In most cases, cancer develops as a result of non-inheritable somatic mutations (epimutations), acquired by the individual adult cell, during the evolution of the cell, and propagated into an expanding clone of progen...In most cases, cancer develops as a result of non-inheritable somatic mutations (epimutations), acquired by the individual adult cell, during the evolution of the cell, and propagated into an expanding clone of progeny of the cells by natural selection [1]. The role of microenvironment in selection for such acquired mutations, or epimutations, is a focus of scientific research in carcinogenesis [2]. Here we describe a defective DNA response to hypoxia due to epigenetic aberrancies, in cancer cellular biology [3]. We also summarize a literature review on hypoxia mediated epigenetic responses, and its role in carcinogenesis and metastasis. Further, we review a novel method of treating hypoxic solid tumors with a combination of epigenetic modifiers with both in vitro and in vivo results in human, translating to an improved prognosis and clinical outcome. We propose that this approach both independently and synergistically (with the current standard of care) can provide an improved outcome.展开更多
Objective To establish C57BL/6J embryonic stem （ES） cell lines with potential germ- line contribution Methods ES cells were isolated from blastocyst inner cell mass of C5 7BL/6J mice, and cultured for 15 passages, a...Objective To establish C57BL/6J embryonic stem （ES） cell lines with potential germ- line contribution Methods ES cells were isolated from blastocyst inner cell mass of C5 7BL/6J mice, and cultured for 15 passages, and then injected into blastococels of ICR mice blastocysts to establish chimeric mice. Results Three ES cell lines （mC57ES1,mC57ES3, mC57ES7） derived from the inner cell mass of C57BL/6J mice blastocysts were established. They were characteristic of undifferentiated state, including normal XY karyotype, expression of a specific cell surface marker “stage-specific embryonic antigen-I” and alkaline phosphatase in continuous passage. When injected into immunodeficient mice, mC57ES1 cells consistently differentiated into derivatives of all three embryonic germ layers. When mC57ES1 cells were transferred into ICR mice blastocysts, 4 chimeric mice have been obtained. One male of them revealed successful germ-line transmission. Conclussion We have obtained C57BL/6J ES cell lines with a potential germ-line contribution, which can be used to generate transgenic and gene knock-out mice.展开更多
DNA methylation plays a critical role in hematopoietic differentiation.Epimutation is a stochastic variation in DNA methylation that induces epigenetic heterogeneity.However,the effects of epimutations on normal hemat...DNA methylation plays a critical role in hematopoietic differentiation.Epimutation is a stochastic variation in DNA methylation that induces epigenetic heterogeneity.However,the effects of epimutations on normal hematopoiesis and hematopoietic diseases remain unclear.In this study,we developed a Julia package called EpiMut that enabled rapid and accurate quantification of epimutations.EpiMut was used to evaluate and provide an epimutation landscape in steady-state hematopoietic differentiation involving 13 types of blood cells ranging from hematopoietic stem/progenitor cells to mature cells.We showed that substantial genomic regions exhibited epigenetic variations rather than significant differences in DNA methylation levels between the myeloid and lymphoid lineages.Stepwise dynamics of epimutations were observed during the differentiation of each lineage.Importantly,we found that epimutation significantly enriched signals associated with lineage differentiation.Furthermore,epimutations in hematopoietic stem cells(HSCs)derived from various sources and acute myeloid leukemia were related to the function of HSCs and malignant cell disorders.Taken together,our study comprehensively documented an epimutation map and uncovered its important roles in human hematopoiesis,thereby offering insights into hematopoietic regulation.展开更多
Liver fibrosis continues to be a major health problem worldwide due to lack of effective therapy.If the etiology cannot be eliminated,liver fibrosis progresses to cirrhosis and eventually to liver failure or malignanc...Liver fibrosis continues to be a major health problem worldwide due to lack of effective therapy.If the etiology cannot be eliminated,liver fibrosis progresses to cirrhosis and eventually to liver failure or malignancy;both are associated with a fatal outcome.Liver transplantation,the only curative therapy,is still mostly unavailable.Liver fibrosis was shown to be a reversible process;however,complete reversibility remains debatable.Recently,the molecular markers of liver fibrosis were shown to be transmitted across generations.Epigenetic mechanisms including DNA methylation,histone posttranslational modifications and noncoding RNA have emerged as major determinants of gene expression during liver fibrogenesis and carcinogenesis.Furthermore,epigenetic mechanisms have been shown to be transmitted through mitosis and meiosis to daughter cells and subsequent generations.However,the exact epigenetic regulation of complete liver fibrosis resolution and inheritance has not been fully elucidated.This communication will highlight the recent advances in the search for delineating the mechanisms governing resolution of liver fibrosis and the potential for multigenerational and transgenerational transmission of fibrosis markers.The fact that epigenetic changes,unlike genetic mutations,are reversible and can be modulated pharmacologically underscores the unique opportunity to develop effective therapy to completely reverse liver fibrosis,to prevent the development of malignancy and to regulate heritability of fibrosis phenotype.展开更多
