Tissue-specific gene expression in mouse hepatocytes cultured in growth-restricting medium.

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A hybrid gene was made by fusing the 2.2-kilobase 5' promoter region of a mouse group 1 major urinary protein (Mup) gene to the coding region of the herpes simplex virus type 1 thymidine kinase gene (HSV tk) and introduced into the genomes of mice by microinjection. Transgenic Go males were sterile, or when fertile did not transmit the foreign gene, and the transgenic male descendants of Go females were also sterile. Seven "lines" were established by breeding from Go females and their transgenic female descendants. Six lines expressed HSV thymidine kinase activity in the liver, and activity correlated perfectly with the presence of HSV tk RNA. In three of four lines examined, expression was lower in female than in male liver, and in these lines the same sex difference was observed in the rate of run-on transcription of the foreign genes in liver nuclei. When females of one of the sexually dimorphic lines were treated with testosterone, the levels of HSV tk RNA and thymidine kinase activity were increased, although not to male levels. In these aspects of liver expression, and also in a lack of expression in seven other tissues, the hybrid gene exhibits many of the characteristics of an endogenous group 1 Mup gene. However, the gene was also expressed (at high levels) in the preputial gland and testis, two tissues in which Mup genes are not expressed. The gene, when introduced into five of the seven lines, carried a copy of the Escherichia coli supF gene attached beyond the 3' end of the HSV tk gene, but this did not affect the overall expression pattern. All of the lines were male sterile and expressed HSV thymidine kinase in the testis, but one line showed no activity in the liver, and another showed none in the preputial gland. Testicular expression is therefore the likely cause of sterility. Data are described which suggest that the causes of misexpression in the preputial gland and testis are different. Since expression in each tissue occurred in several lines, the structure of the hybrid gene must be responsible in each case. Five intensively studied lines showed at least four consistently different patterns of relative expression in preputial gland, testis, male liver, and female liver. These differences do not correlate in any way with the copy number of the foreign gene in the different lines and must be due to some other aspect of line-specific integration.The mouse major urinary proteins (MUPs) are coded for by a family of about 35 genes clustered on chromosome 4 (3, 12, 23). Four groups of Mup genes have been identified, the largest of which are the group 1 genes and the probably inert group 2 pseudogenes (3, 12, 25, 35, 51; R. Al-Shawi, P. Ghazal, A. J. Clark, and J. 0. Bishop, submitted for publication). Most group 1 genes are expressed exclusively in the liver, while a small subset are also expressed in the mammary glands of pregnant females (50, 52). Of the two smaller groups, the group 3 genes are also expressed in the liver, but under hormonal control different from the group 1 genes (35), and...

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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A Mup promoter-thymidine kinase reporter gene shows relaxed tissue-specific expression and confers male sterility upon transgenic mice.

Al‐Shawi¹,

Burke²,

Jones³

et al. 1988

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“…Although several previous studies have shown that variations in apoAl gene expression in response to developmental (17,22), differentiation (2,32), and hormonal (1, 34) factors and various pharmacologic (33) and toxic (21) transactivates the basal promoter, binding of ARP-1 does not; it is important to emphasize that RXRa/ARP-1 heterodimers also bind to site A with an affinity (Kd = 0.6 nM) approximately 10 times greater than the affinity of either ARP-1 (Kd = 5.2 nM) or RXRa (Kd = 8.8 nM) alone. Furthermore, under cotransfection conditions favoring a greater intracellular concentration of RXRa/ARP-1 heterodimers over the concentration of ARP-1, the expression of the site A containing basal apoAI promoter in response to RXRoa and RA is significantly reduced compared with its expression in the absence of ARP-1.…”

Section: Discussionmentioning

confidence: 99%

Repression by ARP-1 sensitizes apolipoprotein AI gene responsiveness to RXR alpha and retinoic acid.

