Lin28A and Lin28B are homologous RNA-binding proteins that participate in the development of primordial germ cells. The mechanisms underlying expression and regulation of Lin28A have been well documented, but such inf...Lin28A and Lin28B are homologous RNA-binding proteins that participate in the development of primordial germ cells. The mechanisms underlying expression and regulation of Lin28A have been well documented, but such information for Lin28B is limited. In this study, a fragment of the Lin28B promoter was cloned, the pEGFP-pLin28B vector was constructed. DF-1 chicken fibroblasts were transfected and the expression of green fluorescent protein (GFP) was measured. Furtherly, Lin28B promoter of different lengths fragments was cloned using the chromosome-walking method and the fragments were ligated into the PGL3-Basic vector, and transfected into DF-1 cells. Results of dual-luciferase reporter assay showed that the core of the Lin28B promoter was included in the sequence from –1 431 to –1 034 bp. The binding sites of the transcription factor TCF7L2 was showed within this sequence by bioinformatics analysis. The promoter activity of Lin28B was downregulated (P<0.05) when the TCF7L2 binding site was mutated. Further experiments suggested that Lin28B promoter activity responded to the activation or inhibition of Wnt signaling. Results of chromatin immunoprecipitation and quantitative PCR showed that β-catenin-TCF7L2 may be enriched in the Lin28B promoter core area. In vivo and in vitro activation or inhibition of Wnt signaling significantly up- or down-regulated (P<0.05) Lin28B expression. H3K4me2 enriched in the promoter of Lin28B, which affected the regulation of Wnt signaling to Lin28B. In conclusion, our results showed that H3K4me2 and Wnt5a/β-catenin/TCF7L2 were the positive regulators of Lin28B expression. Findings of this study may lay a theoretical foundation for illuminating the mechanism underlying Lin28B expression.展开更多
In a growing follicle,the survival and maturation of the oocyte largely depend on support from somatic cells to facilitate FSH-induced mutual signaling and chemical communication.Although apoptosis and autophagy in so...In a growing follicle,the survival and maturation of the oocyte largely depend on support from somatic cells to facilitate FSH-induced mutual signaling and chemical communication.Although apoptosis and autophagy in somatic cells are involved in the process of FSH-induced follicular development,the underlying mechanisms require substantial study.According to our study,along with FSH-induced antral follicles(AFs)formation,both lysine-specific demethylase 1(LSD1)protein levels and autophagy increased simultaneously in granulosa cells(GCs)in a time-dependent manner,we therefore evaluated the importance of LSD upon facilitating the formation of AFs correlated to autophagy in GCs.Conditional knockout of Lsdl in GCs resulted in significantly decreased AF number and subfertility in females,accompanied by marked suppression of the autophagy in GCs.On the one hand,depletion of Lsd1 resulted in accumulation of Wilms tumor 1 homolog(WT1),at both the protein and mRNA levels.WT1 prevented the expression of FSH receptor(Fshr)in GCs and thus reduced the responsiveness of the secondary follicles to FSH induction.On the other hand,depletion of LSD1 resulted in suppressed level of autophagy by upregulation of ATG16L2 in GCs.We finally approved that LSD1 contributed to these sequential activities in GCs through its H3K4me2 demethylase activity.Therefore,the importance of LSD1 in GCs is attributable to its roles in both accelerating autophagy and suppressing WT1 expression to ensure the responsiveness of GCs to FSH during AFs formation.展开更多
Histone methylation is believed to provide binding sites for specific reader proteins, which translate histone code into biological function. Here we show that a family of acidic domain-containing proteins including n...Histone methylation is believed to provide binding sites for specific reader proteins, which translate histone code into biological function. Here we show that a family of acidic domain-containing proteins including nucleophosmin （NPM 1）, pp32, SET/TAF 113, nucleolin （NCL） and upstream binding factor （UBF） are novel H3K4me2-binding proteins. These proteins exhibit a unique pattern of interaction with methylated H3K4, as their binding is stimulated by H3K4me2 and inhibited by H3K4mel and H3K4me3. These proteins contain one or more acidic domains consisting mainly of aspartic and/or glutamic residues that are necessary for preferential binding of H3K4me2. Furthermore, we demonstrate that the acidic domain with sufficient length alone is capable of binding H3K4me2 in vitro and in vivo. NPM1, NCL and UBF require their acidic domains for association with and transcriptional activation ofrDNA genes. Interestingly, by defining acidic domain as a sequence with at least 20 acidic residues in 50 continuous amino acids, we identified 655 acidic domain-containing protein coding genes in the human genome and Gene Ontology （GO） analysis showed that many of the acidic domain proteins have chromatin-related functions. Our data suggest that acidic domain is a novel histone binding motif that can differentially read the status of H3K4 methylation and is broadly present in chromatin-associated proteins.展开更多
基金We thank the Experimental Poultry Farm of the Poultry Institute,Chinese Academy of Agricultural Sciences,for providing experimental materialsThis work was supported by the Key Research and Development Program of China(2017YFE0108000)+1 种基金the National Natural Science Foundation of China(31872341,31572390)the High-Level Talent Support Program of Yangzhou University,China.