Deficiencies in DNA repair due to inherited germ-line mutations in DNA repair genes cause increased risk of gastrointestinal(GI) cancer. In sporadic GI cancers, mutations in DNA repair genes are relatively rare. Howev...Deficiencies in DNA repair due to inherited germ-line mutations in DNA repair genes cause increased risk of gastrointestinal(GI) cancer. In sporadic GI cancers, mutations in DNA repair genes are relatively rare. However, epigenetic alterations that reduce expression of DNA repair genes are frequent in sporadic GI cancers. These epigenetic reductions are also found in field defects that give rise to cancers. Reduced DNA repair likely allows excessive DNA damages to accumulate in somatic cells. Then either inaccurate translesion synthesis past the un-repaired DNA damages or error-prone DNA repair can cause mutations. Erroneous DNA repair can also cause epigenetic alterations(i.e., epimutations, transmitted through multiple replication cycles). Some of these mutations and epimutations may cause progression to cancer. Thus, deficient or absent DNA repair is likely an important underlying cause of cancer. Whole genome sequencing of GI cancers show that between thousands to hundreds of thousands of mutations occur in these cancers. Epimutations that reduce DNA repair gene expression and occur early in progression to GI cancers are a likely source of this high genomic instability. Cancer cells deficient in DNA repair are more vulnerable than normal cells to inactivation by DNA damaging agents. Thus, some of the most clinically effective chemotherapeutic agents in cancer treatment are DNA damaging agents, and their effectiveness often depends on deficient DNA repair in cancer cells. Recently, at least 18 DNA repair proteins, each active in one of six DNA repair pathways, were found to be subject to epigenetic reduction of expression in GI cancers. Different DNA repair pathways repair different types of DNA damage. Evaluation of which DNA repair pathway(s) are deficient in particular types of GI cancer and/or particular patients may prove useful in guiding choice of therapeutic agents in cancer therapy.展开更多
RNA helicases of the DEAD-box and related families are involved in various cellular processes including DNA replication, DNA repair, and RNA processing. However, the function of DEAD-box proteins in aquaculture specie...RNA helicases of the DEAD-box and related families are involved in various cellular processes including DNA replication, DNA repair, and RNA processing. However, the function of DEAD-box proteins in aquaculture species is poorly understood at molecular level. We obtained the full-length cDNA sequences of two genes encoding helicase-related proteins, Fc-vasa and Fc-PL10a, from the testes of Chinese shrimp, Fenneropenaeus chinensis. The two predicted amino acid sequences contain all the conserved motifs characterized by the DEAD-box family and several RGG repeats in the N-terminal regions. Homology and phylogenetic analyses indicate that they belong to the vasa and PLIO subfamilies. The three-dimensional structures of the two proteins were predicted with a homology modeling approach. Both core proteins consist of two tandem RecA-like domains similar to those of the DEAD-box RNA helicase. Using reverse transcription-polymerase chain reaction （RT-PCR） and real-time PCR we found that Fc-vasa was expressed specifically in the adult gonads. Transcription decreased in the ovary but increased in the testis during gonadal development. Fc-PL10a expression was widely distributed in the tissues we examined. Using in situ hybridization, we demonstrated that the Fc-vasa transcript is localized to the cytoplasm of the spermatogonia and oocytes. Thus, our results suggest that Fc-wasa plays an important role in germ-line development, and has utility as a germ cell lineage marker which will help to generate new insight into the origin and differentiation of germ cells as well as the regulation of reproduction in F. chinensis.展开更多