Widom¹,

Rhee²,

Karathanasis³

1992

136

The gene coding for apolipoprotein AI (apoAl), a lipid binding protein involved in the transport of cholesterol and other lipids in the plasma, is expressed in mammals predominantly in the liver and the intestine.Liver-specific expression is controlled by synergistic interactions between transcription factors bound to three separate sites, sites A (-214 to -192), B (-169 to -146), and C (-134 to -119), within a powerful liver-specific enhancer located between nucleotides -222 and -110 upstream of the apoAl gene transcription start site (+1). Previous studies in our laboratory have shown that ARP-1, a member of the nuclear receptor superfamily whose ligand is unknown (orphan receptor), binds to site A and represses transcription of the apoAI gene in liver cells. In a more recent series of experiments, we found that site A is a retinoic acid (RA) response element that responds preferentially to the recently identified RA-responsive receptor RXRea over the previously characterized RA receptors RARae and RARI3. In this study we investigated the combined effects of ARP-1 and RXRa on apoAl gene expression in liver cells. Transient transfection assays showed that site A is necessary and sufficient for RXRa-mediated transactivation of the apoAl gene basal promoter in human hepatoma HepG2 cells in the presence of RA and that this transactivation is abolished by increasing amounts of cotransfected ARP-1. Electrophoretic mobility shift assays and subsequent Scatchard analysis of the data revealed that ARP-1 and RXRoa bind to site A with similar affinities. These assays also revealed that ARP-1 and RXRai bind to site A as heterodimers with an affinity approximately 10 times greater than that of either ARP-1 or RXRa alone. Further transfection assays in HepG2 cells, using as a reporter a construct containing the apoAl gene basal promoter and its upstream regulatory elements (including site A) in their natural context, revealed that RXRai has very little effect on the levels of expression regardless of the presence or absence of RA. However, while ARP-1 alone or ARP-1 and RXRa together dramatically repress expression in the absence of RA, the repression by ARP-1 and RXRai together, but not ARP-1 alone, is almost completely alleviated in the presence of RA. These results indicate that transcriptional repression by ARP-1 sensitizes apoAl gene responsiveness to RXRa and RA and suggest that the magnitude of this responsiveness is regulated by the intracellular ratio of ARP-1 to RXRa. These observations raise the possibility that transcriptional repression is a general mechanism for switching gene transcription between alternative transcription activation pathways.A large body of epidemiologic, genetic, pharmacologic, and biochemical evidence suggests that high-density lipoprotein (HDL) levels in plasma play an important role in regulation of cellular cholesterol homeostasis and atherosclerosis progression and regression. Although changes in plasma HDL levels have been associated with a diverse number of dietary, hormonal, and str...

“…The group 1 liver MUPs are secreted into the plasma and when excreted make up most if not all of the urinary MUP. The expression of the group 1 genes is strongly dependent on testosterone, thyroxine, and growth hormone (9,17,23,29,30). The dependency on testosterone leads to sexual dimorphism in Mup expression, and female expression can be induced to male levels by testosterone induction (9).…”

mentioning

confidence: 99%

Differential expression in male and female mouse liver of very similar mRNAs specified by two group 1 major urinary protein genes.

McIntosh

Bishop

1989

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The major urinary proteins of the mouse are encoded by a large multigene family composed of several distinct groups of genes distinguished by differences in sequence and expression characteristics. The genes in the largest group (group 1) show greater than 99% pairwise similarity in their exons. By hybridization between RNA and a specifically designed oligonucleotide, we confirmed that genes of this group are expressed mainly in the liver. By using additional gene-specific oligonucleotide probes, we have been able to distinguish between the species of mRNA corresponding to two of these genes and to measure their abundance in male and female liver. Both mRNAs are present in male liver at high but different levels. Both are also present in female liver, one at a much lower level than in the male and the second at a very low level indeed. Both are present at male levels in the livers of females induced with testosterone. These results show unequivocally that the expression of different group 1 Mup genes is differentially influenced by the hormonal status of the mouse.