Project description:A series of dual-channel gene expression profiles obtained using Rosetta/Agilent Whole Mouse Genome oligonucleotide microarrays, 4 x 44K format, was used to identify sex-dependent and HNF4alpha-dependent differences in gene expression in adult mouse liver. This series is comprised of four sex-genotype combinations: adult male wild-type liver (M-WT), adult female wild-type liver (F-WT), adult male liver-specific HNF4alpha knockout liver (M-KO) and adult female liver-specific HNF4alpha knockout liver (F-KO). Four pools, each comprised of 4 randomly selected individual liver RNAs, were prepared for each sex-genotype combination. The pools were paired randomly to generate 4 separate experimental comparisons: M-WT:F-WT (first array comparison), M-WT:M-KO (second array comparison), F-WT:F-KO (third array comparison), and M-KO:F-KO (fourth array comparison). A total of 4994 HNF4alpha-dependent genes were identified, of which ~1000 fewer genes responded to the loss of HNF4alpha in female liver as compared to male liver. Moreover, 90% of the genes showing sex-specific expression in the liver were shown to lose sex specificity in HNF4alpha-deficient liver. Keywords: genetic knockout and sex response
Project description:Sex differences in liver gene expression are dictated by sex-differences in circulating growth hormone (GH) profiles. Presently, the pituitary hormone dependence of mouse liver gene expression was investigated on a global scale to discover sex-specific early GH response genes that might contribute to sex-specific regulation of downstream GH targets and to ascertain whether intrinsic sex-differences characterize hepatic responses to plasma GH stimulation. RNA expression analysis using 41,000-feature microarrays revealed two distinct classes of sex-specific mouse liver genes: genes subject to positive regulation (class-I) and genes subject to negative regulation by pituitary hormones (class-II). Genes activated or repressed in hypophysectomized (Hypox) mouse liver within 30-90min of GH pulse treatment at a physiological dose were identified as direct targets of GH action (early response genes). Intrinsic sex-differences in the GH responsiveness of a subset of these early response genes were observed. Notably, 45 male-specific genes, including five encoding transcriptional regulators that may mediate downstream sex-specific transcriptional responses, were rapidly induced by GH (within 30min) in Hypox male but not Hypox female mouse liver. The early GH response genes were enriched in 29 male-specific targets of the transcription factor Mef2, whose activation in hepatic stellate cells is associated with liver fibrosis leading to hepatocellular carcinoma, a male-predominant disease. Thus, the rapid activation by GH pulses of certain sex-specific genes is modulated by intrinsic sex-specific factors, which may be associated with prior hormone exposure (epigenetic mechanisms) or genetic factors that are pituitary-independent, and could contribute to sex-differences in predisposition to liver cancer or other hepatic pathophysiologies.
Project description:A series of dual-channel gene expression profiles obtained using Rosetta/Agilent Whole Mouse Genome oligonucleotide microarrays, 4 x 44K format, was used to identify sex-dependent and HNF4alpha-dependent differences in gene expression in adult mouse liver. This series is comprised of four sex-genotype combinations: adult male wild-type liver (M-WT), adult female wild-type liver (F-WT), adult male liver-specific HNF4alpha knockout liver (M-KO) and adult female liver-specific HNF4alpha knockout liver (F-KO). Four pools, each comprised of 4 randomly selected individual liver RNAs, were prepared for each sex-genotype combination. The pools were paired randomly to generate 4 separate experimental comparisons: M-WT:F-WT (first array comparison), M-WT:M-KO (second array comparison), F-WT:F-KO (third array comparison), and M-KO:F-KO (fourth array comparison). A total of 4994 HNF4alpha-dependent genes were identified, of which ~1000 fewer genes responded to the loss of HNF4alpha in female liver as compared to male liver. Moreover, 90% of the genes showing sex-specific expression in the liver were shown to lose sex specificity in HNF4alpha-deficient liver. Experiment Overall Design: An Alexa555-labeled cDNA sample is co-hybridized with an Alexa647-labeled cDNA sample. The samples are then dye-swapped and compared again on a second microarray chip. Together, these two mixed cDNA samples are considered a fluorescent reverse pair (dye swap). Similarly, a second fluorescent reverse pair is generated and the two pairs are averaged. The normalized expression ratio for each array is reported along with the two separate intensities. In this way, dye swaps were carried out for each of the four experimental comparisons. Thus, four microarrays, one for each mixed cDNA sample, were hybridized for each of the four fluorescent reverse pairs, giving a total of 16 microarrays.
