Map of benzoapyrene metabolites-DNA adducts in human bronchial epithelial-like cells: Based on chromatin immunoprecipitation followed by sequencing technology / 环境与职业医学

Background The active metabolite of benzo[a]pyrene (BaP), 7,8-dihydroxy-9,10-epoxybenzo[a]pyrene (BPDE), can form adducts with DNA, but the spectrum of BPDE-DNA adducts is unclear. Objective To identify the distribution of BPDE adduct sites and associated genes at the whole-genome level by chromatin immunoprecipitation followed by sequencing (ChIP-Seq), and serve as a basis for further exploring the toxicological mechanisms of BaP. Methods Human bronchial epithelial-like cells (16HBE) were cultured to the fourth generation inthe logarithmic growth phase. Cells were harvested and added to chromatin immunoprecipitation lysis buffer. The lysate was divided into experimental and control groups. The experimental group received a final concentration of 20 μmol·L−1 BPDE solution, while the control group received an equivalent volume of dimethyl sulfoxide solution. The cells were then incubated at 37 °C for 24 h. Chromatin fragments of 100-500 bp were obtained through sonication. BPDE-specific antibody (anti-BPDE 8E11) was used to enrich DNA fragments with BPDE adducts. High-throughput sequencing was conducted to detect BPDE adduct sites. The top 1000 peak sequences were subjected to motif analysis using MEME and DREME software. BPDE adduct target genes at the whole-genome level were annotated, and Gene Ontology (GO) functional analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of BPDE adduct target genes were conducted using bioinformatics techniques. Results The high-throughput sequencing detected a total of 842 BPDE binding sites, distributed across various chromosomes. BPDE covalently bound to both coding and non-coding regions of genes, with 73.9% binding sites located in intergenic regions, 19.6% in intronic regions, and smaller proportions in upstream 2 kilobase, exonic, downstream 2 kilobase, and 5' untranslated regions. Regarding the top 1000 peak sequences, four reliable motifs were identified, revealing that sites rich in adenine (A) and guanine (G) were prone to binding. Through the enrichment analysis of binding sites, a total of 199 BPDE-adduct target genes were identified, with the majority located on chromosomes 1, 5, 7, 12, 17, and X. The GO analysis indicated that these target genes were mainly enriched in nucleic acid and protein binding, participating in the regulation of catalytic activity, transport activity, translation elongation factor activity, and playing important roles in cell division, differentiation, motility, substance transport, and information transfer. The KEGG analysis revealed that these target genes were primarily enriched in pathways related to cardiovascular diseases, cancer, and immune-inflammatory responses. Conclusion Using ChIP-Seq, 199 BPDE adduct target genes at genome-wide level are identified, impacting biological functions such as cell division, differentiation, motility, substance transport, and information transfer. These genes are closely associated with cardiovascular diseases, tumors, and immune-inflammatory responses.

WSB1 regulates c-Myc expression through β-catenin signaling and forms a feedforward circuit

The dysregulation of transcription factors is widely associated with tumorigenesis. As the most well-defined transcription factor in multiple types of cancer, c-Myc can transform cells by transactivating various downstream genes. Given that there is no effective way to directly inhibit c-Myc, c-Myc targeting strategies hold great potential for cancer therapy. In this study, we found that WSB1, which has a highly positive correlation with c-Myc in 10 cancer cell lines and clinical samples, is a direct target gene of c-Myc, and can positively regulate c-Myc expression, which forms a feedforward circuit promoting cancer development. RNA sequencing results from Bel-7402 cells confirmed that WSB1 promoted c-Myc expression through the β-catenin pathway. Mechanistically, WSB1 affected β-catenin destruction complex-PPP2CA assembly and E3 ubiquitin ligase adaptor β-TRCP recruitment, which inhibited the ubiquitination of β-catenin and transactivated c-Myc. Of interest, the effect of WSB1 on c-Myc was independent of its E3 ligase activity. Moreover, overexpressing WSB1 in the Bel-7402 xenograft model could further strengthen the tumor-driven effect of c-Myc overexpression. Thus, our findings revealed a novel mechanism involved in tumorigenesis in which the WSB1/c-Myc feedforward circuit played an essential role, highlighting a potential c-Myc intervention strategy in cancer treatment.

