scholarly journals The interaction of modified histones with the bromodomain testis-specific (BRDT) gene and its mRNA level in sperm of fertile donors and subfertile men

Reproduction ◽  
2010 ◽  
Vol 140 (3) ◽  
pp. 435-443 ◽  
Author(s):  
Cornelia Steilmann ◽  
Márcia C O Cavalcanti ◽  
Marek Bartkuhn ◽  
Jörn Pons-Kühnemann ◽  
Hans-Christian Schuppe ◽  
...  
Keyword(s):  
Real Time ◽  
Histone Modifications ◽  
Chromatin Condensation ◽  
Mrna Level ◽  
Regulation Of Gene Expression ◽  
Transcript Level ◽  
Sperm Chromatin ◽  
Epigenetic Mark ◽  
Mrna Levels ◽  
Infertile Patients

As histone modifications have been suggested to be involved in the regulation of gene expression after fertilisation, the present study aimed to analyze the interaction between the bromodomain testis-specific (BRDT) gene and differentially modified histones in human spermatozoa. The BRDT transcript level was studied to identify possible correlations between epigenetic changes, mRNA level and subfertility associated with impaired sperm chromatin condensation. Chromatin immunoprecipitation (ChIP) was performed with ejaculates from fertile and subfertile men using antibodies against specifically acetylated and methylated histone H3. Immunoprecipitated DNA was analysed by real-time quantitative PCR with primer pairs for BRDT. The BRDT mRNA level was screened by real-time RT-PCR. ChIP assay revealed co-localisation of acetylated and methylated histones within promoter and exon regions of the BRDT gene in fertile men. Interestingly, reduced binding of investigated modified histone modifications was observed in the BRDT promoter of subfertile patients. Different mRNA levels of BRDT have been detected in a group of infertile patients, as well as in fertile men. Enrichment of methylated histones within the BRDT promoter of fertile sperm suggests that this epigenetic mark may cause repression of BRDT after fertilisation, and may be changed in infertile patients. Our data suggest that reduced histone methylation in the promoter of BRDT may be associated with increased transcript levels in subfertile patients.

Monitoring CML28 mRNA Levels in Patients before and Post HSCT by Real-Time Quantitative RT-PCR.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4478-4478
Author(s):  
Donghua Zhang ◽  
Min Dai ◽  
Hongsheng Zhou ◽  
Yaya Wang ◽  
Lu Zhang ◽  
...  
Keyword(s):  
Real Time ◽  
Mrna Level ◽  
Conditioning Regimen ◽  
Sybr Green ◽  
Sybr Green I ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Standard Samples ◽  
Hematopoietic Stem ◽  
High Level

Abstract A SYBR Green I real-time quantitative RT-PCR method was established for investigating the correlation between CML28 mRNA expressing levels and relapse of leukemia after allogeneic hematopoietic stem cell transplantation (allo-HSCT). pcDNA3.1HisA-CML28 plasmid had been constructed as the standard template. Serial monitorting of CML28 mRNA levels by SYBR Green I real-time quantitative RT-PCR technique was performed in 14 patients, including 10 patients with CML and 3 patients with AML, 1 patient with Ph+ ALL. The sensitivity of the established method was at 10−4 level, with interassay variation and intraassay variation of standard samples both < 10%. The CML28 was highly expressed in AML and CML-BP or AP. In newly diagnosed group, CML28 was (6.58±2.34)×10−2. In pre-conditioning regimen group was (2.19±0.32)×10−2, in group that 1 month after allo-HSCT was (1.35±1.28)×10−2, in group that 3 months after allo-HSCT was (4.57±6.39)×10−3. CML28 can be detected 3months after HSCT in 1 patient with CML-CP and 3 patients with CML-AP or BC. 2 of them with low level (<2×10−2) survived without relapse, the other 2 patients with high level (>2×10−2) relapsed within one year,1 died and1 received the second time allo-HSCT, CML28 level decreased rapidly after HSCT, but still higher than 2×10−2 and relapsed again. CML28 mRNA level was obviously correlated with the development of diseases. Serial quantification of CML28 mRNA levels were necessary for allo-HSCT recipients, and more informative than a single detection. Use of this assay to evaluate MRD in the patients performed allo-HSCT was helpful for predicition of relapse.

