The involvement of upstream stimulatory factor 1 in Dutch patients with familial combined hyperlipidemia

Recently, the upstream stimulatory factor 1 gene (USF1) was proposed as a candidate gene for familial combined hyperlipidemia (FCH). In this study, we examined the previously identified risk haplotype of USF1 with respect to FCH and its related phenotypes in 36 Dutch FCH families. The diagnosis of FCH was based on both the traditional diagnostic criteria and a nomogram. The two polymorphisms, USF1s1 and USF1s2, were in complete linkage disequilibrium. No association was found for the individual single nucleotide polymorphisms (SNPs) with FCH defined by the nomogram (USF1s1, P = 0.53; USF1s2, P = 0.53), whereas suggestive associations were found when using the traditional diagnostic criteria for FCH (USF1s1, P = 0.08; USF1s2, P = 0.07). USF1 was associated with total cholesterol (USF1s1, P = 0.05; USF1s2, P = 0.04) and apolipoprotein B (USF1s1, P = 0.06; USF1s2, P = 0.04). Small dense LDL showed a suggestive association (USF1s1, P = 0.10; USF1s2, P = 0.09). The results from the haplotype analyses supported the results obtained for the individual SNPs. In conclusion, the previously identified risk haplotype of USF1 showed a suggestive association with FCH and contributed to the related lipid traits in our Dutch FCH families.     

Upstream stimulatory factor induces Nr5a1 and Shbg gene expression during the onset of rat Sertoli cell differentiation

Within the testis, each Sertoli cell can support a finite number of developing germ cells. During development, the cessation of Sertoli cell proliferation and the onset of differentiation establish the final number of Sertoli cells and, thus, the total number of sperm that can be produced. The upstream stimulatory factors 1 and 2 (USF1 and USF2, respectively) differentially regulate numerous Sertoli cell genes during differentiation. To identify genes that are activated by USF proteins during differentiation, studies were conducted in Sertoli cells isolated from 5- and 11-day-old rats, representing proliferating and differentiating cells, respectively. Usf1 mRNA and USF1 protein levels were increased between 5 and 11 days after birth. In vitro studies revealed that USF1 and USF2 DNA-binding activity also increased at 11 days for the promoters of four potential target genes, Fshr, Gata4, Nr5a1, and Shbg. Chromatin immunoprecipitation assays confirmed that USF recruitment increased in vivo between 5 and 11 days after birth at the Fshr, Gata4, and Nr5a1 promoters. Expression of Nr5a1 and Shbg, but not of Fshr or Gata4, mRNAs was elevated in 11-day-old Sertoli cells compared with 5-day-old Sertoli cells. Transient transfection of USF1 and USF2 expression vectors up-regulated Nr5a1 and Shbg promoter activity. RNA interference assays demonstrated that USF1 and USF2 contribute to Nr5a1 and Shbg expression in differentiating cells. Together, these data indicate that increased USF levels induce the expression of Nr5a1 and Shbg during the differentiation of Sertoli cells, whereas Fshr and Gata4 expression is not altered by USF proteins during differentiation.     

SREBP-1 dimerization specificity maps to both the helix-loop-helix and leucine zipper domains: use of a dominant negative

The mammalian SREBP family contains two genes that code for B-HLH-ZIP proteins that bind sequence-specific DNA to regulate the expression of genes involved in lipid metabolism. We have designed a dominant negative (DN), termed A-SREBP-1, that inhibits the DNA binding of either SREBP protein. A-SREBP-1 consists of the dimerization domain of B-SREBP-1 and a polyglutamic acid sequence that replaces the basic region. A-SREBP-1 heterodimerizes with either B-SREBP-1 or B-SREBP-2, and both heterodimers are more stable than B-SREBP-1 bound to DNA. Circular dichroism thermal denaturation studies show that the B-SREBP-1.A-SREBP-1 heterodimer is -9.8 kcal mol(-1) dimer(-1) more stable than the B-SREBP-1 homodimer. EMSA assays demonstrate that A-SREBP-1 can inhibit the DNA binding of either B-SREBP-1 or B-SREBP-2 in an equimolar competition but does not inhibit the DNA binding of the three B-HLH-ZIP proteins MAX, USF, or MITF, even at 100 molar eq. Chimeric proteins containing the HLH domain of SREBP-1 and the leucine zipper from either MAX, USF, or MITF indicate that both the HLH and leucine zipper regions of SREBP-1 contribute to its dimerization specificity. Transient co-transfection studies demonstrate that A-SREBP-1 can inhibit the transactivation of SREBP-1 and SREBP-2 but not USF. A-SREBP-1 may be useful in metabolic diseases where SREBP family members are overexpressed.