Project description:We identified genes expressed in mouse liver that are regulated by Cux2, a highly female-specific liver transcription factor whose expression is regulated by sex-dependent plasma GH patterns. Using siRNA to knockdown Cux2 expression in female liver, we show that female specific genes are predominantly repressed by Cux2 knockdown. In contrast, similar numbers of male-biased genes are repressed as are induced by Cux2 knockdown. A scrambled, non-specific siRNA was used as a control. (Published in: TL Conforto et al 2012, Mol Cell Biol. 2012, 32:4611-4627. PubMed PMID: 22966202; PMCID: PMC3486175)
Project description:We identified genes expressed in mouse liver that are regulated by Cux2, a highly female-specific liver transcription factor whose expression is regulated by sex-dependent plasma GH patterns. Using adenovirus to overexpress Cux2 (Adeno-Cux2) in male liver, we show that Cux2 represses ~35% of male-biased genes and induces/de-represses ~35% of female-biased genes. Adeno-CMV was used as a control for adenoviral infection. (Published in: TL Conforto et al 2012, Mol Cell Biol. 2012, 32:4611-4627. PubMed PMID: 22966202; PMCID: PMC3486175)
Project description:Microarray analysis of male and female CD-1 mouse liver was carried out at 3, 4, and 8 wk of age to elucidate developmental changes in gene expression from the pre-pubertal period to young adulthood. A large number of sex-biased and sex-independent genes showed significant changes during this developmental period. Notably, sex-independent genes involved in cell cycle, chromosome condensation, and DNA replication were down regulated from 3 wk to 8 wk, while genes associated with metal ion binding, ion transport and kinase activity were up regulated. A majority of genes showing sex differential expression in adult liver did not display sex differences prior to puberty, at which time extensive changes in sex-specific gene expression were seen, primarily in males. Thus, in male liver, 76% of male-specific genes were up regulated and 47% of female-specific genes were down regulated from 3 to 8 wk of age, whereas in female liver 67% of sex-specific genes showed no significant change in expression. In both sexes, genes up regulated from 3 to 8 wk were significantly enriched (p < E-76) in the set of genes positively regulated by the liver transcription factor HNF4α, as determined in a liver-specific HNF4α knockout mouse model, while genes down regulated during this developmental period showed significant enrichment (p < E-65) for negative regulation by HNF4α. Significant enrichment of the developmentally regulated genes in genes subject to positive and negative regulation by pituitary hormone was also observed. Nine sex-specific transcription factors showed pubertal changes in expression and may contribute to the developmental changes that onset after 3-4 wk. Overall, the observed changes in gene expression during postnatal liver development reflect the deceleration of liver growth and the induction of specialized liver functions, with widespread changes in sex-specific gene expression primarily occurring in male liver.
Project description:PURPOSE: To provide a detailed gene expression profile of the normal postnatal mouse cornea. METHODS: Serial analysis of gene expression (SAGE) was performed on postnatal day (PN)9 and adult mouse (6 week) total corneas. The expression of selected genes was analyzed by in situ hybridization. RESULTS: A total of 64,272 PN9 and 62,206 adult tags were sequenced. Mouse corneal transcriptomes are composed of at least 19,544 and 18,509 unique mRNAs, respectively. One third of the unique tags were expressed at both stages, whereas a third was identified exclusively in PN9 or adult corneas. Three hundred thirty-four PN9 and 339 adult tags were enriched more than fivefold over other published nonocular libraries. Abundant transcripts were associated with metabolic functions, redox activities, and barrier integrity. Three members of the Ly-6/uPAR family whose functions are unknown in the cornea constitute more than 1% of the total mRNA. Aquaporin 5, epithelial membrane protein and glutathione-S-transferase (GST) omega-1, and GST alpha-4 mRNAs were preferentially expressed in distinct corneal epithelial layers, providing new markers for stratification. More than 200 tags were differentially expressed, of which 25 mediate transcription. CONCLUSIONS: In addition to providing a detailed profile of expressed genes in the PN9 and mature mouse cornea, the present SAGE data demonstrate dynamic changes in gene expression after eye opening and provide new probes for exploring corneal epithelial cell stratification, development, and function and for exploring the intricate relationship between programmed and environmentally induced gene expression in the cornea. Keywords: other