Expression of Rgmc, the murine ortholog of hemojuvelin gene, is modulated by development and inflammation, but not by iron status or erythropoietin

Blood ◽  
2004 ◽  
Vol 104 (13) ◽  
pp. 4308-4310 ◽  
Author(s):  
Jan Krijt ◽  
Martin Vokurka ◽  
Ko-Tung Chang ◽  
Emanuel Nečas
Keyword(s):  
Polymerase Chain Reaction ◽  
Iron Overload ◽  
Real Time ◽  
Postnatal Development ◽  
Iron Status ◽  
Mrna Level ◽  
Mrna Levels ◽  
Chain Reaction ◽  
Juvenile Hemochromatosis ◽  
Polymerase Chain

Abstract Mutations of hepcidin (HAMP) and hemo-juvelin (HJV) genes have been recently demonstrated to result in juvenile hemochromatosis. Expression of HAMP is regulated by iron status or infection, whereas regulation of HJV is yet unknown. Using quantitative real-time polymerase chain reaction, we compared expression of Hamp and Rgmc (the murine ortholog of HJV) in livers of mice treated with iron, erythropoietin, or lipopolysaccharide (LPS), as well as during fetal and postnatal development. Iron overload increased Hamp expression without effect on Rgmc mRNA. Erythropoietin decreased Hamp mRNA, but Rgmc expression was unchanged. Hamp mRNA level decreased after birth by 4 orders of magnitude, without significant changes in Rgmc expression. Administration of LPS elevated Hamp mRNA levels, while markedly decreasing hepatic Rgmc mRNA levels (to ∼5% after 6 hours). The responses of Hamp and Rgmc were quite different and suggested that human HJV expression could be modulated by inflammation.

scholarly journals Gene expression of the three members of hepatocyte nuclear factor-3 is differentially regulated by nutritional and hormonal factors

2000 ◽  
Vol 167 (1) ◽  
pp. R1-R5 ◽  
Author(s):  
M Imae ◽  
Y Inoue ◽  
Z Fu ◽  
H Kato ◽  
T Noguchi
Keyword(s):  
Gene Expression ◽  
Mrna Level ◽  
Regulation Of Gene Expression ◽  
Diabetic Rats ◽  
Physiological Status ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Protein Free Diet

Hepatocyte nuclear factor-3 (HNF-3) belongs to a large family of forkhead transcription factors and is made up of three members (HNF-3alpha, -3beta and -3gamma). It has been shown that HNF-3 regulates a number of metabolically important genes. However, the mechanisms underlying this regulation of HNF-3 activity by hormones and nutrition have not yet been well elucidated. In attempting to explore the regulation of gene expression of HNF-3 members by physiological status, we analyzed the effects of insulin, dexamethasone and protein malnutrition on the hepatic mRNA level of each member. Male Wistar rats were fed on a 12% casein diet, 12% gluten diet (deficient in lysine and threonine) or a protein-free diet for 1 week. The protein-free diet and gluten diet caused a 3. 7-fold increase in HNF-3g mRNA in the liver and did not affect the mRNA level of either HNF-3alpha or HNF-3beta. Daily administration of dexamethasone caused the mRNA levels of HNF-3alpha and HNF-3beta to increase (2.3- and 1.4-fold, respectively), but had no effect on the HNF-3gamma mRNA level. In diabetic rats that had been injected with streptozotocin, an elevation of the hepatic mRNA levels of HNF-3beta and HNF-3gamma was observed (1.6-and 1.9-fold, respectively). Insulin replacement in the diabetic rats decreased both mRNA levels in a dose-dependent manner. HNF-3alpha mRNA was not affected by insulin status. These results show that the genes of the three members of the HNF-3 family respond differently to hormonal and nutritional factors suggesting that the activities of HNF-3 members are regulated, at least in part, by the levels of their gene expression.

P–341 Epigenetic role of the H2BK5ac histone modification on the expression of ALX1 and PDHX genes in the endometriotic tissues and normal endometrium

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
S Sarshomar ◽  
S Sarshomar ◽  
F Chitsazian ◽  
F Ghaffari ◽  
M Shahhoseini ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Chromatin Immunoprecipitation ◽  
Mrna Level ◽  
Rna Extraction ◽  
Migration And Invasion ◽  
Epigenetic Mark ◽  
Promoter Regions ◽  
Normal Endometrium

