertal. RESULTS: Analyzing the methylome changes associated with normal puberty, we identified 120 differentially methylated regions (DMRs) when comparing pre- and post-pubertal healthy girls. Most of these DMRs were hypermethylated in the pubertal group (99%) and located on the X chromosome (74%). Only one genomic region, containing the promoter of ZFP57, was hypomethylated in the pubertal group. ZFP57 is a transcriptional repressor required for both methylation and imprinting of multiple genomic loci. ZFP57 expression in the hypothalamus of female rhesus monkeys increased during peripubertal development, suggesting enhanced repression of downstream ZFP57 target genes. Fourteen other zinc finger (ZNF) genes were related to the hypermethylated DMRs at normal puberty. Analyzing the methylome changes associated with CPP, we demonstrated that the patients with CPP exhibited more hypermethylated CpG sites compared to both pre-pubertal (81%) and pubertal (89%) controls. Forty-eight ZNF genes were identified as having hypermethylated CpG sites in CPP. CONCLUSION: Methylome profiling of girls at normal and precocious puberty revealed a widespread pattern of DNA hypermethylation, indicating that the pubertal process in humans is associated with specific changes in epigenetically driven regulatory control. Moreover, changes in methylation of several ZNF genes appear to be a distinct epigenetic modification underlying the initiation of human puberty.
Assuntos
Metilação de DNA , Proteínas de Ligação a DNA/genética , Estudo de Associação Genômica Ampla/métodos , Puberdade Precoce/genética , Fatores de Transcrição/genética , Animais , Estudos de Casos e Controles , Criança , Epigênese Genética , Feminino , Impressão Genômica , Humanos , Macaca mulatta , Linhagem , Regiões Promotoras Genéticas , Proteínas Repressoras , Dedos de ZincoRESUMO
Daily exposure to low doses of 3-methylcholanthrene (3MC) during the pubertal period in rats disrupts both follicular growth and ovulation. Thus, to provide new insights into the toxicity mechanism of 3MC in the ovary, here we investigated the effect of daily exposure to 3MC on selected ovarian genes, the role of the aryl hydrocarbon receptor (AhR) and the level of epigenetic remodeling of histone post-transcriptional modifications. Immature rats were daily injected with 3MC (0.1 or 1 mg/kg) and mRNA expression of genes involved in different ovarian processes were evaluated. Of the 29 genes studied, 18 were up-regulated, five were down-regulated and six were not altered. To assess whether AhR was involved in these changes, we used the chromatin immunoprecipitation assay. 3MC increased AhR binding to promoter regions of genes involved in Notch signaling (Hes1, Jag1), activation of primordial follicles (Cdk2), cell adhesion (Icam1), stress and tumor progression (Dnajb6), apoptosis (Bax, Caspase-9) and expression of growth and transcription factors (Igf2, Sp1). Studying the trimethylation and acetylation of histone 3 (H3K4me3 and H3K9Ac, respectively) of these genes, we found that 3MC increased H3K4me3 in Cyp1a1, Jag1, Dnajb6, Igf2, Notch2, Adamts1, Bax and Caspase-9, and H3K9Ac in Cyp1a1, Jag1, Cdk2, Dnajb6, Igf2, Icam1, and Sp1. Co-treatment with α-naphthoflavone (αNF), a specific antagonist of AhR, prevented almost every 3MC-induced changes. Despite the low dose used in these experiments, daily exposure to 3MC induced changes in both gene expression and epigenomic remodeling, which may lead to premature ovarian failure.
Assuntos
Benzoflavonas/farmacologia , Metilcolantreno/toxicidade , Folículo Ovariano/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/metabolismo , Acetilação , Animais , Imunoprecipitação da Cromatina , Regulação para Baixo , Epigênese Genética , Feminino , Histonas/química , Metilação , Regiões Promotoras Genéticas , Ligação Proteica , Processamento de Proteína Pós-Traducional , Ratos , Ratos Sprague-Dawley , Regulação para CimaRESUMO
In the present study, we investigated the effect of 3-methylcholanthrene (3MC) on sexual maturity and the ability of α-naphthoflavone (αNF) to prevent this action. To this end, immature rats were daily injected intraperitoneally with 3MC (0.1 or 1mg/kg) and/or αNF (80mg/kg). Body weight, vaginal opening and estrous cycle were recorded and ovaries were obtained on the day of estrus. Ovarian weight, ovulation rate (measured by the number of oocytes within oviducts), and follicular development (determined by histology) were studied. No differences were found in body weight, ovarian weight, day of vaginal opening, or the establishment of the estrous cycle among the different groups of rats. However, animals treated with 3MC, at both doses, exhibited a lower number of primordial, primary, preantral and antral follicles than controls. Also, 3MC inhibited the ovulation rate and induced an overexpression of both the Cyp1a1 and Cyp1b1 genes, measured by chromatin immunoprecipitation assay. The daily treatment with αNF alone increased the number of follicles in most of the stages analyzed when compared with controls. Moreover, the αNF treatment prevented completely not only the 3MC-induced decrease in all types of follicles but also the 3MC-induced overexpression of Cyp enzymes and the genetic damage in bone marrow cells and oocytes. These results suggest that (i) daily exposure to 3MC during the pubertal period destroys the follicle reserve and alters the ovulation rate; (ii) the 3MC action seems to be mediated by an aryl hydrocarbon receptor-dependent mechanism; (iii) daily administration of αNF has a clear stimulatory action on the ovarian function; and (iv) αNF may prevent both the systemic and gonadal 3MC-induced toxicity.
