DNA methyltransferases（Dnmts）comprise a family of proteins which involved in the establishment and maintenance of DNA methylation patterns.In pig,the molecular characterization and tissue expression profile of Dnmt ...DNA methyltransferases（Dnmts）comprise a family of proteins which involved in the establishment and maintenance of DNA methylation patterns.In pig,the molecular characterization and tissue expression profile of Dnmt gene family are not clear.To solve this problem,reverse transcriptase PCR and rapid amplification of c DNA ends were used to clone the sequences of the porcine Dnmt2 and Dnmt3b genes.Furthermore,the m RNA expression profiles of Dnmt1,Dnmt2,Dnmt3a and Dnmt3b genes from 54 adult tissues and 2 entire fetuses of Rongchang pig were analyzed by quantitative real-time PCR（q RT-PCR）.As a result,the lengths of porcine Dnmt2 and Dnmt3b gene c DNAs were 1 227 and 2 559 bp with cytosine-C5specific DNA methylase domain,respectively.The four Dnmt genes were highly expressed in longissimus dorsi muscle（P〈0.01）.Dnmt1 is highly expressed in heart（P〈0.01）and Dnmt 2 shows its preference in liver and seminal vesicle tissue（P〈0.01）.Dnmt3a and Dnmt3b are highly expressed in the two fetus stages（P〈0.01）.All these results suggested that each gene has its specific expression profile,and deeper study is required to dig more details between the methylation level and Dnmt family m RNA expressions in different tissues.展开更多
Objective:DNA methylation is one of the most important epigenetic modulation,which is catalyzed primarily by three methyltransferases,DNMT1,DNMT3a and DNMT3b.The aim of our study is to investigate the modulatory effec...Objective:DNA methylation is one of the most important epigenetic modulation,which is catalyzed primarily by three methyltransferases,DNMT1,DNMT3a and DNMT3b.The aim of our study is to investigate the modulatory effect of DNMTs on hippocampus-dependent memory formation,and to explore the underlying molecular,cellular and synaptic mechanisms.Methods:Dnmt1,3a and 3b were selectively deleted in the CA1 region of dorsal hippocampus by Cre/LoxP recombinase systerm,either with virus-mediated Cre expression in CA1 of Dnmtsflox/flox mice or conditionalαCaMKII-Cre;Dnmtsflox/flox mice.Hippocampus-dependent and hippocampus-independent memory performance were evaluated in adult KO and control mice.RNA-seq analysis was conducted to screen differential expression genes in the hippocampus after conditional Dnmts knockout.Real-time qPCR and Western blot analysis were used to confirm the differential expression.We also analyzed the alteration in DNA methylation by Whole Genome Bisulfite Sequencing.Neuronal excitability,synaptic transmission and plasticity were measured in CA1 pyramidal neurons of hippocampal slices.Finally,we checked whether virus-mediated shRNA expression in hippocampal CA1 could ameliorate abnormal synaptic function and memory deficit observed inαCaMKII-Cre;Dnmstflox/flox mice.Results:We found that both Dnmt1flox/floxDnmt3aflox/flox mice and Dnmt3bflox/flox mice receiving AAV-Cre virus infection into CA1 region displayed recognition memory deficit to object place,but normal memory to novel object.All mice showed similar performance in fear memory tests.Also,virus infection and Dnmts deletion did not changed anxiety-or depression-like behavior.The object place recognition memory deficit was also observed in bothαCaMKII-Cre;Dnmt1flox/floxDnmt3aflox/flox mice andαCaMKII-Cre;Dnmt3bflox/flox mice.The Cre expression inαCaMKII-Cre mice was verified to be dominant in hippocampal CA1.RNA-seq based gene expression and followed real-time qPCR and western blot analysis confirmed significant upregulation of certain genes after Dnmts deletion in aCaMKII-expression excitatory neurons in the hippocampus.WGBS analysis showed differentiated DNA methylation in related genes.Normal basal synaptic transmission but impaired LTP was observed in SCCA1 path of bothαCaMKII-Cre;Dnmt1flox/floxDnmt3aflox/flox mice andαCaMKII-Cre;Dnmt3bflox/flox mice.AAV-virus mediated specific shRNA expression in CA1 region of dorsal hippocampus interfered upregulation of candidate genes,rescued abnormal synaptic function,and ameliorated object place cognition impairment in bothαCaMKII-Cre;Dnmt1flox/floxDnmt3aflox/flox mice andαCaMKII-Cre;Dnmt3bflox/flox mice.Virus-mediated shRNA expression in CA1 region of dorsal hippocampus did not affect recognition memory to novel object.Conclusion:In conclusion,our findings suggest that Dnmts in CA1 excitatory neurons plays an important role in regulating synaptic function and hippocampus-dependent recognition memory process by control the expression of certain target genes.展开更多
