Comprehensive understanding of mechanisms of epigenetic regulation requires identification of molecules bound to genomic regions of interest in vivo. We have developed a novel method, insertional chromatin immunopreci...Comprehensive understanding of mechanisms of epigenetic regulation requires identification of molecules bound to genomic regions of interest in vivo. We have developed a novel method, insertional chromatin immunoprecipitatin (iChIP), to isolate specific genomic regions retaining molecular interaction in order to perform non-biased identification of interacting molecules in vivo. Here, we developed a second-generation tagged LexA DNA-binding domain, 3xFNLDD, for the iChIP analysis. 3xFNLDD consists of 3 x FLAG tags, a nuclear localization signal (NLS), the DNA-binding domain (DB) and the dimerization domain of the LexA protein. Expression of 3xFNLDD can be detected by immunoblot analysis as well as flowcytometry. We showed that iChIP using 3xFNLDD is able to consistently isolate more than 10% of input genomic DNA, several-fold more efficient compared to the first-generation tagged LexA DB. 3xFNLDD would be a useful tool to perform the iChIP analysis for locus-specific biochemical epigenetics.展开更多
LexA and RecA play a crucial role in SOS response in Escherichia coli.In classical SOS response,LexA functions as a repressor to regulate the expression of many genes including RecA after DNA damage occurs.Previous st...LexA and RecA play a crucial role in SOS response in Escherichia coli.In classical SOS response,LexA functions as a repressor to regulate the expression of many genes including RecA after DNA damage occurs.Previous studies showed that LexA1 or LexA2 is not involved in RecA induction in Deinococ- cus radiodurans.In this study,we constructed a double mutant of lexA1 and lexA2.Results showed that mutation of lexA1 and lexA2 resulted in an apparent decrease in growth rate and an increased re- sistance to ultraviolet and hydrogen peroxide compared with wild type R1 and two single mutants. However,this double mutant did not exhibit any remarkably increased constitutive expression of RecA under normal conditions,just like wild type R1 and two single mutants,suggesting that neither LexA1 nor LexA2 is involved in the induced expression of RecA.Further transcriptional assay showed that LexA1 and LexA2 together participated in many regulatory pathways involved in cell division,protein synthesis,antioxidant process,transcription regulation and other mechanisms.These results indicate that there should be a new mechanism by which these damage-induced proteins are regulated in D. radiodurans.展开更多
Phytochromes are a family of photoreceptors in plants that perceive the red(R)and far-red(FR)components of their light environment.Phytochromes exist in vivo in two forms,the inactive Pr form and the active Pfr form,t...Phytochromes are a family of photoreceptors in plants that perceive the red(R)and far-red(FR)components of their light environment.Phytochromes exist in vivo in two forms,the inactive Pr form and the active Pfr form,that are interconvertible by treatments with R or FR light.It is believed that phytochromes transduce light signals by interacting with their signaling partners.A GAL4-based lightswitchable yeast two-hybrid(Y2H)system was developed two decades ago and has been successfully employed in many studies to determine phytochrome interactions with their signaling components.However,several pairs of interactions between phytochromes and their interactors,such as the phyACOP1 and phyA-TZP interactions,were demonstrated by other assay systems but were not detected by this GAL4 Y2H system.Here,we report a modified LexA Y2H system,in which the LexA DNA-binding domain is fused to the C-terminus of a phytochrome protein.The conformational changes of phytochromes in response to R and FR light are achieved in yeast cells by exogenously supplying phycocyanobilin(PCB)extracted from Spirulina.The well-defined interaction pairs,including phyA-FHY1 and phyB-PIFs,are well reproducible in this system.Moreover,we show that our system is successful in detecting the phyA-COP1 and phyA-TZP interactions.Together,our study provides an alternative Y2H system that is highly sensitive and reproducible for detecting light-switchable interactions of phytochromes with their interacting partners.展开更多
文摘Comprehensive understanding of mechanisms of epigenetic regulation requires identification of molecules bound to genomic regions of interest in vivo. We have developed a novel method, insertional chromatin immunoprecipitatin (iChIP), to isolate specific genomic regions retaining molecular interaction in order to perform non-biased identification of interacting molecules in vivo. Here, we developed a second-generation tagged LexA DNA-binding domain, 3xFNLDD, for the iChIP analysis. 3xFNLDD consists of 3 x FLAG tags, a nuclear localization signal (NLS), the DNA-binding domain (DB) and the dimerization domain of the LexA protein. Expression of 3xFNLDD can be detected by immunoblot analysis as well as flowcytometry. We showed that iChIP using 3xFNLDD is able to consistently isolate more than 10% of input genomic DNA, several-fold more efficient compared to the first-generation tagged LexA DB. 3xFNLDD would be a useful tool to perform the iChIP analysis for locus-specific biochemical epigenetics.
基金Supported by the National Natural Science Foundation of China(Grant No.30330020),National Basic Research Program of China(Grant No.2004CB19604)and the State Funds for Distinguished Young Scientists of China(Grant No.30425038)
文摘LexA and RecA play a crucial role in SOS response in Escherichia coli.In classical SOS response,LexA functions as a repressor to regulate the expression of many genes including RecA after DNA damage occurs.Previous studies showed that LexA1 or LexA2 is not involved in RecA induction in Deinococ- cus radiodurans.In this study,we constructed a double mutant of lexA1 and lexA2.Results showed that mutation of lexA1 and lexA2 resulted in an apparent decrease in growth rate and an increased re- sistance to ultraviolet and hydrogen peroxide compared with wild type R1 and two single mutants. However,this double mutant did not exhibit any remarkably increased constitutive expression of RecA under normal conditions,just like wild type R1 and two single mutants,suggesting that neither LexA1 nor LexA2 is involved in the induced expression of RecA.Further transcriptional assay showed that LexA1 and LexA2 together participated in many regulatory pathways involved in cell division,protein synthesis,antioxidant process,transcription regulation and other mechanisms.These results indicate that there should be a new mechanism by which these damage-induced proteins are regulated in D. radiodurans.
基金by grants from the National Natural Science Foundation of China(31970262 and 31770321)Beijing Outstanding University Discipline Program,and the Recruitment Program of Global Youth Experts of China。
文摘Phytochromes are a family of photoreceptors in plants that perceive the red(R)and far-red(FR)components of their light environment.Phytochromes exist in vivo in two forms,the inactive Pr form and the active Pfr form,that are interconvertible by treatments with R or FR light.It is believed that phytochromes transduce light signals by interacting with their signaling partners.A GAL4-based lightswitchable yeast two-hybrid(Y2H)system was developed two decades ago and has been successfully employed in many studies to determine phytochrome interactions with their signaling components.However,several pairs of interactions between phytochromes and their interactors,such as the phyACOP1 and phyA-TZP interactions,were demonstrated by other assay systems but were not detected by this GAL4 Y2H system.Here,we report a modified LexA Y2H system,in which the LexA DNA-binding domain is fused to the C-terminus of a phytochrome protein.The conformational changes of phytochromes in response to R and FR light are achieved in yeast cells by exogenously supplying phycocyanobilin(PCB)extracted from Spirulina.The well-defined interaction pairs,including phyA-FHY1 and phyB-PIFs,are well reproducible in this system.Moreover,we show that our system is successful in detecting the phyA-COP1 and phyA-TZP interactions.Together,our study provides an alternative Y2H system that is highly sensitive and reproducible for detecting light-switchable interactions of phytochromes with their interacting partners.