The laboratory rat is a valuable mammalian model organism for basic research and drug discovery. Here we demonstrate an efficient methodology by applying transcription activator-like effector nucleases(TALENs) technol...The laboratory rat is a valuable mammalian model organism for basic research and drug discovery. Here we demonstrate an efficient methodology by applying transcription activator-like effector nucleases(TALENs) technology to generate Leptin receptor(Lepr) knockout rats on the Sprague Dawley(SD) genetic background. Through direct injection of in vitro transcribed m RNA of TALEN pairs into SD rat zygotes, somatic mutations were induced in two of three resulting pups. One of the founders carrying bi-allelic mutation exhibited early onset of obesity and infertility. The other founder carried a chimeric mutation which was efficiently transmitted to the progenies. Through phenotyping of the resulting three lines of rats bearing distinct mutations in the Lepr locus, we found that the strains with a frame-shifted or premature stop codon mutation led to obesity and metabolic disorders. However, no obvious defect was observed in a strain with an in-frame 57 bp deletion in the extracellular domain of Lepr. This suggests the deleted amino acids do not significantly affect Lepr structure and function. This is the first report of generating the Lepr mutant obese rat model in SD strain through a reverse genetic approach. This suggests that TALEN is an efficient and powerful gene editing technology for the generation of disease models.展开更多
Identification of germ cell markers in fishes is crucial to track the germ cell differentiation and migration for manipulation of the cells to study sexual differentiation as well as to carry out transgenic transplant...Identification of germ cell markers in fishes is crucial to track the germ cell differentiation and migration for manipulation of the cells to study sexual differentiation as well as to carry out transgenic transplantation techniques.Several germ cell-specific markers such as vasa,cnbp,dnd,nanos3,cbx2,amh,dmrt1,Ly75/CD205 have been characterized so far in fishes using localization and expression analysis,which have highlighted the spatio-temporal pattern of expression in early gonadal development.Incidentally,seasonal breeders show dramatic changes during gonadal recrudescence,which might also influence germ cell differentiation and growth to entrain the reproductive cycle.Hence,an in-depth analysis of the gonadal cycle is required to delineate germ cell progress,differentiation,and maturation explicitly.In this context,fishes undergoing gonadal recrudescence for the seasonal cycle show germ cell proliferation differentially.Most of these germ cell markers belong to the DEAD-box protein family of ATP-dependent RNA helicases sharing consensus motifs and clustering in phylogenetic analysis.These markers were found to be well-conserved throughout evolution.In situ hybridization approaches confirmed the germ cell specific distribution of these molecular markers.In addition,several genes such as fgf and gsdf seem to facilitate germ cell development and differentiation.Hence,more detailed studies on these factors will facilitate a better understanding of germ cell development.This review highlights various germ cell markers in fishes and their immense potential to use these cells for germ cell transplantation.The extensive knowledge of the germ cell markers can also be exploited to carry out other biotechnological experiments aiming at the preservation of genetic information of endangered species or the analytical study of gonadogenesis.展开更多
文摘In most cases, cancer develops as a result of non-inheritable somatic mutations (epimutations), acquired by the individual adult cell, during the evolution of the cell, and propagated into an expanding clone of progeny of the cells by natural selection [1]. The role of microenvironment in selection for such acquired mutations, or epimutations, is a focus of scientific research in carcinogenesis [2]. Here we describe a defective DNA response to hypoxia due to epigenetic aberrancies, in cancer cellular biology [3]. We also summarize a literature review on hypoxia mediated epigenetic responses, and its role in carcinogenesis and metastasis. Further, we review a novel method of treating hypoxic solid tumors with a combination of epigenetic modifiers with both in vitro and in vivo results in human, translating to an improved prognosis and clinical outcome. We propose that this approach both independently and synergistically (with the current standard of care) can provide an improved outcome.
文摘Objective To establish C57BL/6J embryonic stem （ES） cell lines with potential germ- line contribution Methods ES cells were isolated from blastocyst inner cell mass of C5 7BL/6J mice, and cultured for 15 passages, and then injected into blastococels of ICR mice blastocysts to establish chimeric mice. Results Three ES cell lines （mC57ES1,mC57ES3, mC57ES7） derived from the inner cell mass of C57BL/6J mice blastocysts were established. They were characteristic of undifferentiated state, including normal XY karyotype, expression of a specific cell surface marker “stage-specific embryonic antigen-I” and alkaline phosphatase in continuous passage. When injected into immunodeficient mice, mC57ES1 cells consistently differentiated into derivatives of all three embryonic germ layers. When mC57ES1 cells were transferred into ICR mice blastocysts, 4 chimeric mice have been obtained. One male of them revealed successful germ-line transmission. Conclussion We have obtained C57BL/6J ES cell lines with a potential germ-line contribution, which can be used to generate transgenic and gene knock-out mice.
基金supported by the National Science Foundation of China(82022002,82131430173,81900117,81890993)the National Key Research and Development Program of China(2021YFA1102800)+2 种基金the Distinguished Young Scholars of Tianjin(21JCJQJC00070)the CAMS Initiative for Innovative Medicine(2021-I2M-1-040)the Foundation of Haihe Laboratory(HH22KYZX0018).