Inflammation is closely related to stroke prognosis, and high inflammation status leads to poor functional outcome in stroke. DNA methylation is involved in the pathogenesis and prognosis of stroke. However, the effec...Inflammation is closely related to stroke prognosis, and high inflammation status leads to poor functional outcome in stroke. DNA methylation is involved in the pathogenesis and prognosis of stroke. However, the effect of DNA methylation on stroke at high levels of inflammation is unclear. In this study, we constructed a hyperinflammatory cerebral ischemia mouse model and investigated the effect of hypomethylation and hypermethylation on the functional outcome. We constructed a mouse model of transient middle cerebral artery occlusion and treated the mice with lipopolysaccharide to induce a hyperinflammatory state. To investigate the effect of DNA methylation on stroke, we used small molecule inhibitors to restrain the function of key DNA methylation and demethylation enzymes. 2,3,5-Triphenyltetrazolium chloride staining, neurological function scores, neurobehavioral tests, enzyme-linked immunosorbent assay, quantitative reverse transcription PCR and western blot assay were used to evaluate the effects after stroke in mice. We assessed changes in the global methylation status by measuring DNA 5-mc and DNA 5-hmc levels in peripheral blood after the use of the inhibitor. In the group treated with the DNA methylation inhibitor, brain tissue 2,3,5-triphenyltetrazolium chloride staining showed an increase in infarct volume, which was accompanied by a decrease in neurological scores and worsening of neurobehavioral performance. The levels of inflammatory factors interleukin 6 and interleukin-1 beta in ischemic brain tissue and plasma were elevated, indicating increased inflammation. Related inflammatory pathway exploration showed significant overactivation of nuclear factor kappa B. These results suggested that inhibiting DNA methylation led to poor functional outcome in mice with high inflammation following stroke. Further, the effects were reversed by inhibition of DNA demethylation. Our findings suggest that DNA methylation regulates the inflammatory response in stroke and has an important role in the functional outcome of hyperinflammatory stroke.展开更多
外周T细胞淋巴瘤是一组罕见的强侵袭性的非霍奇金淋巴瘤,绝大部分目前治疗效果欠佳。近些年的证据表明表观遗传失调对于PTCL的发生发展起着驱动作用,特别是DNA甲基化。已发现在PTCL中存在DNA甲基化异常以及DNA甲基化修饰酶DNMT、TET、ID...外周T细胞淋巴瘤是一组罕见的强侵袭性的非霍奇金淋巴瘤,绝大部分目前治疗效果欠佳。近些年的证据表明表观遗传失调对于PTCL的发生发展起着驱动作用,特别是DNA甲基化。已发现在PTCL中存在DNA甲基化异常以及DNA甲基化修饰酶DNMT、TET、IDH的频繁突变,且与患者不良预后相关。随着对PTCL基因组学认识的深入,DNA甲基化作为表观遗传学的一个重要组成部分,有望为治疗PTCL提供新方向。Peripheral T-cell lymphoma (PTCL) is a group of rare, aggressive non-Hodgkin’s lymphomas, and most of them have poor treatment outcomes. Recent evidence suggests that epigenetic dysregulation plays a driving role in the occurrence and development of PTCL, especially DNA methylation. Abnormal DNA methylation and frequent mutations of DNA methylation modification enzyme DNMT, TET, and IDH have been found in PTCL, which are associated with poor prognosis of patients. With the deepening understanding of the genomics of PTCL, DNA methylation, as an important component of epigenetics, is expected to provide new directions for the treatment of PTCL.展开更多
The advances in the research on the structure and function of DNMT3A and the role of DNMT3A in tumorigenesis and development were reviewed,including gynecologic cancers,hematologic tumors,melanoma,amelanoma,colorectal...The advances in the research on the structure and function of DNMT3A and the role of DNMT3A in tumorigenesis and development were reviewed,including gynecologic cancers,hematologic tumors,melanoma,amelanoma,colorectal cancer and other cancers,to provide new ideas for the treatment of cancer.展开更多
基金supported by the grants from the Specialized Doctor Research Fund of Southwestern University of China (2013Bsr8)the Youth Fund of Rongchang Campus of Southwest University, China (20700429)+2 种基金the Chongqing Fund of Application and Development, China (cstc2013yykf C80003)the Chongqing Fund of Basic Research, China (15428, 14440)the National Biological Breeding Capacity Building and Industrialization Projects, China (2014-2573)
