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Sexually Dimorphic Expression of eGFP Transgene in the Akr1A1 Locus of Mouse Liver Regulated by Sex Hormone-Related Epigenetic Remodeling.

Lai CW, Chen HL, Tsai TC, Chu TW, Yang SH, Chong KY, Chen CM - Sci Rep (2016)

Bottom Line: However, the mechanism of sexually dimorphic expression is still not fully understood.In this study, a pCAG-eGFP transgenic mouse strain with a specific transgene integration site in the Akr1A1 locus presented male-biased EGFP expression in the liver, and the expression was activated by testosterone during puberty.The integration of the pCAG-eGFP transgene altered the epigenetic regulation of the adjacent chromatin, including increased binding of STAT5b, a sexually dimorphic expression regulator, and the transformation of DNA methylation from hypermethylation into male-biased hypomethylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, and Agricultural Biotechnology Center, National Chung Hsing University, Taichung 402, Taiwan.

ABSTRACT
Sexually dimorphic gene expression is commonly found in the liver, and many of these genes are linked to different incidences of liver diseases between sexes. However, the mechanism of sexually dimorphic expression is still not fully understood. In this study, a pCAG-eGFP transgenic mouse strain with a specific transgene integration site in the Akr1A1 locus presented male-biased EGFP expression in the liver, and the expression was activated by testosterone during puberty. The integration of the pCAG-eGFP transgene altered the epigenetic regulation of the adjacent chromatin, including increased binding of STAT5b, a sexually dimorphic expression regulator, and the transformation of DNA methylation from hypermethylation into male-biased hypomethylation. Through this de novo sexually dimorphic expression of the transgene, the Akr1A1(eGFP) mouse provides a useful model to study the mechanisms and the dynamic changes of sexually dimorphic gene expression during either development or pathogenesis of the liver.

No MeSH data available.


Related in: MedlinePlus

The integration of the pCAG-eGFP transgene activated sexually dimorphic DNA methylation and binding of STAT5b in the 5′ adjacent sequence of the transgene.(A) A proportional map of the CpG sites (vertical lines) in the 4 kb sequence upstream of the eGFP translation start site (+1). The CpG sites are divided into 5 regions, with R1-R4 located in the endogenous mouse genome and R5 located in the CAG promoter of the transgene. The vertical arrowheads represent the locations of the putative STAT5b binding motifs. (B) DNA methylation of the R1-R5 regions was analyzed by bisulfite sequencing. Scaled lollipop diagrams show the DNA methylation of the CpG sites in the R1-R5 regions of Akr1A1eGFP/+ mice and the R4 region of Akr1A1eGFP/eGFP mice. The black and white circles represent methylated and unmethylated CpG sites, respectively, and the gray circles represent indiscriminate sequencing signals. The triangles represent the CpG sites (at −2,178 nt and −2,185 nt) that exhibit sexually dimorphic DNA methylation in the Tg mouse livers. (C) The percentage of the CpG site (−2,178 nt and −2,185 nt) methylation in the livers of WT and the Tg mice. F: female; M: male. The data were analyzed by Fisher’s exact test; #P < 0.01 between male and female or Akr1A1eGFP/+ and Akr1A1eGFP/eGFP male mice. (D) The STAT5b binding in the R4 and R5 regions in the Akr1A1eGFP/+ mouse livers. The bars show the mean ± s.e.m. of five animals per group (n = 5); data were analyzed by a one-way ANOVA with post hoc comparisons using Duncan’s new multiple range test, and *represents significant differences (P < 0.05). (E,F) The STAT5b binding in the R4 and R5 regions in WT, Akr1A1eGFP/eGFP and Akr1A1eGFP/+ mice. The bars show the mean ± s.e.m. of six animals (n = 6), including three males and three females; data were analyzed by one-tailed Student’s t-test; *P < 0.05 and **P < 0.01 vs. WT or the Akr1A1eGFP/+ group.
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f7: The integration of the pCAG-eGFP transgene activated sexually dimorphic DNA methylation and binding of STAT5b in the 5′ adjacent sequence of the transgene.(A) A proportional map of the CpG sites (vertical lines) in the 4 kb sequence upstream of the eGFP translation start site (+1). The CpG sites are divided into 5 regions, with R1-R4 located in the endogenous mouse genome and R5 located in the CAG promoter of the transgene. The vertical arrowheads represent the locations of the putative STAT5b binding motifs. (B) DNA methylation of the R1-R5 regions was analyzed by bisulfite sequencing. Scaled lollipop diagrams show the DNA methylation of the CpG sites in the R1-R5 regions of Akr1A1eGFP/+ mice and the R4 region of Akr1A1eGFP/eGFP mice. The black and white circles represent methylated and unmethylated CpG sites, respectively, and the gray circles represent indiscriminate sequencing signals. The triangles represent the CpG sites (at −2,178 nt and −2,185 nt) that exhibit sexually dimorphic DNA methylation in the Tg mouse livers. (C) The percentage of the CpG site (−2,178 nt and −2,185 nt) methylation in the livers of WT and the Tg mice. F: female; M: male. The data were analyzed by Fisher’s exact test; #P < 0.01 between male and female or Akr1A1eGFP/+ and Akr1A1eGFP/eGFP male mice. (D) The STAT5b binding in the R4 and R5 regions in the Akr1A1eGFP/+ mouse livers. The bars show the mean ± s.e.m. of five animals per group (n = 5); data were analyzed by a one-way ANOVA with post hoc comparisons using Duncan’s new multiple range test, and *represents significant differences (P < 0.05). (E,F) The STAT5b binding in the R4 and R5 regions in WT, Akr1A1eGFP/eGFP and Akr1A1eGFP/+ mice. The bars show the mean ± s.e.m. of six animals (n = 6), including three males and three females; data were analyzed by one-tailed Student’s t-test; *P < 0.05 and **P < 0.01 vs. WT or the Akr1A1eGFP/+ group.