SMYD3-PARP16 axis accelerates unfolded protein response and mediates neointima formation

Neointimal hyperplasia after vascular injury is a representative complication of restenosis. Endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) is involved in the pathogenesis of vascular intimal hyperplasia. PARP16, a member of the poly(ADP-ribose) polymerases family, is correlated with the nuclear envelope and the ER. Here, we found that PERK and IRE1

Three-dimensional chromosome conformation capture and its derived technologies / 生物工程学报

Linear chromatin is compacted into eukaryotic nucleus through a complex and multi-layered architecture. Consequently, chromatin conformation in a local or long-distance manner is strongly correlated with gene expression. Chromosome conformation capture (3C) technology, together with its variants like 4C/5C/Hi-C, has been well developed to study chromatin looping and whole genome structure. In this review, we introduce new technologies including chromosome capture combined with immunoprecipitation, nuclei acid-based hybridization, single cell and genome sequencing, as well as their application.

Effect of acetylation modification on microenvironment adaptability of mesenchymal stem cells / 解放军医学杂志

Objective To study the changes of acetylation level of smooth muscle marker gene after the differentiation of bone marrow mesenchymal stem cells (BMSCs) in smooth muscle microenvironment, and explore the role and mechanism of histone acetylation modification in the differentiation of stem cells. Methods BMSCs and bladder smooth muscle cells (BSMCs) were cultured in vitro. The third generation BMSCs of the same batch were selected, and BMSCs co-cultured with BSMCs for 3 days were set as the experimental group and the BMSCs without co-culture as the control group. RT-PCR was performed to compare the expression abundance between the two groups of α-smooth muscle actin (α-SMA), calponin and smooth muscle myosin heavy chain (SM-MHC) in BMSCs. Ultrasound of power 80%, 20 times, 0.5 s and 8 cycles were used to break the BMSCs DNA of both experimental and control group. Antibody H3K9 was used to bind to the specific acetylation sites. The acetylation site genes of histone in BMSCs were precipitated by chromatin immunoprecipitation (ChIP). The genes obtained was amplified by adaptor PCR. The expression levels of the three kinds of target genes (α-SMA, calponin, SM-MHC) were detected by Real-time PCR. Results RT-PCR showed that the expression levels of mRNA of smooth muscle marker genes (α-SMA, calponin, SM-MHC) in BMSCs were significantly higher in experimental group than in control group (0.176±0.003 vs. 0.070±0.002; 0.079±0.002 vs. 0.051±0.003 and 0.091±0.004 vs. 0.034±0.001, respectively) with significant differences. Test results of spectrophotometer showed that the amount of DNA obtained by precipitation of H3K9 acetylated antibody was higher than that of IgG antibody, and was higher in experimental group than in control group (P<0.05). Real-time PCR used to analyze the acetylation level of H3K9 before and after the differentiation of BMSCs showed that the mRNA transcription levels of smooth muscle marker genes (α-SMA, calponin, SM-MHC) were significantly higher in experimental group than in the control group (9.26±5.03 vs. 1.01±0.05, 2.33±0.65 vs. 0.99±0.05, 2.63±0.37 vs. 1.00±0.03, respectively) after BMSCs differentiation with statistically significant differences. Conclusion The increase of acetylation level of H3K9 specific site of BSMCs in smooth muscle microenvironment promotes the differentiation of BMSCs into BSMCs.

