AN ETS SITE IN THE WHEY ACIDIC PROTEIN GENE PROMOTER MEDIATES TRANSCRIPTIONAL ACTIVATION IN THE MAMMARY GLAND OF PREGNANT MICE BUT IS DISPENSABLE DURING LACTATION.

Authors: MCKNIGHT, ROBERT - RANGOS RESEARCH CENTER; Spencer, Mark; DITTMER, JURGEN - NIH; BRADY, JOHN - NIH; Wall, Robert; HENNIGHAUSEN, LOTHAR - NIH

Submitted to: Molecular Endocrinology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/16/1995
Publication Date: N/A
Citation: N/A

Interpretive Summary: The success of the fledgling bioreactor industry, which hopes to produce pharmacueticals in milk of farm animals, will rely heavily on a detailed understanding of a genetic control of milk protein genes. The research reported here is a continuation of our efforts to understand the genetic control of the whey acidic protein (WAP) gene. A sequence in the putative genetic control region of the WAP gene has the characteristics of binding site for a transcription factor known as Ets. Binding of Ets to other gene regulatory elements is involved in controlling timing of gene activation during devlopment and in controlling cell growth. In these experiments we demonstrated that the putative regulatory sequence in the WAP gene does specifically bind an Ets transcription facator protein, Ets1. We then mutated the WAP gene ETs1 binding site so it could not loner bind Ets1 and introduced that mutated gene into mice. Twenty-one transgenic lines were producted and we analyzed 9 of them in detail. Mutating the Ets1 binding site had no affect on the function of the transgene during lactaction. However, the mutation silenced the transgene during pregnancy. Normally WAP transgenes are "turned on" by day 13 of the pregnancy in transgenic mice. But the Ets1 mutant transgene remained "turned off" until at least day 15 of pregnancy. These findings support the notion that different parats of the WAP gene reglatory region are responsible for controlling its expression during pregnancy and lactation. This experiment shows for the first time that Ets transcription factors participates in the developmental regulation of genes in an organ system. The information will assist in designing genes for the bioreactor industry.

Technical Abstract: The whey acidic protein (WAP) gene is specifically expressed in mammary tissue, and its transcription is induced several thousand-fold during pregnancy and remains high throughout lactation. A purine rich sequence (PRS) located around -110 of the WAP gene promoter is conserved between mice, rats and rabbits, suggesting that it features a regulatory element. This PRS contains an invariant "GGAA/T" core motif characteristic of the binding site for Ets transcription factors. Electromobility shift assays demonstrate that Ets1 binding specifically to the PRS. Experiments in transgenic mice further demonstrate that this PRS/Ets site plays a critical role in the activation of WAP transgenes during pregnancy, but that its presence is not required for high expression throughout lactation. Transgenes with an intact PRS/Ets site are not expressed at mid-pregnancy, but their transcriptional activity is not affected during lactation. These results demonstrate that Ets signaling pathways can function as stage specific transcriptional activators of milk protein genes in the developing mammary gland. In addition, this work extends earlier findings that activation during pregnancy and lactation is mediated in part by different mechanisms.