The gonadotropin alpha-gene contains multiple protein binding domains that interact to modulate basal and cAMP-responsive transcription.
DNA sequences that modulate basal and cAMP-stimulated transcription are located within the initial 169 base pairs of the alpha-gene 5'-flanking sequence. Using DNase I protection analyses and gel-mobility shift assays, we examined in vitro the domains in the human alpha-gene 5'-flanking sequence that bind nuclear proteins extracted from JEG-3 choriocarcinoma cells. DNase I protection studies of the sequences between -236 and -100 demonstrate two major protected regions: -178 to -156 corresponding to an upstream regulatory element (URE) and -146 to -112 corresponding to 18-base pair repeated sequences that contain cAMP-responsive enhancers (CREs). Nuclear proteins extracted from JEG-3 choriocarcinoma cells bind specifically to oligonucleotides corresponding to both the URE and CRE domains as well as to a downstream domain (-99 to -72) that contains consensus CCAAT motifs on both the sense and antisense strands. Binding to a DNA fragment (-236 to -100) that contains both the URE and CRE domains was 10-fold more effective than that using either fragment alone. Binding to this multisite DNA fragment is readily disrupted using the URE sequence, but not the CRE sequence as a competitor, suggesting that the URE binding factor may stabilize DNA-protein interactions in these adjacent complexes. The amount of protein binding to each of the alpha-gene 5'-flanking domains was unaffected by treatment with 8-bromo-cAMP. These studies indicate that there are multiple adjacent protein binding domains in human alpha-gene 5'-flanking sequence that correspond to cis-acting regulatory elements including an upstream element that activates basal expression, repeated cAMP-response elements, and a downstream sequence containing consensus CCAAT box elements. Interactions between regulatory domains facilitate protein binding and synergistically stimulate alpha-gene transcription.
Jameson
J L
JL
Thyroid Unit, Massachusetts General Hospital, Boston 02114.
Jaffe
R C
RC
Deutsch
P J
PJ
Albanese
C
C
Habener
J F
JF
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000123:Acetyltransferases / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D015500:Chloramphenicol O-Acetyltransferase; D002822:Choriocarcinoma / Q000032:analysis; D000242:Cyclic AMP / Q000494:pharmacology*; D004247:DNA / Q000378:metabolism*; D004262:DNA Restriction Enzymes; D003850:Deoxyribonuclease I / Q000378:metabolism; D015292:Glycoprotein Hormones, alpha Subunit; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D010908:Pituitary Hormones, Anterior / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012091:Repetitive Sequences, Nucleic Acid; D014158:Transcription, Genetic / Q000187:drug effects*; D014407:Tumor Cells, Cultured
D015292:Glycoprotein Hormones, alpha Subunit; D009687:Nuclear Proteins; D010908:Pituitary Hormones, Anterior; D015124:8-Bromo Cyclic Adenosine Monophosphate; D004247:DNA; D000242:Cyclic AMP; D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase; D004262:DNA Restriction Enzymes; D003850:Deoxyribonuclease I