文摘DNA methylation plays a critical role in hematopoietic differentiation.Epimutation is a stochastic variation in DNA methylation that induces epigenetic heterogeneity.However,the effects of epimutations on normal hematopoiesis and hematopoietic diseases remain unclear.In this study,we developed a Julia package called EpiMut that enabled rapid and accurate quantification of epimutations.EpiMut was used to evaluate and provide an epimutation landscape in steady-state hematopoietic differentiation involving 13 types of blood cells ranging from hematopoietic stem/progenitor cells to mature cells.We showed that substantial genomic regions exhibited epigenetic variations rather than significant differences in DNA methylation levels between the myeloid and lymphoid lineages.Stepwise dynamics of epimutations were observed during the differentiation of each lineage.Importantly,we found that epimutation significantly enriched signals associated with lineage differentiation.Furthermore,epimutations in hematopoietic stem cells(HSCs)derived from various sources and acute myeloid leukemia were related to the function of HSCs and malignant cell disorders.Taken together,our study comprehensively documented an epimutation map and uncovered its important roles in human hematopoiesis,thereby offering insights into hematopoietic regulation.
基金Egyptian Science and Technology Development Fund under Project 1550
文摘Liver fibrosis continues to be a major health problem worldwide due to lack of effective therapy.If the etiology cannot be eliminated,liver fibrosis progresses to cirrhosis and eventually to liver failure or malignancy;both are associated with a fatal outcome.Liver transplantation,the only curative therapy,is still mostly unavailable.Liver fibrosis was shown to be a reversible process;however,complete reversibility remains debatable.Recently,the molecular markers of liver fibrosis were shown to be transmitted across generations.Epigenetic mechanisms including DNA methylation,histone posttranslational modifications and noncoding RNA have emerged as major determinants of gene expression during liver fibrogenesis and carcinogenesis.Furthermore,epigenetic mechanisms have been shown to be transmitted through mitosis and meiosis to daughter cells and subsequent generations.However,the exact epigenetic regulation of complete liver fibrosis resolution and inheritance has not been fully elucidated.This communication will highlight the recent advances in the search for delineating the mechanisms governing resolution of liver fibrosis and the potential for multigenerational and transgenerational transmission of fibrosis markers.The fact that epigenetic changes,unlike genetic mutations,are reversible and can be modulated pharmacologically underscores the unique opportunity to develop effective therapy to completely reverse liver fibrosis,to prevent the development of malignancy and to regulate heritability of fibrosis phenotype.
文摘Deficiencies in DNA repair due to inherited germ-line mutations in DNA repair genes cause increased risk of gastrointestinal(GI) cancer. In sporadic GI cancers, mutations in DNA repair genes are relatively rare. However, epigenetic alterations that reduce expression of DNA repair genes are frequent in sporadic GI cancers. These epigenetic reductions are also found in field defects that give rise to cancers. Reduced DNA repair likely allows excessive DNA damages to accumulate in somatic cells. Then either inaccurate translesion synthesis past the un-repaired DNA damages or error-prone DNA repair can cause mutations. Erroneous DNA repair can also cause epigenetic alterations(i.e., epimutations, transmitted through multiple replication cycles). Some of these mutations and epimutations may cause progression to cancer. Thus, deficient or absent DNA repair is likely an important underlying cause of cancer. Whole genome sequencing of GI cancers show that between thousands to hundreds of thousands of mutations occur in these cancers. Epimutations that reduce DNA repair gene expression and occur early in progression to GI cancers are a likely source of this high genomic instability. Cancer cells deficient in DNA repair are more vulnerable than normal cells to inactivation by DNA damaging agents. Thus, some of the most clinically effective chemotherapeutic agents in cancer treatment are DNA damaging agents, and their effectiveness often depends on deficient DNA repair in cancer cells. Recently, at least 18 DNA repair proteins, each active in one of six DNA repair pathways, were found to be subject to epigenetic reduction of expression in GI cancers. Different DNA repair pathways repair different types of DNA damage. Evaluation of which DNA repair pathway(s) are deficient in particular types of GI cancer and/or particular patients may prove useful in guiding choice of therapeutic agents in cancer therapy.