Mentions: The DNA methylation status of the 4-kb sequence upstream of the eGFP translation start site, which includes the CAG promoter of the transgene, was analyzed by bisulfite sequencing in the Tg mouse liver (Fig. 7A). A total of 32 CpG sites, which were divided into 5 regions (R1-R5), were analyzed, and the results showed that the hypermethylated CpG sites were mainly present in the 5′ adjacent regions (R1-R4) and the hypomethylated CpG sites were in the CAG promoter (R5) (Fig. 7B) in both the male and female mice. In addition, two CpG sites at −2,185 nt and −2,178 nt in the R4 region, which are close to the CAG promoter, display sexually dimorphic methylation (Fig. 7B,C). The DNA methylation status of the two CpG sites was 55% and 25% in the Akr1A1eGFP/+ and Akr1A1eGFP/eGFP male mice, respectively; these values were significantly lower than the approximately 100% methylation status of the CpG sites in both sexes of the wild-type (WT) and in the female Tg mice (P < 0.01) (Fig. 7C). Furthermore, the sexually dimorphic DNA methylation was also abolished in the mice that were gonadectomized before puberty and 100% methylation was found in both CX and OVX Tg mouse livers (Fig. 7C). Thus, combining the results of the EGFP expression with the DNA methylation status, the sex hormones, most likely testosterone, influenced the epigenetic environment of the transgene integration region in male mice during puberty and may have consequently activated the sexually dimorphic EGFP expression in the Tg mouse liver.


Sexually Dimorphic Expression of eGFP Transgene in the Akr1A1 Locus of Mouse Liver Regulated by Sex Hormone-Related Epigenetic Remodeling.

Lai CW, Chen HL, Tsai TC, Chu TW, Yang SH, Chong KY, Chen CM - Sci Rep (2016)

