POU同源域蛋白的结构及发育中的功能

POU同源域蛋白含有两个保守的亚结构域以及它们之间有变化的连接区.两个亚结构域与DNA相互作用,在连接区可塑性和辅因子的帮助下,POU蛋白作为调控因子和转录因子,显示出错综复杂的DNA结合和识别能力.在脊椎动物和无脊椎动物中,POU蛋白参与胚胎发生的早期过程,在细胞谱系的分化和神经发育中起调控作用.     

POU1F1-mediated activation of hGH-N by deoxyribonuclease I hypersensitive site II of the human growth hormone locus control region

The human growth hormone gene (hGH-N) is regulated by a distal locus control region (LCR) composed of five deoxyribonuclease I hypersensitive sites (HSs). The region encompassing HSI and HSII contains the predominant pituitary somatotrope-specific hGH-N activation function of the LCR. This activity was attributed primarily to POU1F1 (Pit-1) elements at HSI, as linkage to HSI was sufficient for properly regulated hGH-N expression in transgenic mice, while HSII alone had no activity. However, the presence of HSII in conjunction with HSI further enhanced hGH-N transgene expression, indicating additional determinants of pituitary hGH-N activation in the HSII region, but limitations of transgenic models and previous ex vivo systems have prevented the characterization of HSII. In the present study, we employ a novel minichromosome model of the hGH-N regulatory domain and show that HSII confers robust POU1F1-dependent activation of hGH-N in this system. This effect was accompanied by POU1F1-dependent histone acetylation and methylation throughout the minichromosome LCR/hGH-N domain. A series of in vitro DNA binding experiments revealed that POU1F1 binds to multiple sites at HSII, consistent with a direct role in HSII function. Remarkably, POU1F1 binding was localized in part to the 3' untranslated region of a primate-specific LINE-1 (long interspersed nuclear element 1) retrotransposon, suggesting that its insertion during primate evolution may have conferred function to the HSII region in the context of pituitary GH gene regulation. These observations clarify the function of HSII, expanding the role of POU1F1 in hGH LCR activity, and provide insight on the molecular evolution of the LCR.     

Purification of a protein doublet that binds to six TGG-containing sequences in the promoter for hamster 3-hydroxy-3-methylglutaryl-coenzyme A reductase

The gene for 3-hydroxy-3-methylglutaryl-coenzyme A reductase, the rate-controlling enzyme of cholesterol biosynthesis, is transcribed at a relatively high level when cellular sterols are depleted and is repressed when sterols accumulate. We have previously reported that the regulatory region of the hamster reductase gene contains eight different sequences that bind nuclear proteins as determined by DNase I footprinting assays. We here report the purification of a single activity that accounts for six of these footprints. This activity was found in a doublet of proteins (designated reductase promoter factor 1, RPF-1) that have apparent molecular weights of 33,000 and 35,000. They were isolated by DNA affinity chromatography using oligonucleotides corresponding to either of two footprinted sequences. The 33- and 35-kDa species were present as monomers, as indicated by gel filtration and gradient ultracentrifugation. Oligonucleotides corresponding to any one of the six footprinted sequences prevented the binding of RPF-1 to all of the other sequences, indicating that all six bind to a single site in RPF-1. The only sequence shared by all six footprinted sequences is the trinucleotide, TGG, both of whose guanosines made contact with RPF-1, as determined by methylation interference assays. The footprinted sequence that binds RPF-1 with highest affinity contains the palindrome, TGG(N7)CCA, which conforms to the consensus sequence for binding NF-1, a nuclear protein that stimulates replication of adeno-virus-2. Purified RPF-1 was shown to bind to the adenovirus NF-1 binding site with high affinity. Although the apparent molecular weight of the RPF-1 doublet was lower than the molecular weight range for NF-1 proteins (52,000-66,000), it is likely that the 33-35-kDa doublet is derived from a larger NF-1-like protein as a result of proteolysis. We conclude that RPF-1 belongs to a group of TGG-binding proteins that includes NF-1 and other proteins previously described as CCAAT binding proteins. This protein binds to six sites in the promoter region for hamster 3-hydroxy-3-methylglutaryl CoA reductase, where its function remains to be determined.