Assuntos
Benzoflavonas/administração & dosagem , Benzoflavonas/farmacologia , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1B1/genética , Metilcolantreno/toxicidade , Animais , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/patologia , Imunoprecipitação da Cromatina , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Oócitos/patologia , Folículo Ovariano/efeitos dos fármacos , Ovulação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de Hidrocarboneto Arílico/metabolismoRESUMO
OBJECTIVE: In the present work, we evaluated the effect of exposing the submandibular glands (SMG) to radiation, studying different functional parameters such as salivary secretion, nitric oxide (NO) production, reactive oxygen species formation, prostaglandin (PGE) content and apoptosis. METHODS: We irradiated rats in the head and neck region with a single dose of gamma-ray radiation of 15 Gy. Two hours after radiation, we measured norepinephrine-induced salivary secretion. After that, the SMG were dissected, and in this tissue, we measured the activity of NO synthase (NOS), the PGE content, the amount of reactive oxygen species, apoptotic cells and mitochondrial inducible NOS (iNOS) expression. RESULTS: We found that radiation decreased salivary secretion when 10 and 30 microg/kg of norepinephrine was administered via the right femoral vein. We observed that iNOS activity was reduced and PGE content increased after radiation in SMG, indicating that NO and PGEs may participate in salivary secretion. The expression of mitochondrial NOS was increased after radiation leading to the formation of large amounts of NO that acts as a proapoptotic signal. In fact, we observed an augmentation in apoptotic cells. In this study, we also observed an increase in lipid peroxidation induced by radiation that may contribute to tissue damage. CONCLUSIONS: Our results indicate that radiation induced a decrease in salivary secretion and SMG iNOS activity, meanwhile the PGE content, the lipid peroxidation and apoptosis increased in the tissue. These modifications decrease salivary secretion.
Assuntos
Óxido Nítrico/efeitos da radiação , Prostaglandinas/efeitos da radiação , Radioterapia/efeitos adversos , Glândula Submandibular/metabolismo , Glândula Submandibular/efeitos da radiação , Xerostomia/fisiopatologia , Animais , Apoptose/fisiologia , Apoptose/efeitos da radiação , Modelos Animais de Doenças , Regulação para Baixo/fisiologia , Regulação para Baixo/efeitos da radiação , Células Epiteliais/metabolismo , Células Epiteliais/efeitos da radiação , Feminino , Neoplasias de Cabeça e Pescoço/radioterapia , Peroxidação de Lipídeos/fisiologia , Peroxidação de Lipídeos/efeitos da radiação , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/metabolismo , Óxido Nítrico Sintase Tipo II/efeitos da radiação , Estresse Oxidativo/fisiologia , Estresse Oxidativo/efeitos da radiação , Prostaglandinas/metabolismo , Ratos , Saliva/metabolismo , Glândula Submandibular/fisiopatologia , Xerostomia/etiologia , Xerostomia/metabolismoRESUMO
BACKGROUND/OBJECTIVE: Nutritional dwarfing (ND) consists of a decrease in weight and height gain and delayed onset of puberty. The aim of the present investigation was to study the modifications induced in male rats by the nutritional stress of a mere 20% reduction in food intake which, however, started immediately after weaning. MATERIALS AND METHODS: At weaning, male Wistar rats were divided into two groups: Control (C) and ND. C rats were fed ad libitum with a balanced rodent diet. ND received 80% of the diet consumed by C for 4 weeks (T4); then they were fed ad libitum for another 4 (T8) and 8 weeks (T12). The rats were studied at T0, T4, T8 and T12 for the effects of nutritional stress and refeeding on nutritional status, body composition, hypothalamic-pituitary-gonadal axis, and sperm morphology and concentration. RESULTS: ND body weight and length diminished vs. C (p < 0.001). ND body fat percentage decreased 40% (p < 0.001) without change in the percentage of body protein content. The hypothalamic content of LHRH did not change. However, FSH, LH and testosterone serum levels had significantly decreased (p < 0.001) at T4 in ND rats. A 48.4 % decrease in serum leptin in the ND group was observed at T4 (p < 0.05). The absolute testicular and seminal vesicle weight was significantly decreased by ND at T4 (p < 0.001). At T4 the percentage of anomalies of caudal spermatozoa increased in about 64% (p < 0.001) of ND vs. C rats, despite the unchanged sperm concentrations. All parameters normalized during refeeding. CONCLUSION: In this model, a decrease in leptin due to nutritional stress could be responsible, at least in part, for the inhibition of reproductive function. Refeeding normalized all parameters studied.