The integration of the pCAG-eGFP transgene activated sexually dimorphic DNA methylation and binding of STAT5b in the 5′ adjacent sequence of the transgene.(A) A proportional map of the CpG sites (vertical lines) in the 4 kb sequence upstream of the eGFP translation start site (+1). The CpG sites are divided into 5 regions, with R1-R4 located in the endogenous mouse genome and R5 located in the CAG promoter of the transgene. The vertical arrowheads represent the locations of the putative STAT5b binding motifs. (B) DNA methylation of the R1-R5 regions was analyzed by bisulfite sequencing. Scaled lollipop diagrams show the DNA methylation of the CpG sites in the R1-R5 regions of Akr1A1eGFP/+ mice and the R4 region of Akr1A1eGFP/eGFP mice. The black and white circles represent methylated and unmethylated CpG sites, respectively, and the gray circles represent indiscriminate sequencing signals. The triangles represent the CpG sites (at −2,178 nt and −2,185 nt) that exhibit sexually dimorphic DNA methylation in the Tg mouse livers. (C) The percentage of the CpG site (−2,178 nt and −2,185 nt) methylation in the livers of WT and the Tg mice. F: female; M: male. The data were analyzed by Fisher’s exact test; #P < 0.01 between male and female or Akr1A1eGFP/+ and Akr1A1eGFP/eGFP male mice. (D) The STAT5b binding in the R4 and R5 regions in the Akr1A1eGFP/+ mouse livers. The bars show the mean ± s.e.m. of five animals per group (n = 5); data were analyzed by a one-way ANOVA with post hoc comparisons using Duncan’s new multiple range test, and *represents significant differences (P < 0.05). (E,F) The STAT5b binding in the R4 and R5 regions in WT, Akr1A1eGFP/eGFP and Akr1A1eGFP/+ mice. The bars show the mean ± s.e.m. of six animals (n = 6), including three males and three females; data were analyzed by one-tailed Student’s t-test; *P < 0.05 and **P < 0.01 vs. WT or the Akr1A1eGFP/+ group.
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f7: The integration of the pCAG-eGFP transgene activated sexually dimorphic DNA methylation and binding of STAT5b in the 5′ adjacent sequence of the transgene.(A) A proportional map of the CpG sites (vertical lines) in the 4 kb sequence upstream of the eGFP translation start site (+1). The CpG sites are divided into 5 regions, with R1-R4 located in the endogenous mouse genome and R5 located in the CAG promoter of the transgene. The vertical arrowheads represent the locations of the putative STAT5b binding motifs. (B) DNA methylation of the R1-R5 regions was analyzed by bisulfite sequencing. Scaled lollipop diagrams show the DNA methylation of the CpG sites in the R1-R5 regions of Akr1A1eGFP/+ mice and the R4 region of Akr1A1eGFP/eGFP mice. The black and white circles represent methylated and unmethylated CpG sites, respectively, and the gray circles represent indiscriminate sequencing signals. The triangles represent the CpG sites (at −2,178 nt and −2,185 nt) that exhibit sexually dimorphic DNA methylation in the Tg mouse livers. (C) The percentage of the CpG site (−2,178 nt and −2,185 nt) methylation in the livers of WT and the Tg mice. F: female; M: male. The data were analyzed by Fisher’s exact test; #P < 0.01 between male and female or Akr1A1eGFP/+ and Akr1A1eGFP/eGFP male mice. (D) The STAT5b binding in the R4 and R5 regions in the Akr1A1eGFP/+ mouse livers. The bars show the mean ± s.e.m. of five animals per group (n = 5); data were analyzed by a one-way ANOVA with post hoc comparisons using Duncan’s new multiple range test, and *represents significant differences (P < 0.05). (E,F) The STAT5b binding in the R4 and R5 regions in WT, Akr1A1eGFP/eGFP and Akr1A1eGFP/+ mice. The bars show the mean ± s.e.m. of six animals (n = 6), including three males and three females; data were analyzed by one-tailed Student’s t-test; *P < 0.05 and **P < 0.01 vs. WT or the Akr1A1eGFP/+ group.
Mentions: The DNA methylation status of the 4-kb sequence upstream of the eGFP translation start site, which includes the CAG promoter of the transgene, was analyzed by bisulfite sequencing in the Tg mouse liver (Fig. 7A). A total of 32 CpG sites, which were divided into 5 regions (R1-R5), were analyzed, and the results showed that the hypermethylated CpG sites were mainly present in the 5′ adjacent regions (R1-R4) and the hypomethylated CpG sites were in the CAG promoter (R5) (Fig. 7B) in both the male and female mice. In addition, two CpG sites at −2,185 nt and −2,178 nt in the R4 region, which are close to the CAG promoter, display sexually dimorphic methylation (Fig. 7B,C). The DNA methylation status of the two CpG sites was 55% and 25% in the Akr1A1eGFP/+ and Akr1A1eGFP/eGFP male mice, respectively; these values were significantly lower than the approximately 100% methylation status of the CpG sites in both sexes of the wild-type (WT) and in the female Tg mice (P < 0.01) (Fig. 7C). Furthermore, the sexually dimorphic DNA methylation was also abolished in the mice that were gonadectomized before puberty and 100% methylation was found in both CX and OVX Tg mouse livers (Fig. 7C). Thus, combining the results of the EGFP expression with the DNA methylation status, the sex hormones, most likely testosterone, influenced the epigenetic environment of the transgene integration region in male mice during puberty and may have consequently activated the sexually dimorphic EGFP expression in the Tg mouse liver.

Bottom Line: However, the mechanism of sexually dimorphic expression is still not fully understood.In this study, a pCAG-eGFP transgenic mouse strain with a specific transgene integration site in the Akr1A1 locus presented male-biased EGFP expression in the liver, and the expression was activated by testosterone during puberty.The integration of the pCAG-eGFP transgene altered the epigenetic regulation of the adjacent chromatin, including increased binding of STAT5b, a sexually dimorphic expression regulator, and the transformation of DNA methylation from hypermethylation into male-biased hypomethylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, and Agricultural Biotechnology Center, National Chung Hsing University, Taichung 402, Taiwan.

ABSTRACT
Sexually dimorphic gene expression is commonly found in the liver, and many of these genes are linked to different incidences of liver diseases between sexes. However, the mechanism of sexually dimorphic expression is still not fully understood. In this study, a pCAG-eGFP transgenic mouse strain with a specific transgene integration site in the Akr1A1 locus presented male-biased EGFP expression in the liver, and the expression was activated by testosterone during puberty. The integration of the pCAG-eGFP transgene altered the epigenetic regulation of the adjacent chromatin, including increased binding of STAT5b, a sexually dimorphic expression regulator, and the transformation of DNA methylation from hypermethylation into male-biased hypomethylation. Through this de novo sexually dimorphic expression of the transgene, the Akr1A1(eGFP) mouse provides a useful model to study the mechanisms and the dynamic changes of sexually dimorphic gene expression during either development or pathogenesis of the liver.

No MeSH data available.


Related in: MedlinePlus