Regulatory effect of grainyhead-like 2 gene on proliferation of epithelial ovarian cancer cells by nuclear factor erythroid-2-related factor 2 transcription / 解剖学报

Objective: To study the effect of nuclear factor erythroid-2-related factor 2(Nrf2) and the downstream gene grainyhead-like 2(GRHL2) on epithelial ovarian cancer cell lines and the interaction of Nrf2 and GRHL2. Methods: We conducted ChlP-PCR assay to test the binding of Nrf2 with six candidate genes including GRHL2. Furthermore, we proved whether Nrf2 could bind with the GRHL2 promoter and transcriptionally activate the GRHL2 gene or not. Moreover, if the overexpression and knockdown of Nrf2 could increase and decrease the GRHL2 protein respectively. Results: We discovered that fallopian tube epithelial cells taken from epithelial ovarian cancer patients and ovarian cancer cell lines highly expressed the Nrf2 and GRHL2 at mRNA level. The overexpression and knockdown of Nrf2 could increase and decrease the cells activity, respectively. However, the knockdown of GRHL2 could inhibit the influence of Nrf2 overexpression. Conclusion: Nrf2 promotes the activity of epithelial ovarian cancer cells via modulating the GRHL2 gene transcriptionally.

The improvewment of DNA library construction in non-crosslinked chromatin immunoprecipitation coupled with next-generation sequencing / 南方医科大学学报

OBJECTIVE@#To optimize DNA library construction in non-crosslinked chromatin immunoprecipitation coupled with next-generation sequencing (Native ChIP-seq) to obtain high-quality Native ChIP-seq data.@*METHODS@#Human nasopharyngeal carcinoma HONE1 cell lysate was digested with MNase for release of the nucleosomes, and the histone-DNA complexes were immunoprecipitated with specific antibodies. The protein component in the precipitate was digested with proteinase K followed by DNA purification; the DNA library was constructed for sequence analysis.@*RESULTS@#Compared with the conventional DNA library construction, Tn5 transposase method allowed direct enrichment of the target DNA after Tn5 fragmentation, which was simple, time-saving and more efficient. The IGV visualized map showed that the information obtained by the two library construction methods was consistent. The sequencing data obtained by the two methods revealed more signal enrichment with Tn5 transposase library construction than with the conventional approach. H3K4me3 ChIP results showed a good reproducibility after Tn5 transposase library construction with a signal-to-noise ratio above 50%.@*CONCLUSIONS@#Tn5 transposase method improves the efficiency of DNA library construction and the results of subsequent sequence analysis, and is especially suitable for detecting histone modification in the DNA to provide a better technical option for epigenetic studies.

A ChIP-Seq Data Analysis Pipeline Based on Bioconductor Packages

Nowadays, huge volumes of chromatin immunoprecipitation-sequencing (ChIP-Seq) data are generated to increase the knowledge on DNA-protein interactions in the cell, and accordingly, many tools have been developed for ChIP-Seq analysis. Here, we provide an example of a streamlined workflow for ChIP-Seq data analysis composed of only four packages in Bioconductor: dada2, QuasR, mosaics, and ChIPseeker. ‘dada2’ performs trimming of the high-throughput sequencing data. ‘QuasR’ and ‘mosaics’ perform quality control and mapping of the input reads to the reference genome and peak calling, respectively. Finally, ‘ChIPseeker’ performs annotation and visualization of the called peaks. This workflow runs well independently of operating systems (e.g., Windows, Mac, or Linux) and processes the input fastq files into various results in one run. R code is available at github: https://github.com/ddhb/Workflow_of_Chipseq.git.

Study on the regulation of ERαon NK1R-Tr in breast cancer cells / 天津医药

Objective To analyze the regulation of estrogen receptor α (ERα) on truncated neurokinin-1 receptor (NK1R-Tr), and the influence of this regulation on cell proliferation in estrogen receptor-positive breast cancer cell lines. Methods The chromatin immune coprecipitation (CHIP) was used to observe the transcriptional regulation function of ERαon NK1R-Tr in breast cancer cells. Luciferase reporter gene assay was used to verify whether ERα played a positive regulatory role in the expression of NK1R-Tr. Western blot assay and real-time-PCR were used to detect the expression of ERα and NK1R-Tr in breast cancer cells, MCF-7 and T47D, as well as the expression of NK1R-Tr protein and mRNA level. NK1R-Tr levels were also detected after using estradiol (E2, ERα agonist) and small interfering RNA (knock out ERα). CCK-8 and clone formation experimen were used to detect the proliferation ability of breast cancer cells after knocking out NK1R-Tr with small interfering RNAs. Results CHIP test and Luciferase reporter gene assay proved that ERα can positively regulate the expression of NK1R-Tr via the ERα sequences in the upstream of the NK1R-Tr gene promoter. The expression of NK1R-Tr at both protein level and mRNA level dropped in the estrogen receptor-positive breast cancer cell line MCF-7 upon knocking out ERα. After knocking out NK1R-Tr, the proliferation ability of estrogen receptor-positive breast cancer cells was lower than that of the control group. Conclusion The ERα positively regulates the expression of NK1R-Tr, resulting in the increased cell proliferation in estrogen positive breast cancer cells.

