Histone modification including H3 lysine 79 methylation (H3K79me) plays a key role during gene transcription and DNA damage repair. DOT1L, the sole methyltransferase for three states of H3K79me, is implicated in leuke...Histone modification including H3 lysine 79 methylation (H3K79me) plays a key role during gene transcription and DNA damage repair. DOT1L, the sole methyltransferase for three states of H3K79me, is implicated in leukemia, colorectal cancer, and dilated cardiomyopathy. However, understanding of DOT1L and H3K79me in these pathways and disease pathogenesis has been limited due to the difficulty of working with DOT1L protein. For instance, locus-specific or genome-wide binding sites of DOT1L revealed by chromatin immunoprecipitation (ChIP)-based methods are necessary for inferring its functions, but high-quality ChIP-grade antibodies are currently not available. Herein we have developed a knock-in approach to tag endogenous DOT1L with 3 × Flag at its C-terminal domain to follow functional analyses. The knock-in was facilitated by using TALENs to induce a targeted double-strand break at the endogenous DOTIL to stimulate local homologous recombination at that site. The single cell colonies with successful knock-in were isolated and verified by different methods. We also demonstrated that tagged DOT1L maintains its normal function in terms of methylation and that the engineered cells would be very useful for further studies.展开更多
Background:Delayed wound healing is one of the major complications of diabetes mellitus and is characterized by prolonged inflammation,delayed re-epithelialization and consistent oxidative stress,although the detailed...Background:Delayed wound healing is one of the major complications of diabetes mellitus and is characterized by prolonged inflammation,delayed re-epithelialization and consistent oxidative stress,although the detailed mechanism remains unknown.In this study,we aimed to investigate the potential role and effect of pterostilbene(PTE)and hematopoietic stem cells(HSCs)on diabetic wound healing.Methods:Diabetic rats were used to measure the epigenetic changes in both HSCs and peripheral blood mononuclear cells(PBMCs).A cutaneous burn injury was induced in the rats and PTE-treated diabetic HSCs were transplanted for evaluation of wound healing.In addition,several biomedical parameters,including gene expression,oxidative stress,mitochondrial function and inflammation in macrophages,were also measured.Results:Our data showed that PTE had a much stronger effect than resveratrol on accelerating diabetic wound healing,likely because PTE can ameliorate diabetes-induced epigenetic changes to estrogen receptorβpromoter in HSCs,while resveratrol cannot.Further investigation showed that bone marrow transplantation of PTE-treated diabetic HSCs restores diabetes-induced suppression of estrogen receptorβand its target genes,including nuclear respiratory factor-1 and superoxide dismutase 2,and protects against diabetes-induced oxidative stress,mitochondrial dysfunction and elevated pro-inflammatory cytokines in both PBMCs and macrophages,subsequently accelerating cutaneous wound healing.Conclusions:HSC may play an important role in wound healing through transferring epigenetic modifications to subsequent PBMCs and macrophages by differentiation,while PTE accelerates diabetic wound healing by modulating diabetes-induced epigenetic changes in HSCs.Thus,PTE may be a novel therapeutic strategy for diabetic wound healing.展开更多
文摘Histone modification including H3 lysine 79 methylation (H3K79me) plays a key role during gene transcription and DNA damage repair. DOT1L, the sole methyltransferase for three states of H3K79me, is implicated in leukemia, colorectal cancer, and dilated cardiomyopathy. However, understanding of DOT1L and H3K79me in these pathways and disease pathogenesis has been limited due to the difficulty of working with DOT1L protein. For instance, locus-specific or genome-wide binding sites of DOT1L revealed by chromatin immunoprecipitation (ChIP)-based methods are necessary for inferring its functions, but high-quality ChIP-grade antibodies are currently not available. Herein we have developed a knock-in approach to tag endogenous DOT1L with 3 × Flag at its C-terminal domain to follow functional analyses. The knock-in was facilitated by using TALENs to induce a targeted double-strand break at the endogenous DOTIL to stimulate local homologous recombination at that site. The single cell colonies with successful knock-in were isolated and verified by different methods. We also demonstrated that tagged DOT1L maintains its normal function in terms of methylation and that the engineered cells would be very useful for further studies.
基金supported by the National Natural Science Foundation of China Project(81772097)the National Key Disease Preventive Project for Wound Healing(2018-ZX-01S-001).
文摘Background:Delayed wound healing is one of the major complications of diabetes mellitus and is characterized by prolonged inflammation,delayed re-epithelialization and consistent oxidative stress,although the detailed mechanism remains unknown.In this study,we aimed to investigate the potential role and effect of pterostilbene(PTE)and hematopoietic stem cells(HSCs)on diabetic wound healing.Methods:Diabetic rats were used to measure the epigenetic changes in both HSCs and peripheral blood mononuclear cells(PBMCs).A cutaneous burn injury was induced in the rats and PTE-treated diabetic HSCs were transplanted for evaluation of wound healing.In addition,several biomedical parameters,including gene expression,oxidative stress,mitochondrial function and inflammation in macrophages,were also measured.Results:Our data showed that PTE had a much stronger effect than resveratrol on accelerating diabetic wound healing,likely because PTE can ameliorate diabetes-induced epigenetic changes to estrogen receptorβpromoter in HSCs,while resveratrol cannot.Further investigation showed that bone marrow transplantation of PTE-treated diabetic HSCs restores diabetes-induced suppression of estrogen receptorβand its target genes,including nuclear respiratory factor-1 and superoxide dismutase 2,and protects against diabetes-induced oxidative stress,mitochondrial dysfunction and elevated pro-inflammatory cytokines in both PBMCs and macrophages,subsequently accelerating cutaneous wound healing.Conclusions:HSC may play an important role in wound healing through transferring epigenetic modifications to subsequent PBMCs and macrophages by differentiation,while PTE accelerates diabetic wound healing by modulating diabetes-induced epigenetic changes in HSCs.Thus,PTE may be a novel therapeutic strategy for diabetic wound healing.
