Functional similarity of Knirps CtBP-dependent and CtBP-independent transcriptional repressor activities

Abstract

Short-range transcriptional repressors are locally acting factors that play important roles in developmental gene expression in Drosophila. To effect repression, Knirps and other short-range repressors bind the CtBP corepressor, but these repressors also function via CtBP-independent pathways. Possible mechanistic differences between CtBP-dependent and -independent repression activities are poorly understood. The distinct activities might provide qualitatively different activities necessary in different promoter contexts, or they might combine to give quantitatively different effects. We analyze separately the CtBP-dependent and CtBP-independent domains of Knirps previously characterized in the embryo to determine possible functional distinctions of the two repression activities. Both domains are active in cell culture and are dependent on the same residues required for activity in the embryo. The domains have similar properties with respect to distance-dependent repression and resistance to inhibition by the deacetylase inhibitor trichostatin A. In tests of repressor-activator specificity, the extent of repression was related not to the chemical nature of the activation domain but to the total activation potential. This result indicates that the balance of competing activation and repression signals is decisive in determining the effectiveness of repressors on genetic switches, suggesting that multiple repression activities are utilized to provide quantitatively, rather than qualitatively, distinct outputs.

Quantitative contributions of CtBP-dependent and -independent repression activities of Knirps

“…CtBP-independent activity can in some cases be directly attributed to direct competition with activator for DNA binding (Hoch et al, 1992;Nibu et al, 2003); however, the CtBPindependent activity of Knirps can repress activators on elements where sites are not overlapping (Keller et al, 2000;Ryu and Arnosti, 2003), and overexpression of the DNAbinding domain of Knirps (Knirps1-105) is insufficient to mediate repression of endogenous eve enhancers (data not shown). Cell culture and transgenic embryo assays indicate that both CtBP-dependent and independent repression activities of Knirps have very similar characteristics with respect to activator specificity, distance dependence and overall potency, thus the targets and molecular mechanisms might well be similar in each case (Ryu and Arnosti, 2003;Sutrias-Grau and Arnosti, 2004). Key to a deeper understanding of the molecular circuitry controlled by short-range repressors such as Knirps will be biochemical knowledge of the mechanisms of repression employed on these developmentally regulated enhancers.…”

“…The higher levels of Bicoid activator in anterior regions of the embryo might necessitate additional repression activities beyond those afforded by CtBP-independent pathways, suggesting that CtBP might contribute quantitatively to overall repressor output. In cell culture studies, CtBP-dependent and CtBP-independent repression activities of Knirps possess similar functional attributes, including distance dependence, trichostatin A insensitivity and activator specificity, suggesting that their quantitative effects might be mediated through similar pathways (Ryu and Arnosti, 2003).…”

“…In a number of well-studied cases in the Drosophila embryo, multiple enhancers act independently on a single promoter, in part due to the action of so-called short-range repressors, proteins whose inhibitory action is restricted to ranges of ~100 bp from the factor binding site (Gray and Levine, 1996a). In cell culture and transgenic embryo assays, short-range repressors can selectively inhibit individual enhancers, or entirely silence a gene if bound close to the basal promoter (Arnosti et al, 1996;Gray and Levine, 1996b;Ryu and Arnosti, 2003).…”

Tinman is a direct activator of midline in the drosophila dorsal vessel

“…The mid4.3, mid1.7, and mid1.0 fragments were also cloned into the p2TLuc vector for S2 cell luciferase reporter assays (Ryu and Arnosti, 2003).…”

“…Drosophila S2 cells were plated in 12-well plates at a density of 1 Â 10 6 cells per well and transient transfection was performed as described previously (Ryu and Arnosti, 2003). Briefly, each transfection cocktail contained 100 ng of mid CRE-Luc, 100 ng of Actin-expressor, and 20 ng of pAc/ lacZ internal control for each well.…”