基金Supported by the Specialized Research Fund for the Doctoral Program of Higher Education,China (No. 200804230015)the National High Technology Research and Development Program of China (863 Program) (No. 2006AA10A401)
文摘RNA helicases of the DEAD-box and related families are involved in various cellular processes including DNA replication, DNA repair, and RNA processing. However, the function of DEAD-box proteins in aquaculture species is poorly understood at molecular level. We obtained the full-length cDNA sequences of two genes encoding helicase-related proteins, Fc-vasa and Fc-PL10a, from the testes of Chinese shrimp, Fenneropenaeus chinensis. The two predicted amino acid sequences contain all the conserved motifs characterized by the DEAD-box family and several RGG repeats in the N-terminal regions. Homology and phylogenetic analyses indicate that they belong to the vasa and PLIO subfamilies. The three-dimensional structures of the two proteins were predicted with a homology modeling approach. Both core proteins consist of two tandem RecA-like domains similar to those of the DEAD-box RNA helicase. Using reverse transcription-polymerase chain reaction （RT-PCR） and real-time PCR we found that Fc-vasa was expressed specifically in the adult gonads. Transcription decreased in the ovary but increased in the testis during gonadal development. Fc-PL10a expression was widely distributed in the tissues we examined. Using in situ hybridization, we demonstrated that the Fc-vasa transcript is localized to the cytoplasm of the spermatogonia and oocytes. Thus, our results suggest that Fc-wasa plays an important role in germ-line development, and has utility as a germ cell lineage marker which will help to generate new insight into the origin and differentiation of germ cells as well as the regulation of reproduction in F. chinensis.
基金supported by the State Key Development Programs of China (2012CB910400 to Mingyao Liu)the National Natural Science Foundation of China (31371455, 31171318 and 81330049)the Science and Technology Commission of Shanghai Municipality (14140900300)
文摘The laboratory rat is a valuable mammalian model organism for basic research and drug discovery. Here we demonstrate an efficient methodology by applying transcription activator-like effector nucleases(TALENs) technology to generate Leptin receptor(Lepr) knockout rats on the Sprague Dawley(SD) genetic background. Through direct injection of in vitro transcribed m RNA of TALEN pairs into SD rat zygotes, somatic mutations were induced in two of three resulting pups. One of the founders carrying bi-allelic mutation exhibited early onset of obesity and infertility. The other founder carried a chimeric mutation which was efficiently transmitted to the progenies. Through phenotyping of the resulting three lines of rats bearing distinct mutations in the Lepr locus, we found that the strains with a frame-shifted or premature stop codon mutation led to obesity and metabolic disorders. However, no obvious defect was observed in a strain with an in-frame 57 bp deletion in the extracellular domain of Lepr. This suggests the deleted amino acids do not significantly affect Lepr structure and function. This is the first report of generating the Lepr mutant obese rat model in SD strain through a reverse genetic approach. This suggests that TALEN is an efficient and powerful gene editing technology for the generation of disease models.
基金The research work mentioned in this review was supported by a grant-in-aid(Ref.No.37(1708)/18/EMR-II)from the Council of Scientific and Industrial Research,India,to BS.NA is thankful to the University of Hyderabad for the Non-NET fellowshipAuthors acknowledge BUILDER Grant from DBT(Ref No.BUILDER-DBT-BT/INF/22/SP41176/2020),India,to School of Life Sciences,University of Hyderabad.Authors also acknowledge Ms.Sonika Kar for proofreading the manuscript.
文摘Identification of germ cell markers in fishes is crucial to track the germ cell differentiation and migration for manipulation of the cells to study sexual differentiation as well as to carry out transgenic transplantation techniques.Several germ cell-specific markers such as vasa,cnbp,dnd,nanos3,cbx2,amh,dmrt1,Ly75/CD205 have been characterized so far in fishes using localization and expression analysis,which have highlighted the spatio-temporal pattern of expression in early gonadal development.Incidentally,seasonal breeders show dramatic changes during gonadal recrudescence,which might also influence germ cell differentiation and growth to entrain the reproductive cycle.Hence,an in-depth analysis of the gonadal cycle is required to delineate germ cell progress,differentiation,and maturation explicitly.In this context,fishes undergoing gonadal recrudescence for the seasonal cycle show germ cell proliferation differentially.Most of these germ cell markers belong to the DEAD-box protein family of ATP-dependent RNA helicases sharing consensus motifs and clustering in phylogenetic analysis.These markers were found to be well-conserved throughout evolution.In situ hybridization approaches confirmed the germ cell specific distribution of these molecular markers.In addition,several genes such as fgf and gsdf seem to facilitate germ cell development and differentiation.Hence,more detailed studies on these factors will facilitate a better understanding of germ cell development.This review highlights various germ cell markers in fishes and their immense potential to use these cells for germ cell transplantation.The extensive knowledge of the germ cell markers can also be exploited to carry out other biotechnological experiments aiming at the preservation of genetic information of endangered species or the analytical study of gonadogenesis.
