Thyroid hormone-responsive elements of the prolactin gene: evidence for both positive and negative regulation

To analyze the regulation of PRL gene expression by thyroid hormone (T3), fusion gene constructs containing various lengths of the rat PRL gene 5'-flanking sequence linked to the bacterial chloramphenicol acetyltransferase (CAT) gene were transfected into the GH3 cell line. Thyroid hormone had no effect on basal or cAMP-stimulated CAT expression in constructs containing more than 1.7 kilobasepairs of the 5'-sequence. However, deletion to 1.5 or 0.6 kilobasepairs resulted in an inhibition of both basal and cAMP-stimulated expression by T3. A construct containing the proximal enhancer region (positions -292 to -38 basepairs) linked to the herpes simplex thymidine kinase promoter (TK) and the CAT reporter gene also responded to T3 with inhibition of basal and cAMP-induced CAT expression. The distal enhancer region (positions -1714 to -1495) linked to thymidine kinase promoter CAT responded to T3 with a stimulation of CAT expression, and the response was additive with the stimulatory response to cAMP. Deletion analysis of the distal enhancer region revealed that the sequence between positions -1530 and -1565 was required for the stimulatory response to T3. The stimulatory response to T3 was additive with the response to estradiol, suggesting distinct elements, but deletion to position -1565 abolished the response to estradiol and permitted an inhibitory response to T3. Mutation of the estrogen response element prevents the response to estradiol, but only blunted the response to T3. Mutation of the sequence GGTCA at positions -1555 to -1551 resulted in an inhibitory response to T3, implicating this sequence in the stimulatory response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of potential protein regulators bound to the tissue-specific positive and negative cis-acting elements of a green tissue-specific promoter in rice

Although some tissue-specific cis-acting elements have been identified, the molecular mechanisms of tissue-specific gene expression remain elusive. Here, we report the identification by a yeast one-hybrid screen of five proteins, Os10g31330/glycine-rich, Os01g10400/metallothionein-like, Os05g51180/nucleic acid-binding, Os05g37930/unknown and Os01g01689/phosphatidylinositol kinase that bound to either the positive or negative tissue-specific cis elements of a rice promoter from the green tissue-specific D54O gene. These proteins are localised in the nucleus and the genes encoding them are differentially expressed in different tissues, further suggesting their putative roles in regulating gene expression. These results suggest that the green tissue-specific expression of the D54O gene may be regulated by the interaction of multiple proteins with cis elements in the promoter region.     

Characterization of two distinct positive cis-acting elements in the mouse alpha 1 (III) collagen promoter

We have identified two distinct sequence elements in the mouse alpha 1(III) collagen promoter which are protected from DNase I digestion by the binding of factors present in crude nuclear extracts of NIH 3T3 fibroblasts. Small substitution mutations were introduced into these promoter elements and shown by the gel retardation (gel mobility shift) and DNase I protection assays to decrease or eliminate factor binding to the mutated element but not to the remaining wild-type element, indicating that two distinct factors recognize these separate promoter regions. Region A appears to bind a factor related to the Jun/AP-1 protein, whereas the factor binding to region B remains as yet unidentified. Mutagenesis of either region decreased the activity of the alpha 1(III) collagen promoter in DNA transfection assays by about 3-fold for the A region (located between - 122 and - 106) and about 5-fold for the B region (located between -83 and -61). These results indicate that regions A and B in the mouse alpha 1(III) collagen promoter are positive cis-regulatory elements, independently binding two distinct trans-activating factors.