文摘Lin28A and Lin28B are homologous RNA-binding proteins that participate in the development of primordial germ cells. The mechanisms underlying expression and regulation of Lin28A have been well documented, but such information for Lin28B is limited. In this study, a fragment of the Lin28B promoter was cloned, the pEGFP-pLin28B vector was constructed. DF-1 chicken fibroblasts were transfected and the expression of green fluorescent protein (GFP) was measured. Furtherly, Lin28B promoter of different lengths fragments was cloned using the chromosome-walking method and the fragments were ligated into the PGL3-Basic vector, and transfected into DF-1 cells. Results of dual-luciferase reporter assay showed that the core of the Lin28B promoter was included in the sequence from –1 431 to –1 034 bp. The binding sites of the transcription factor TCF7L2 was showed within this sequence by bioinformatics analysis. The promoter activity of Lin28B was downregulated (P<0.05) when the TCF7L2 binding site was mutated. Further experiments suggested that Lin28B promoter activity responded to the activation or inhibition of Wnt signaling. Results of chromatin immunoprecipitation and quantitative PCR showed that β-catenin-TCF7L2 may be enriched in the Lin28B promoter core area. In vivo and in vitro activation or inhibition of Wnt signaling significantly up- or down-regulated (P<0.05) Lin28B expression. H3K4me2 enriched in the promoter of Lin28B, which affected the regulation of Wnt signaling to Lin28B. In conclusion, our results showed that H3K4me2 and Wnt5a/β-catenin/TCF7L2 were the positive regulators of Lin28B expression. Findings of this study may lay a theoretical foundation for illuminating the mechanism underlying Lin28B expression.
基金supported by the National Key Research&Developmental Program of China(2022YFC2703803,2018YFC1003700,2018YFC1003801,and 2023YFD1300501)the National Natural Science Foundation of China(32071132,31872792,32270904,32070839,32100913,82260291,and 32100686)+2 种基金China Postdoctoral Science Foundation(2021M700972)Institution of Higher Education Projects of Building First-class Discipline Construction in Ningxia Region(Biology)(NXYLXK2017B05)Doctoral Startup Foundation of Guizhou Medical University([2020]038)。
文摘In a growing follicle,the survival and maturation of the oocyte largely depend on support from somatic cells to facilitate FSH-induced mutual signaling and chemical communication.Although apoptosis and autophagy in somatic cells are involved in the process of FSH-induced follicular development,the underlying mechanisms require substantial study.According to our study,along with FSH-induced antral follicles(AFs)formation,both lysine-specific demethylase 1(LSD1)protein levels and autophagy increased simultaneously in granulosa cells(GCs)in a time-dependent manner,we therefore evaluated the importance of LSD upon facilitating the formation of AFs correlated to autophagy in GCs.Conditional knockout of Lsdl in GCs resulted in significantly decreased AF number and subfertility in females,accompanied by marked suppression of the autophagy in GCs.On the one hand,depletion of Lsd1 resulted in accumulation of Wilms tumor 1 homolog(WT1),at both the protein and mRNA levels.WT1 prevented the expression of FSH receptor(Fshr)in GCs and thus reduced the responsiveness of the secondary follicles to FSH induction.On the other hand,depletion of LSD1 resulted in suppressed level of autophagy by upregulation of ATG16L2 in GCs.We finally approved that LSD1 contributed to these sequential activities in GCs through its H3K4me2 demethylase activity.Therefore,the importance of LSD1 in GCs is attributable to its roles in both accelerating autophagy and suppressing WT1 expression to ensure the responsiveness of GCs to FSH during AFs formation.
基金supported by the Ministry of Science and Technology of China(2015CB910402)to Jiemin Wongthe National Natural Science Foundation of China(91419303)+1 种基金The Science and Technology Commission of Shanghai Municipality(14XD1401700,11DZ2260300)the National Science&Technology Major Project“Key New Drug Creation and Manufacturing Program”of China(2014ZX09507002-002)
文摘Histone methylation is believed to provide binding sites for specific reader proteins, which translate histone code into biological function. Here we show that a family of acidic domain-containing proteins including nucleophosmin （NPM 1）, pp32, SET/TAF 113, nucleolin （NCL） and upstream binding factor （UBF） are novel H3K4me2-binding proteins. These proteins exhibit a unique pattern of interaction with methylated H3K4, as their binding is stimulated by H3K4me2 and inhibited by H3K4mel and H3K4me3. These proteins contain one or more acidic domains consisting mainly of aspartic and/or glutamic residues that are necessary for preferential binding of H3K4me2. Furthermore, we demonstrate that the acidic domain with sufficient length alone is capable of binding H3K4me2 in vitro and in vivo. NPM1, NCL and UBF require their acidic domains for association with and transcriptional activation ofrDNA genes. Interestingly, by defining acidic domain as a sequence with at least 20 acidic residues in 50 continuous amino acids, we identified 655 acidic domain-containing protein coding genes in the human genome and Gene Ontology （GO） analysis showed that many of the acidic domain proteins have chromatin-related functions. Our data suggest that acidic domain is a novel histone binding motif that can differentially read the status of H3K4 methylation and is broadly present in chromatin-associated proteins.
