Reconstitution of cell-type-specific transcription of the rat prolactin gene in vitro.

Abstract

We present evidence for the existence of prolactin upstream factor I (PUF-I) in rat pituitary-derived cells and demonstrate its interaction with a symmetrical DNA element located in the 5' flanking region of the gene. An in vitro expression system developed from pituitary-derived Gil3 cells was used to determine that 420 base pairs (bp) of 5' flanking DNA was sufficient for cell-specific, accurate, and efficient RNA polymerase II transcription of the rat prolactin gene. Reconstitution of in vitro transcription with pituitary and nonpituitary nuclear extracts suggested that the presence of GH3 cell-specific factors mediated the activation of prolactin gene expression. We also demonstrated that a functionally stable transcription complex assembled on the prolactin promoter. Using DNase I protection procedures, we have identified the DNA-protein binding area in the prolactin 5' flanking region. Gil3 nuclear extracts contain a cell-specific protein (PUF-I) that binds to a 28.bp region (-63 to -36) which contains an 18-bp imperfect palindrome (-63 to -46). The role that the interaction between PUF-I and the imperfect palindrome plays in in vitro pituitary-specific prolactin gene expression is discussed.The prolactin gene is expressed primarily in mammalian pituitary lactotrophs and is regulated by inhibitory and stimulatory hormones from the brain and gonads (6) plus several other classes of factors including Ca2+ (12), phorbol esters (22), and cyclic AMP (16). Previous studies have suggested that most of these factors modulate prolactin production at the level of gene transcription (2,15,16,19,21). Regulation of gene activity is presumably mediated by the interaction of cis-acting promoter elements with transacting nuclear protein transcription factors (8). The 5' flanking prolactin DNA contains homologous sequences for several eucaryotic cis control elements including two TATA boxes at positions -28 to -23 and -142 to -136 and a CAAAT sequence from -192 to -187. In addition, there is an interesting A+T-rich 18-base-pair (bp) imperfect symmetrical sequence from -63 to -46 (4, 17).An investigation of the prolactin cis-acting elements by Nelson et al. (20), using gene transfer methods, suggested the presence of two discrete, separable sequences which act in a position and orientation independent manner to enhance prolactin gene transcription. It was proposed that these two regions (from -1768 to -1712 and from -1653 to -1531) are cell-specific enhancer elements which mediate the activation of these genes in lactotrophs and somatotrophs.We have used a different approach to study the interaction of cis-acting DNA sequences and trans-acting elements leading to an activated rat prolactin gene. Cell-free nuclear extracts prepared from a rat pituitary-derived cell line, GH3, and nonpituitary cells were used to study the cell-specific in vitro transcription and DNA protein-binding interactions of the prolactin gene. We combined transcriptional and binding assays to identify DNA sequences upstream from the prolactin gene whic...

Estrogen-receptor-α exchange and chromatin dynamics are ligand- and domain-dependent

“…entirely mammalian regulatory elements). Advantages of PRL control elements for this purpose are: (1) they are well characterized in terms of binding and transcriptional activation by Pit-1 and ER (Barron et al, 1989;Cao et al, 1987;Crenshaw et al, 1989;Day et al, 1990;Howard and Maurer, 1995;Ingraham et al, 1990;Mangalam et al, 1989;Nelson et al, 1988;Smith et al, 1995), (2) they are responsive to 17-␤ estradiol (E2) through transcriptional synergy by Pit-1 and ER (Day et al, 1990;Day and Maurer, 1989;Holloway et al, 1995;Nowakowski and Maurer, 1994;Seyfred and Gorski, 1990), (3) they were useful in some of the original work to characterize transcriptional downregulation by the antiestrogen 4-hyrdroxytamoxifen (4HT) (Gothard et al, 1996;Jordan et al, 1988;Lieberman et al, 1983;Seyfred and Gorski, 1990) and, (4) alterations of their chromatin structure in response to E2 (Cullen et al, 1993;Durrin and Gorski, 1985;Durrin et al, 1984;Gothard et al, 1996;Malayer and Gorski, 1995;Maurer, 1985;Seyfred and Gorski, 1990;Willis and Seyfred, 1996) and 4HT (Gothard et al, 1996;Liu and Bagchi, 2004;Seyfred and Gorski, 1990) are well studied.…”

An avian muscle factor related to MyoD1 activates muscle-specific promoters in nonmuscle cells of different germ-layer origin and in BrdU-treated myoblasts.

“…Chicken and mouse S1 probes were prepared as described (De Ponti-Zilli et al 1988). Primer extensions were carried out as described (Cao et al 1987), using MMLV reverse transcriptase (BRL) with 20-40 ~g of total RNA and a kinased CAT primer [Maniatis et al 1982) from position 4921-4944 in PSV2CAT (Gorman 1985). The c~-cardiac actin and 13-actin CAT promoter constructs are as described elsewhere (Quitschke et al 1989a, b).…”

Pituitary cell phenotypes involve cell-specific Pit-1 mRNA translation and synergistic interactions with other classes of transcription factors.

“…Data from gene-direeted ablation studies in transgenic mice are consistent with the notion that at least most lactotrophs are derived from a presomatotroph lineage (Behringer et al 1988;Borrelli et al 1989). The cell-specific expression of high levels of the rat PRL and GH genes depends on a series of cell-specific cis-active elements (Nelson et al 1986(Nelson et al , 1988Bodner and Karin 1987;Cao et al 1987;GutierrezHartman et al 1987;Lufkin and Bancroft 1987;West et al 1987;Ye and Samuels 1987).…”