Histone acetylation and methylation modification of topoisomerase Ⅱαpromoter regulatory factor Sp1 in patients with chronic benzene poison-ing / 中国病理生理杂志

AIM:To investigate the histone modification changes of topoisomerase Ⅱα( TOPOⅡα) promoter regulatory factor Sp1 in the patients with chronic benzene poisoning .METHODS:The bone marrow samples were collect-ed from 25 chronic benzene poisoning cases and 25 controls.The chromatin immunoprecipitation assay was carried out to study the possible mechanism of TOPOⅡαpromoter regulatory factor Sp 1 expression changes .The mRNA expression of Sp1 was detected by RT-PCR.RESULTS:Compared with the controls , the histone H4 acetylation and histone H3 acetyla-tion of Sp1 in the chronic benzene poisoning patients significantly decreased (P0.05). The mRNA expression of Sp1 in the chronic benzene poisoning patients was significantly lower than that in the controls (P<0.05).CONCLUSION:In chronic benzene poisoning patients , the histone acetylation and methylation modification changes of TOPOⅡαpromoter regulatory factor Sp 1 accompanied with the changes of mRNA level are observed .Histone H4 and H3 acetylation and H3K9 methylation modification of Sp1 may play an important role in the benzene ’s hematopoiet-ic toxicities.

Influence of transcription factor Stat5a on proliferation of human breast cancer cells and its epigenetic mechanisms / 重庆医学

Objective To investigate and clarify the effect of Stat5a on proliferation of human breast cancer cells (MCF‐7) and to detect the changes of epigenetic signature on the promoter region of p53 gene .Methods Stat5a was over expressed in human breast cancer cells (MCF‐7) by using adenovirus mediated gene transfer technology .The cell proliferation was examined by MTS assay .ChIP assay was used to check the trimethylation of lysine 27 on histone 3 (H3K27Me3) of p53 gene promoter region .Fur‐thermore ,qRT‐PCR and western blot were also applied to confirm the expression of p53 gene .Results The number of MCF 7 in‐creased in a dose dependent manner .Compared with that of control group ,the cell density of MCF‐7 increased 7 .603 1% , 18 .123 7% and 24 .898 7% when the MOI were 10 ,20 and 30 .Chromatin Immunoprecipitation showed that Stat5a significantly in‐creased H3K27Me3 and down regulated the expression level of p53 gene .Conclusion Stat5a promotes proliferation of breast cancer cells through trimethylation of H3K27 and inhibition of p53 gene expression .

NF-κB participates in hepcidin up-regulation induced by iron overload in HH4 hepatocytes / 中国病理生理杂志

[ ABSTRACT] AIM:To study the effects of nuclear factor kappa B ( NF-κB) on human hepcidin expression in fer-ric ammonium citrate ( FAC)-induced HH4 hepatocytes.METHODS:Non-transformed HH4 cells were exposed to FAC at concentrations of 0.1, 1, 5 and 10 mmol/L for 48 h.The expression of iron regulatory gene hepcidin was determined by semi-quantitative RT-PCR.The effects of NF-κB on hepcidin transcriptional activity were detected using chromatin immuno-precipitation (ChIP), electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter assay system, combined with the inhibition experiments of intracellular NF-κB activity.RESULTS: FAC at concentrations of 5 mmol/L and 10 mmol/L significantly enhanced the expression of hepcidin.The results of ChIP and EMSA showed the binding of NF-κB to the upstream of hepcidin promoter.Treatment with NF-κB inhibitor BAY 11-7082 attenuated hepcidin expression.The lucif-erase activity in the cells transfected with recombinant luciferase reporter plasmid was obviously higher than that in control group.CONCLUSION:NF-κB is the transcription factor that contributes to hepcidin expression in iron overload-induced HH4 cells.