文摘DNA methyltransferases（Dnmts）comprise a family of proteins which involved in the establishment and maintenance of DNA methylation patterns.In pig,the molecular characterization and tissue expression profile of Dnmt gene family are not clear.To solve this problem,reverse transcriptase PCR and rapid amplification of c DNA ends were used to clone the sequences of the porcine Dnmt2 and Dnmt3b genes.Furthermore,the m RNA expression profiles of Dnmt1,Dnmt2,Dnmt3a and Dnmt3b genes from 54 adult tissues and 2 entire fetuses of Rongchang pig were analyzed by quantitative real-time PCR（q RT-PCR）.As a result,the lengths of porcine Dnmt2 and Dnmt3b gene c DNAs were 1 227 and 2 559 bp with cytosine-C5specific DNA methylase domain,respectively.The four Dnmt genes were highly expressed in longissimus dorsi muscle（P〈0.01）.Dnmt1 is highly expressed in heart（P〈0.01）and Dnmt 2 shows its preference in liver and seminal vesicle tissue（P〈0.01）.Dnmt3a and Dnmt3b are highly expressed in the two fetus stages（P〈0.01）.All these results suggested that each gene has its specific expression profile,and deeper study is required to dig more details between the methylation level and Dnmt family m RNA expressions in different tissues.
文摘Objective:DNA methylation is one of the most important epigenetic modulation,which is catalyzed primarily by three methyltransferases,DNMT1,DNMT3a and DNMT3b.The aim of our study is to investigate the modulatory effect of DNMTs on hippocampus-dependent memory formation,and to explore the underlying molecular,cellular and synaptic mechanisms.Methods:Dnmt1,3a and 3b were selectively deleted in the CA1 region of dorsal hippocampus by Cre/LoxP recombinase systerm,either with virus-mediated Cre expression in CA1 of Dnmtsflox/flox mice or conditionalαCaMKII-Cre;Dnmtsflox/flox mice.Hippocampus-dependent and hippocampus-independent memory performance were evaluated in adult KO and control mice.RNA-seq analysis was conducted to screen differential expression genes in the hippocampus after conditional Dnmts knockout.Real-time qPCR and Western blot analysis were used to confirm the differential expression.We also analyzed the alteration in DNA methylation by Whole Genome Bisulfite Sequencing.Neuronal excitability,synaptic transmission and plasticity were measured in CA1 pyramidal neurons of hippocampal slices.Finally,we checked whether virus-mediated shRNA expression in hippocampal CA1 could ameliorate abnormal synaptic function and memory deficit observed inαCaMKII-Cre;Dnmstflox/flox mice.Results:We found that both Dnmt1flox/floxDnmt3aflox/flox mice and Dnmt3bflox/flox mice receiving AAV-Cre virus infection into CA1 region displayed recognition memory deficit to object place,but normal memory to novel object.All mice showed similar performance in fear memory tests.Also,virus infection and Dnmts deletion did not changed anxiety-or depression-like behavior.The object place recognition memory deficit was also observed in bothαCaMKII-Cre;Dnmt1flox/floxDnmt3aflox/flox mice andαCaMKII-Cre;Dnmt3bflox/flox mice.The Cre expression inαCaMKII-Cre mice was verified to be dominant in hippocampal CA1.RNA-seq based gene expression and followed real-time qPCR and western blot analysis confirmed significant upregulation of certain genes after Dnmts deletion in aCaMKII-expression excitatory neurons in the hippocampus.WGBS analysis showed differentiated DNA methylation in related genes.Normal basal synaptic transmission but impaired LTP was observed in SCCA1 path of bothαCaMKII-Cre;Dnmt1flox/floxDnmt3aflox/flox mice andαCaMKII-Cre;Dnmt3bflox/flox mice.AAV-virus mediated specific shRNA expression in CA1 region of dorsal hippocampus interfered upregulation of candidate genes,rescued abnormal synaptic function,and ameliorated object place cognition impairment in bothαCaMKII-Cre;Dnmt1flox/floxDnmt3aflox/flox mice andαCaMKII-Cre;Dnmt3bflox/flox mice.Virus-mediated shRNA expression in CA1 region of dorsal hippocampus did not affect recognition memory to novel object.Conclusion:In conclusion,our findings suggest that Dnmts in CA1 excitatory neurons plays an important role in regulating synaptic function and hippocampus-dependent recognition memory process by control the expression of certain target genes.