Abstract Study question Evolution of the ALX1 and PDHX genes expression and incorporation of H2BK5ac mark on the promoter of these genes in endometriotic tissues versus normal endometrium Summary answer Lower incorporation of H2BK5ac mark in ALX1 and PDHX promoters can because to downregulation of these genes in endometriotic tissues compared to normal endometrium. What is known already Endometriosis is considered as multifactorial disease affected by genetic, hormonal, and environmental factors. Recent evidences suggest the role of epigenetic mechanisms in this disease. Aristaless-like homeobox1 (ALX1) and Pyruvate Dehydrogenase Protein X (PDHX) genes are considered in this study. Studies show that upregulation of the ALX1 gene cause cell proliferation, migration, and invasion in cancer cells. PDHX is involved in cellular metabolism and acts as a tumor suppressor gene while maintaining normal homeostasis. It is Hypothesized that H2BK5ac which is known as a dynamic marker in promoter regions of active genes, may be involved in regulation of these gene expression. Study design, size, duration Ten eutopic and ectopic endometrium tissue, as well as ten normal endometrium, were collected. Ectopic biopsies were obtained using diagnostic laparoscopy, while the endometrial control samples and eutopic samples were collected via pipelle. Participants/materials, setting, methods RNA extraction and cDNA synthesis were done then the expression of ALX1 and PDHX genes evaluated by quantitative real-time PCR. Promoter regions of mentioned genes investigated for the incorporation of the epigenetic mark of H2BK5ac using Chromatin immunoprecipitation (ChIP) followed by real-time PCR. Data analysis performed using One-way ANOVA analysis (SPSS software) considered the significant level of P &lt; 0.05. Main results and the role of chance Results showed that the expression of ALX1 was significantly decreased in eutopic endometrial samples compared to normal endometrium (p = 0.007). Also, there was a significant reduction in PDHX mRNA level in the eutopic and ectopic samples vs. normal endometrium (p = .017 and p =.021, respectively). The chromatin immunoprecipitation real-time PCR (ChIP PCR) analyses showed significantly lower incorporation of H2BK5ac epigenetic mark in ALX1 promoter in eutopic endometrial samples compared to normal endometrium (p = 0.007). Also, reduced incorporation of H2BK5ac at the PDHX promoter region was observed in both eutopic and ectopic endometrial samples compared to normal endometrium (p = 0.004 and p = 0.003, respectively). Limitations, reasons for caution The main limitation of our study is the low number of samples. Wider implications of the findings: Our results suggest that the marked lower levels of H2BK5ac in regulatory regions of ALX1and PDHX might lead to deregulation of these genes in tissue endometriotic samples. Trial registration number ‘not applicable’ for non-clinical trials

scholarly journals Low expression of Talin1 is associated with advanced pathological features in colorectal cancer patients

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Somayeh Vafaei ◽  
Leili Saeednejad Zanjani ◽  
Zohreh Habibi Shams ◽  
Marzieh Naseri ◽  
Fahimeh Fattahi ◽  
...  
Keyword(s):  
Protein Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Mrna Level ◽  
Univariate Analysis ◽  
Survival Rates ◽  
Tnm Stage ◽  
Mrna Levels ◽  
Significant Difference ◽  
Low Expression

Abstract To explore the proper prognostic markers for the likelihood of metastasis in CRC patients. Seventy-seven fresh CRC samples were collected to evaluate the mRNA level of the selected marker using Real-time PCR. Moreover, 648 formalin-fixed paraffin-embedded CRC tissues were gathered to evaluate protein expression by immunohistochemistry (IHC) on tissue microarrays. The results of Real-Time PCR showed that low expression of Talin1 was significantly associated with advanced TNM stage (p = 0.034) as well as gender (p = 0.029) in mRNA levels. Similarly, IHC results indicated that a low level of cytoplasmic expression of Talin1 was significantly associated with advanced TNM stage (p = 0.028) as well as gender (p = 0.009) in CRC patients. Moreover, decreased expression of cytoplasmic Talin1 protein was found to be a significant predictor of worse disease-specific survival (DSS) (p = 0.011) in the univariate analysis. In addition, a significant difference was achieved (p = 0.039) in 5-year survival rates of DSS: 65% for low, 72% for moderate, and 88% for high Talin1 protein expression. Observations showed that lower expression of Talin1 at both the gene and protein level may drive the disparity of CRC patients’ outcomes via worse DSS and provide new insights into the development of progression indicators because of its correlation with increased tumor aggressiveness.

Gene Transcript Assay by Real-Time Rt-PCR in Epithelial Breast Cancer Cells Selected by Laser Microdissection

2004 ◽  
Vol 19 (2) ◽  
pp. 100-108 ◽  
Author(s):  
V. Becette ◽  
S. Vignaud ◽  
C. Régnier ◽  
M. Labroquère ◽  
E. Fourme ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Real Time ◽  
Target Gene ◽  
Laser Microdissection ◽  
Mrna Level ◽  
Cancer Tissue ◽  
Gene Transcript ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Tissue Samples

