Identification of a growth hormone gene promoter repressor element and its cognate double- and single-stranded DNA-binding proteins

In previous investigations, cell fusion was found to silence either the endogenous rat growth hormone (GH) gene or a transfected rat GH gene promoter, implying that repression plays a role in regulation of this gene. To search the rat GH gene promoter for repressor sequences, a series of 5'-deleted GH-CAT constructs was analyzed by transient expression in GH3 rat pituitary cells. Deletion of either a distal region between positions -307/-244 or a proximal sequence between -169/-152 increased CAT enzymatic activity by 3-4-fold. Since the action of the proximal repressor element (PRE) at -169/-152 was serum-independent, and the element is located between two strong positive elements, the PRE and its cognate binding proteins were further analyzed. A 5-base pair sequence centered at -163 is critical for PRE repressor activity, since mutation of this sequence in GH-CAT constructs yielded 6-11-fold increases in expression in GH3 cells. Although the PRE is adjacent to the GH thyroid hormone (T3) response region, they are distinct elements, since the PRE mutation has little effect on the T3 response of GH-CAT constructs. Nuclear extracts of 10 cell lines were searched by DNA mobility shift for protein(s) binding specifically to a double-stranded PRE probe. No such protein was detected in any of four rodent pituitary cell lines or three human cell lines. However, three different rodent non-pituitary cell lines yielded a common shifted band, corresponding to a DNA sequence-specific PRE-binding protein (PREB). Similar analysis with the coding strand of the PRE detected no shifted band in any of these cell lines. However, the PRE noncoding strand yielded a common shifted band in all of the cell lines, corresponding to a ubiquitous, strand-specific, single-stranded PRE-binding protein (ssPREB). Mutation of the PRE permitted ssPREB binding to the coding strand, implying that the wild-type coding strand somehow excludes ssPREB binding. That PREB and ssPREB are distinct proteins was confirmed by the inability of their DNA binding sites to cross-compete binding of the proteins.     

Tissue-specific expression is conferred by a sequence from the 5'' end of the rat albumin gene.

We have constructed a transient expression vector containing 400 bp of rat albumin gene immediate 5'-flanking sequences inserted 5' to the bacterial enzyme chloramphenicol acetyl transferase (CAT). We have transfected various clones of rat hepatoma cells representing different states of expression of the liver phenotype with this vector (pALB-cat) and also with two control vectors containing viral promoters (pSVE-cat and pRSV-cat), and measured activity of the bacterial enzyme CAT in cellular extracts 48 h later. The albumin flanking sequences are able to direct highly efficient CAT expression, compared with the control vectors, only in cells which express their own albumin gene: the albumin-negative hepatoma cells are at least 100 times less efficient in expressing CAT after transfection with the pALB-cat plasmid than are the albumin-positive ones. An unexpected result of our study is the total inability of the rat albumin flanking sequences to direct expression in albumin-producing mouse hepatoma cells.     

Retina-specific expression from the IRBP promoter in transgenic mice is conferred by 212 bp of the 5'-flanking region

IRBP is a photoreceptor-specific glycoprotein that has been suggested as a retinoid carrier in the visual process. Previous research has shown that 1.3 kb of 5'-flanking sequence from the human IRBP gene is sufficient to promote photoreceptor-specific expression of reporter genes in transgenic mice. To define more narrowly the sequences that promote tissue-specific expression, chimeric constructs with shorter promoters were used to generate transgenic mice. The bacterial CAT gene was fused to fragments of 706 bp or 212 bp from the 5' end of the human IRBP gene. Analysis of the three transgenic families bearing the 706 bp IRBP promoter revealed that CAT expression was confined to the neuro-retina and the pineal gland. Analysis of the four transgenic families bearing the 212 bp IRBP promoter revealed the same tissue-specific CAT expression in three families. These results establish that tissue-specific expression of IRBP can be regulated by a short 212 bp promoter which has been conserved between humans and mice.