The applications of chromatin immunoprecipitation in the study on the downstream target genes of androgen receptor in prostate cancer cells / 中华检验医学杂志

Prostate cancer is one of the most common malignant tumor of male population in the West.Via binding to androgen response element in the genome and interacting with co-regulators,androgen receptor can participate in the development of prostate cancer and the convertion from androgen dependent prostate cancer to androgen independent prostate cancer.Chromatin immunoprecipitation ,coupled with PCR , chip,and high-throughput sequencing ,makes a huge progress in the study on the downstream target genes of androgen receptor and provide a new way to understand the molecular mechanisms of castration -resistant prostate cancer.

CpG Array Analysis of Histone H3 Lysine 4 Trimethylation by Chromatin Immunoprecipitation Linked to Microarrays Analysis in Peripheral Blood Mononuclear Cells of IgA Nephropathy Patients

PURPOSE: The purpose of the present study was to investigate the aberrance of histone H3 lysine 4 trimethylation (H3K4me3) in patients with IgA Nephropathy (IgAN). MATERIALS AND METHODS: In this study, H3K4me3 variations in peripheral blood mononuclear cells (PBMCs) from 15 IgAN patients and 15 healthy subjects were analyzed using chromatin immunoprecipitation linked to microarrays analysis (ChIP-chip). ChIP real-time PCR was used to validate the microarray results. Expression analysis by quantitative real-time PCR (qRT-PCR) revealed correlations between mRNA and H3K4me3 levels. DNA methylation status was analyzed by quantitative methylation-specific PCR. RESULTS: We found that 321 probes displayed significant H3K4me3 differences in IgAN patients compared with healthy controls. Among these probes, 154 probes displayed increased H3K4me3 and 167 probes demonstrated decreased H3K4me3. For further validation, we selected 4 key relevant genes (FCRL4, GALK2, PTPRN2 and IL1RAPL1) to study. The results of ChIP real-time PCR coincided well with the microarray data. Quantitative RT-PCR revealed the correlations between the mRNA expression and the methylation levels of H3K4me3. Different degrees of DNA methylation alterations appeared on the selected positive genes. CONCLUSION: Our studies indicated that there were significant alterations in H3K4me3 in IgAN patients. These findings may help to explain the disturbed immunity and abnormal glycosylation involved in IgAN patients.

The study of histone H3 lysine 4 trimethylation in peripheral blood mononuclear cells of systemic lupus erythematosus patients / 中华风湿病学杂志

Objective To study histone H3 lysine 4 trimethylation (H3K4me3) in peripheral blood mononuclear cells (PBMCs) of systemic lupus erythematosus(SLE) patients.Methods PBMCs were isolated by density gradient centrifugation from 10 active SLE patients,7 inactive SLE patients and 8 healthy volunteers.Chromatin immunopreeipitation linked to mieroarrays (ChIP-chip) was used to profile the variations in H3K4me3 in CpG island regions in PBMCs of SLE patients and controls.ChIP-qPCR was used to validate the mieroarray results.To confirm correlations between H3K4me3 and gene expression,expression analysis by qRT-PCR was performed on three randomly selected H3K4me3 candidates.Results 413 (137 increased and 276 decreased H3K4me3) and 393 genes (112 increased and 281 decreased H3K4me3) displayed significant differences in H3K4me3 between active and inactive SLE when compared with healthy SUhjeets.The results of ChiP-qPCR were consistent with microarray.ConclusiOn There are significant differences in H3K4me3 profiling between SLE and healthy subjects.These novel candidate genes may be potential biomarkers for future therapeutic targets.The ChIP-chip technology can help further reveal SLE molecular mechanisms and discover new therapeutic targets.