The cell type heterogeneity within clinical cancer tissue samples may affect the accuracy of gene expression analysis. In order to validate our laser microdissection (LMD) method using the Leica AS LMD system (LEICA Microsystems), we compared the mRNA levels of three major genes involved in breast cancer (ERα, PR, HER2), measured by means of real-time quantitative RT-PCR, in 5000 microdissected malignant epithelial cells and in corresponding bulk tumor ho-mogenates from 14 patients. We also compared the mRNA level results to protein expression measured by immunohistochemistry (IHC) on the same tumors. For the three genes, significant correlations were found between mRNA results obtained on microdissected cells and IHC. Comparison between IHC and mRNA results obtained on microdissected cells and bulk tumors showed that in all cases microdissection enhanced the sensitivity of assessing target gene transcript levels and was essential for their accurate evaluation in heterogeneous tumors.

scholarly journals Molecular Characterization of Paralichthys olivaceus MAF1 and Its Potential Role as an Anti-Viral Hemorrhagic Septicaemia Virus Factor in Hirame Natural Embryo Cells

2021 ◽  
Vol 22 (3) ◽  
pp. 1353
Author(s):  
Julan Kim ◽  
Ja Young Cho ◽  
Ju-Won Kim ◽  
Dong-Gyun Kim ◽  
Bo-Hye Nam ◽  
...  
Keyword(s):  
Immune Response ◽  
Amino Acid ◽  
Paralichthys Olivaceus ◽  
Mrna Level ◽  
Rna Polymerase Iii ◽  
Transcript Level ◽  
Viral Hemorrhagic Septicemia Virus ◽  
Mrna Levels ◽  
Coding Region ◽  
Embryo Cells

MAF1 is a global suppressor of RNA polymerase III-dependent transcription, and is conserved from yeast to human. Growing evidence supports the involvement of MAF1 in the immune response of mammals, but its biological functions in fish are unknown. We isolated and characterized Maf1 from the olive flounder Paralichthys olivaceus (PoMaf1). The coding region of PoMaf1 comprised 738 bp encoding a 245-amino-acid protein. The deduced PoMAF1 amino acid sequence shared features with those of MAF1 orthologues from vertebrates. PoMaf1 mRNA was detected in all tissues examined, and the levels were highest in eye and muscle tissue. The PoMaf1 mRNA level increased during early development. In addition, the PoMaf1 transcript level decreased during viral hemorrhagic septicemia virus (VHSV) infection of flounder hirame natural embryo (HINAE) cells. To investigate the role of PoMaf1 in VHSV infection, single-cell-derived PoMaf1 knockout HINAE cells were generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated-9 (CRISPR/Cas9) system, and cell clones with complete disruption of PoMaf1 were selected. PoMaf1 disruption increased the VHSV glycoprotein (G) mRNA levels during VHSV infection of HINAE cells, implicating PoMAF1 in the immune response to VSHV infection. To our knowledge, this is the first study to characterize fish Maf1, which may play a role in the response to viral infection.

scholarly journals MRG15 is required for pre-mRNA splicing and spermatogenesis

2016 ◽  
Vol 113 (37) ◽  
pp. E5408-E5415 ◽  
Author(s):  
Naoki Iwamori ◽  
Kaoru Tominaga ◽  
Tetsuya Sato ◽  
Kevin Riehle ◽  
Tokuko Iwamori ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Chromatin Condensation ◽  
Somatic Cells ◽  
Round Spermatid ◽  
Mrna Splicing ◽  
Sperm Chromatin ◽  
Splicing Factors ◽  
Chromosome 15 ◽  
Infertile Patients

Splicing can be epigenetically regulated and involved in cellular differentiation in somatic cells, but the interplay of epigenetic factors and the splicing machinery during spermatogenesis remains unclear. To study these interactions in vivo, we generated a germline deletion of MORF-related gene on chromosome 15 (MRG15), a multifunctional chromatin organizer that binds to methylated histone H3 lysine 36 (H3K36) in introns of transcriptionally active genes and has been implicated in regulation of histone acetylation, homology-directed DNA repair, and alternative splicing in somatic cells. Conditional KO (cKO) males lacking MRG15 in the germline are sterile secondary to spermatogenic arrest at the round spermatid stage. There were no significant alterations in meiotic division and histone acetylation. Specific mRNA sequences disappeared from 66 germ cell-expressed genes in the absence of MRG15, and specific intronic sequences were retained in mRNAs of 4 genes in the MRG15 cKO testes. In particular, introns were retained in mRNAs encoding the transition proteins that replace histones during sperm chromatin condensation. In round spermatids, MRG15 colocalizes with splicing factors PTBP1 and PTBP2 at H3K36me3 sites between the exons and single intron of transition nuclear protein 2 (Tnp2). Thus, our results reveal that MRG15 is essential for pre-mRNA splicing during spermatogenesis and that epigenetic regulation of pre-mRNA splicing by histone modification could be useful to understand not only spermatogenesis but also, epigenetic disorders underlying male infertile patients.

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