Pulses of prolactin promoter activity depend on a noncanonical E-box that can bind the circadian proteins CLOCK and BMAL1.
Recent findings from our laboratory and those of others demonstrated that prolactin gene expression (PRL-GE) oscillates in single living mammotropes, but little information is available on the molecular processes that contribute to this phenomenon. To elucidate the source of this activity, we generated a series of constructs containing decreasing lengths of the PRL promoter fused to a luciferase reporter gene. These constructs were injected into single cells and assayed for photonic activity. We found pulse activity with all plasmids tested, even with the smallest promoter fragment of 331 bp. Sequence analysis of this fragment identified two potential E-boxes (elements known to bind CLOCK and BMAL1 circadian proteins). Furthermore, RT-PCR of PRL cells (pituitary, MMQ, and GH(3)) revealed expression of clock and bmal1 as well as five other clock genes (per1, per2, cry1, cry2, and tim), suggesting that the circadian system may function in PRL cells. Next, we mutated the core sequences of both E-boxes within the 2.5-kb PRL promoter and found that only mutation of the E-box133 completely abolished PRL-GE pulses. EMSAs revealed that CLOCK and BMAL1 were able to bind to the E-box133 site in vitro. Our results demonstrate that PRL-GE pulses are dependent on a specific E-box binding site in the PRL promoter. Moreover, the indication that CLOCK/BMAL1 can bind to this site suggests that these circadian proteins, either alone or in conjunction with other factors, may regulate intermittent PRL promoter activity in mammotropes, perhaps by acting as a temporal switch for the on/off expression of PRL.
Leclerc
Gilles M
GM
Laboratory of Molecular Dynamics, Department of Cell Biology and Anatomy, Medical University of South Carolina, 173 Ashley Avenue, Charleston, South Carolina 29425, USA.
Boockfor
Fredric R
FR
D056930:ARNTL Transcription Factors; D000818:Animals; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins; D056926:CLOCK Proteins; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D005260:Female; D005786:Gene Expression Regulation / Q000502:physiology; D007774:Lactation / Q000502:physiology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010507:Periodicity; D010902:Pituitary Gland / Q000166:cytology / Q000502:physiology*; D011388:Prolactin / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics* / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
D056930:ARNTL Transcription Factors; D051792:Basic Helix-Loop-Helix Proteins; D056926:CLOCK Proteins; D004268:DNA-Binding Proteins; D011388:Prolactin; D015534:Trans-Activators; D014157:Transcription Factors; C538945:Clock protein, rat