Estudo do gene GPB2 no carcinoma de células escamosas do esôfago esua relação com a proteína P53

O câncer de esôfago está entre os 10 tipos de câncer mais incidentes no mundo e no Brasil. O carcinoma de células escamosas do esôfago (CCEE) é o tipo histopatológico mais frequente e, em geral, é diagnosticado em estágios avançados, impossibilitando o tratamento curativo. A sobrevida é de menos de 10% dos pacientes após 5 anos do diagnóstico da doença. Para reverter esta situação é fundamental o entendimento de como ocorre a carcinogênese molecular esofagiana. Mutações no gene supressor de tumor TP53 são frequentes no CCEE, sendo relatados na literatura 30-70% de casos mutados. Outra alteração, recentemente descrita por nosso grupo, é o aumento da expressão da proteína ligadora de guanilato-2, GBP2, no tecido tumoral em relação ao epitélio normal adjacente de pacientes com CCEE. Dados de nosso grupo também demonstram a regulação de GBP2 dependente de p53 em uma linhagem celular de CCEE. O presente estudo, portanto, teve como objetivo geral investigar a relação entre GBP2 e p53 no CCEE através de duas etapas: pré-clínica e clínica. O objetivo pré-clínico foi investigar a possível ligação direta da proteína p53 selvagem à região promotora de GBP2 na linhagem TE-1 (CCEE, p53Met272Val) através da bioinformática e do ensaio de imunoprecipitação de cromatina. Foi identificada a ligação direta de p53 selvagem à região promotora de GBP2. Na etapa clínica, foram analisadas amostras de RNA e DNA dos tecidos tumoral e não-tumoral adjacente de pacientes com CCEE. Utilizando a técnica de PCR quantitativa, foi visto em 69,3% (18/26) dos pacientes um aumento da expressão de GBP2 no tecido tumoral em relação ao tecido não- -tumoral adjacente. O rastreamento por alterações nos éxons 5 ao 9 do gene TP53 foi realizado pela técnica de cromatografia líquida desnaturante de alta performance (dHPLC) e pela técnica de polimorfismo conformacional de fita simples (SSCP) seguido pelo sequenciamento automático. Em 56,1% (23/41) dos casos, a mutação em TP53 foi encontrada e em 3 pacientes foram encontrados polimorfismos neste gene. Não foi encontrada qualquer associação com significância estatística entre a expressão de GBP2, o status mutacional de TP53 e os dados clínico-patológicos no grupo de pacientes estudados. Os resultados deste trabalho sugerem que GBP2 é um gene-alvo de p53, resultado esse inédito na literatura, e que GBP2 tem um papel na carcinogênese do esôfago. Além disso, foi observada a ausência de associação entre a expressão de GBP2 e o status mutacional da proteína p53 durante o...

Esophageal cancer is one of the 10 most common incident cancers in the world and in Brazil. Esophageal squamous cell carcinoma (ESCC) is the most frequent histopathological type. In general, ESCC is diagnosed in advanced stages, when curative treatment is not possible. After 5 years of diagnosis, overall survival is less than 10% of patients. To change this situation, it is essential to understand how esophageal molecular carcinogenesis occurs. Mutations in TP53 tumor suppressor gene are frequent in ESCC, appearing in 30-70% of cases according to the literature. Another alteration, recently described by our group, is the increase of guanylate binding protein-2, GBP2, expression in tumoral tissue compared to adjacent normal epithelium of patients with ESCC. Data from our group also show p53-dependent GBP2 regulation on a ESCC cell line. The present study, therefore, had as general objective investigate the relationship between GBP2 and p53 through two parts: preclinical and clinical. The pre-clinical objective was to investigate a possible direct binding of wild type p53 protein to GBP2 promoter region on TE-1 cell line (ESCC, p53Met272Val) through bioinformatics and chromatin immunoprecipitation assay. In this study, it was identified the direct binding of wild type p53 to GBP2 promoter region. On the clinical part of the study, RNA and DNA samples of tumoral and adjacent nontumoral tissues from patients with ESCC were analysed. Through quantitative PCR technique, an increase of GBP2 expression in tumoral tissue in relation to adjacent non-tumoral tissue was seen in 69.3% (18/26) of patients. The screening of alterations on exons 5 to 9 of TP53 gene was performed by denaturing high performance liquid chromatography (dHPLC) and followed by automated sequencing. In 56,09% (23/41) of cases, TP53 mutation was found and in 3 patients polymorphisms were found. No statistically significant association was found between GBP2 expression, TP53 mutational status and clinicopathological data on the studied group. These results suggest that GBP2 is a p53 target-gene, an unpublished data on the literature, and that GBP2 has a role on esophageal carcinogenesis. Also, it was found no association between increased GBP2 gene expression and the presence of wild type p53 during esophageal tumor development. These data encourage a better characterization of GBP2 on ESCC to better understand its participation on the development of this cancer.

