Essential Roles of Da Transactivation Domains in Neurogenesis and in E(spl)-Mediated Repression

Abstract

E proteins are a special class of basic helix-loop-helix (bHLH) proteins that heterodimerize with many bHLH activators to regulate developmental decisions, such as myogenesis and neurogenesis. Daughterless (Da) is the sole E protein in Drosophila and is ubiquitously expressed. We have characterized two transcription activation domains (TADs) in Da, called activation domain 1 (AD1) and loop-helix (LH), and have evaluated their roles in promoting peripheral neurogenesis. In this context, Da heterodimerizes with proneural proteins, such as Scute (Sc), which is dynamically expressed and also contributes a TAD. We found that either one of the Da TADs in the Da/Sc complex is sufficient to promote neurogenesis, whereas the Sc TAD is incapable of doing so. Besides its transcriptional activation role, the Da AD1 domain serves as an interaction platform for E(spl) proteins, bHLH-Orange family repressors which antagonize Da/Sc function. We show that the E(spl) Orange domain is needed for this interaction and strongly contributes to the antiproneural activity of E(spl) proteins. We present a mechanistic model on the interplay of these bHLH factors in the context of neural fate assignment.

The CDK8 Complex and Proneural Proteins Together Drive Neurogenesis from a Mesodermal Lineage

“…We found that the DPY-22 PQ-rich domain selectively interacted with HLH-2 but not HLH-3, whereas the last 129 amino acids truncated in all five dpy-22 alleles were required for this interaction (Figure 4B). Further analysis indicated that the PQ-rich domain interacted with the N-terminal half of HLH-2, the region of a predicted transactivation domain important for gene expression and neurogenesis [36, 37] (Figure 4C). These findings suggest that Mediator physically interacts with and might function in the same pathway as HLH-2 to promote I4 neurogenesis.…”

bHLH proteins involved in Drosophila neurogenesis are mutually regulated at the level of stability

“…We were able to restore degradation of Sc[1–320] by m7 when we fused the AD1 domain of Da in place of the Sc TAD (Supplementary Figure S5F and G), since AD1 also strongly interacts with m7 via the latter's Orange domain (68). Fusion of a non-interacting TAD, the Da LH, did not restore Sc[1–320] degradation (Supplementary Figure S5G).…”

E(spl)

“…Like the HLH domain, the Orange domain consists of two amphipathic alpha-helices and mediates protein-protein interactions, including dimerization of the basic helix-loop-helix-orange (bHLH-O) proteins, where it acts as an "extension" of the HLH domain (Dawson, Turner, Weintraub, & Parkhurst, 1995;Eastwood, Yin, Bandyopadhyay, & Bidwai, 2011;Knust et al, 1992;Taelman et al, 2004;Zarifi et al, 2012). Like the HLH domain, the Orange domain consists of two amphipathic alpha-helices and mediates protein-protein interactions, including dimerization of the basic helix-loop-helix-orange (bHLH-O) proteins, where it acts as an "extension" of the HLH domain (Dawson, Turner, Weintraub, & Parkhurst, 1995;Eastwood, Yin, Bandyopadhyay, & Bidwai, 2011;Knust et al, 1992;Taelman et al, 2004;Zarifi et al, 2012).…”

“…This system explicitly illustrates the antagonism between AS-C proneural proteins and E(spl), for example, by their diametrically opposite lof and gain-of-function (gof ) phenotypes. Whereas the naturally basic-less HLH protein Extramacrochetae (Emc, homolog of vertebrate Id) interacts with Da and proneural proteins using the HLH helices and in doing so averts their DNA binding (Van Doren, Ellis, & Posakony, 1991), E(spl) proteins neither use their HLH helices to interact with proneurals nor do they abolish proneural DNA binding (Giagtzoglou et al, 2003(Giagtzoglou et al, , 2005Zarifi et al, 2012). Reciprocally, overexpression of any of the three AS-C genes causes overproduction of bristles, whereas overexpression of any of the seven E(spl) genes causes reduction or complete elimination of bristles (Giebel & Campos-Ortega, 1997;Hinz, Giebel, & Campos-Ortega, 1994;Ligoxygakis, Bray, Apidianakis, & Delidakis, 1999;Tata & Hartley, 1995).…”

“…Whereas loss of AS-C results in clonal patches which lack all external sensory bristles, loss of E(spl) results in clonal patches with densely crowded supernumerary bristles (Heitzler, Bourouis, Ruel, Carteret, & Simpson, 1996). Interactions between proneural and E(spl) proteins have been mapped to the transactivation domains of the former and the N-terminal and Orange domains of the latter (Giagtzoglou et al, 2005;Zarifi et al, 2012). These defects have been traced back to SOP formation, namely, supernumerary bristles arise from supernumerary SOPs, while loss of bristles is due to failure to generate SOPs.…”