基金supported by the National Natural Science Foundation of China,No.82171270 (to ZL)Public Service Platform for Artificial In telligence Screening and Auxiliary Diagnosis for the Medical and Health Industry,Ministry of Industry and Information Technology of the People's Republic of China,No.2020-0103-3-1 (to ZL)+3 种基金the Natural Science Foundation of Beijing,No.Z200016 (to ZL)Beijing Talents Project,No.2018000021223ZK03 (to ZL)Beijing Municipal Committee of Science and Technology,No.Z201 100005620010 (to ZL)CAMS Innovation Fund for Medical Sciences,No.2019-I2M-5-029 (to YongW)。
文摘Inflammation is closely related to stroke prognosis, and high inflammation status leads to poor functional outcome in stroke. DNA methylation is involved in the pathogenesis and prognosis of stroke. However, the effect of DNA methylation on stroke at high levels of inflammation is unclear. In this study, we constructed a hyperinflammatory cerebral ischemia mouse model and investigated the effect of hypomethylation and hypermethylation on the functional outcome. We constructed a mouse model of transient middle cerebral artery occlusion and treated the mice with lipopolysaccharide to induce a hyperinflammatory state. To investigate the effect of DNA methylation on stroke, we used small molecule inhibitors to restrain the function of key DNA methylation and demethylation enzymes. 2,3,5-Triphenyltetrazolium chloride staining, neurological function scores, neurobehavioral tests, enzyme-linked immunosorbent assay, quantitative reverse transcription PCR and western blot assay were used to evaluate the effects after stroke in mice. We assessed changes in the global methylation status by measuring DNA 5-mc and DNA 5-hmc levels in peripheral blood after the use of the inhibitor. In the group treated with the DNA methylation inhibitor, brain tissue 2,3,5-triphenyltetrazolium chloride staining showed an increase in infarct volume, which was accompanied by a decrease in neurological scores and worsening of neurobehavioral performance. The levels of inflammatory factors interleukin 6 and interleukin-1 beta in ischemic brain tissue and plasma were elevated, indicating increased inflammation. Related inflammatory pathway exploration showed significant overactivation of nuclear factor kappa B. These results suggested that inhibiting DNA methylation led to poor functional outcome in mice with high inflammation following stroke. Further, the effects were reversed by inhibition of DNA demethylation. Our findings suggest that DNA methylation regulates the inflammatory response in stroke and has an important role in the functional outcome of hyperinflammatory stroke.
文摘外周T细胞淋巴瘤是一组罕见的强侵袭性的非霍奇金淋巴瘤,绝大部分目前治疗效果欠佳。近些年的证据表明表观遗传失调对于PTCL的发生发展起着驱动作用,特别是DNA甲基化。已发现在PTCL中存在DNA甲基化异常以及DNA甲基化修饰酶DNMT、TET、IDH的频繁突变,且与患者不良预后相关。随着对PTCL基因组学认识的深入,DNA甲基化作为表观遗传学的一个重要组成部分,有望为治疗PTCL提供新方向。Peripheral T-cell lymphoma (PTCL) is a group of rare, aggressive non-Hodgkin’s lymphomas, and most of them have poor treatment outcomes. Recent evidence suggests that epigenetic dysregulation plays a driving role in the occurrence and development of PTCL, especially DNA methylation. Abnormal DNA methylation and frequent mutations of DNA methylation modification enzyme DNMT, TET, and IDH have been found in PTCL, which are associated with poor prognosis of patients. With the deepening understanding of the genomics of PTCL, DNA methylation, as an important component of epigenetics, is expected to provide new directions for the treatment of PTCL.
基金Supported by the Student Innovation and Entrepreneurship Training Program of Chengde Medical University (2023023)the Natural Science Fund of Hebei Province (H2022406025)+2 种基金the Science and Technology Project of Hebei Provincial Department of Education (BJK2023001)the High-level Talents Research Startup Fund of Chengde Medical University (202201)Discipline Construction Funds of Chengde Medical University.
文摘The advances in the research on the structure and function of DNMT3A and the role of DNMT3A in tumorigenesis and development were reviewed,including gynecologic cancers,hematologic tumors,melanoma,amelanoma,colorectal cancer and other cancers,to provide new ideas for the treatment of cancer.