Temporal regulation of p300 on heart-specific transcription factors during mouse cardiogenesis / 重庆医科大学学报

Objective:Cardiogenesis is a precise process controlled by sequential gene regulatory steps.Heart-specific transcription factors (TFs)GATA4,Nkx2.5,MEF2C and Tbx5 play critical roles in controlling heart development.But the genetic basis for the temporal expression of genes during heart development still remains unclear.Substantial studies displayed gene regulation partly attributes to histone acetylation.However,the functions of individual histone acetyltransferases (HATs)in specific developmental transcription programs are not well elucidated.p300,a histone acetyltransferase and coactivator,plays key role in many physiological processes,moreover our previous study suggests that expression of p300 is developmentally regulated during mouse cardiogenesis,but the underlying molecular mechanism remains elusive.Methods:The whole hearts from embryonic mice on embryonic days (E)10.5 and 16.5 were collected accordingly,and then total RNA was extracted.Heart-specific TFs GATA4,Nkx2.5,MEF2C and Tbx5 mRNA from mouse myocardium at differential embryonic days during cardiac development were analyzed by quantitative (Q)-PCR.The levels of histone H3 acetylation on these heart-specific TFs promoter and transcription factor binding sites of p300 to these genes were identified by chromatin immunoprecipitation assays (ChIP).Results:The present study furnishes a comparative temporal expression map of cardiac transcriptional factors,during murine heart development (E10.5 and E16.5).The mRNA levels of these genes present dynamic change.On E10.5,the expressions of GATA4 and MEF2C were remarkable lower than those on E16.5 (P

Alpha 1-antitrypsin Matrix Attachment Region Enhances RNA PolymeraseⅡ-dependent Transcription / 中国生物工程杂志

Eukaryotic DNA element called Matrix Attachment Regions (MARs) can function on regulating the structure and activity of chromosome. Traditional quantitation in vitro and indirect functional analysis can not always reflect MAR-involved physiological state. In order to study transcription regulation and make a try in methodism,? 1-antitrypsin MAR (?1-AT MAR) is cloned and incorporated into pEGFP-C1 vector. Non-MAR-containing and MAR-containing plasmids were then transfected into HEK-293 cells with LipofectamineTM 2000 respectively. Positive cell clones were assayed after 20 days of selection by G418. Semiquantitative RT-PCR and fluorescence microscope analysis show that this MAR has a positive effect on modulating nearby gene expression. Further, co-localization with newly CMV promoter and RNA polymeraseⅡ(RNAPⅡ) was detected by chromatin immunoprecipitation (ChIP), The PCR result demonstrates that more RNAPⅡwas recruited to the CMV promoter to initiate transcription in presence of MAR. ChIP can be used to confirm the MAR-mediated transcriptional activation and provide more reliable information than RT-PCR in real time. The technology is also providing a platform for our research in gene expression regulation.

Effect of Sodium Butyrate on Phosphorylation of Histone at ?-Globin Gene Promoter Regions in K562 Cells / 实用儿科临床杂志

