Autoregulation controls temporal progression of gene expression during development. Leslie A Dunipace, Angelike Stathopoulos. Biology, California Institute of Technology, Pasadena, CA.

   It is widely accepted that multiple cis-regulatory modules (CRMs) may associate with individual genes to control temporal changes in expression throughout development. However, very little is understood regarding mechanisms of temporal control of gene expression and, in particular, of how the switch from one CRM to the next is accomplished. In order to further our understanding of these dynamics, we identified three CRMs that regulate the expression of brinker (brk) during the first 4 hours of embryonic development. Through the use of standard reporter assays as well as a series of deletions from a 32kb reporter construct, we found that two distal CRMs provide regulatory information for expression of brk; first in a narrow stripe pre-cellularization (5 CRM), and then in a broad lateral band post-cellularization (3 CRM). The third CRM is a promoter proximal element (PPE) which provides no expression when put into a standard reporter construct, but abolishes all early expression of brk when deleted from the large reporter construct. Using this PPE we showed that it is required for the activity of both the 5 and 3 CRMs when they are located at a distance and behind an insulating element. Although there is minimal overlap in the expression of the 5 and 3 CRMs at cellularization, there is a marked switch between the early and late acting CRMs. By expressing the large reporter constructs in a brk mutant background, we discovered that Brk itself is required for the proper timing of this exchange. Furthermore, if the early acting 5 CRM is placed near the promoter it delays the activity of the later acting 3 CRM, suggesting that a physical exchange at the promoter is required for proper timing of expression. Therefore, through analysis of the brk cis-regulatory system we have uncovered (i) that two CRMs control spatially and temporally distinct patterns of brk expression; (ii) that expression of these distantly located CRMs requires the PPE; and (iii) that levels of Brk protein influence the switch from early-enhancer to late acting-enhancer in the early embryo.