Objective To explore the effect of sodium butyrate(NaB) on phosphorylation/ acetylation of histone H3(ph/acH3) at G?-globin gene and A?-globin gene promoter regions in K562 cells.Methods K562 cells were devided into 2 groups:K562 cells were grown in the presence or absence of 0.5 mmol?L-1NaB for 48 h [K562(NaB) group] and untreated K562 cells group(K562 group).Semi-quantitative RT-PCR was employed to measure the levels of G?-globin mRNA and A?-globin mRNA.The real time PCR-based chromatin immunoprecipitation(ChIP) was used to detect the levels of ph/acH3 at G?-globin gene and A?-globin gene promoter regions.Results Compared with the K562 group,there was a 1.4-fold(t=-149.022,P=0.000) and 1.2-fold(t=-13.363,P=0.000) increase in G?-globin mRNA and A?-globin mRNA,respectively,in K562(NaB) group.The level of ph/acH3 at G?-globin gene and A?-globin gene promoter region increased by 2.9-fold(t=-12.833,P=0.006) and 3.2-fold(t=-10.484,P=0.000),respectively,in K562(NaB) group,compared with the K562 group.The %Input value of G?-globin and A?-globin promoter fragment was 10.0-fold(P=0.000) and 9.5-fold(P=0.000) higher than that value of Necdin gene promoter fragment in the K562(NaB) group,while the %Input value of G?-globin and A?-globin promoter fragment was 3.2-fold(P=0.000) and 2.7-fold(P=0.000) higher than that value of necdin gene promoter fragment in K562 group.Conclusions NaB improves the phosphorylation and acetylation of H3 at ?-globin gene promoter regions,and this may be one of the mechanisms of expression of ?-globin genes induced by NaB.

Thyroid Hormone Receptor Dynamics in Thyroid Hormone Response Elements of the Human Type 1 Iodothyronine Deiodinase Gene / 대한내분비학회지

BACKGROUND: Type 1 iodothyronine deiodinase (D1), the product of the hdio1 gene, is involved in thyroid hormone activation by the deiodination of thyroxine (T4) to form 3, 5, 3'-triiodothyronine (T3). Recent studies have identified two thyroid hormone response elements (TREs) in the 5 flanking region of the hdio1 gene. TRE1, proximal to TRE in the hdio1 gene, consists of a direct repeat of thyroid hormone receptor (TR) binding octamers with 10 bp separating the two TR binding sites. The upstream TRE, TRE2, is a classical direct repeat of retinoid X receptor (RXR)/TR binding half-sites with a 4-bp separation. There are few studies clarifying the TR dynamics in the TRE of a specific gene with or without the exposure of activated thyroid hormone. We evaluated TR binding patterns in the proximal and distal TREs of the hdio1 gene before and after T3 stimulation. METHODS: We employed chromatin immunoprecipitation (ChIP) technique to investigate the TR-TRE interaction before and after T3 stimulation in human hepatocellular carcinoma HepG2 cell line.Following cross-linking and sonication of the cells, immunoprecipitation was performed overnight at 4degrees C with TR 1, TR 1 and TR 2 antibodies. We analyzed the binding patterns and amounts of TR 1, TR 1 and TR 2 to TRE1 and TRE2 before and after 12 hours stimulation with 100 nM T3 by using conventional and quantitative real-time polymerase chain reactions (RQ-PCR). Reverse transcriptional PCR (RT-PCR) and Western blot with TR 1, TR 1 and TR 2 antibodies were performed to measure the levels of hdio1 mRNA and TR 1, TR 1 and TR 2 proteins before and after 12 hours exposure to 100 nM T3. RESULTS: In TRE1, TR 1 binding was significantly decreased after 12 hours stimulation with 100nM T3 (3.74-->1.97, delta=-47.3%, p3.01, delta=-71.1%, p 2.93, delta=-76.7%, p 9.84, delta=+7.3%). Total TR bindings in TRE2 were significantly decreased after 12 hours stimulation with 100 nM T3 (32.14 --> 15.78, delta=-50.9%, p<0.05). The TR bindings to TRE1 and TRE2 were not significantly different by the amounts of TR antibodies used during ChIP assays. The levels of hdio1 mRNA were significantly increased, 2.03 times, after 12 hours exposure to 100nM T3 (p<0.001). Western blot showed no significant change of the level of each TR isoform protein before and after 12 hours exposure to 100 nM T3. CONCLUSION: Our results demonstrate the dynamics of TR 1 at proximal TRE (TRE1) and the switching phenomenon of TR isoforms at distal TRE (TRE2) of the hdio1 gene after T3 stimulation. Further investigation, however, is needed to clarify the mechanisms of these observations.