Transcriptional regulation by CHIP/LDB complexes

Abstract

It is increasingly clear that transcription factors play versatile roles in turning genes "on" or "off" depending on cellular context via the various transcription complexes they form. This poses a major challenge in unraveling combinatorial transcription complex codes. Here we use the powerful genetics of Drosophila combined with microarray and bioinformatics analyses to tackle this challenge. The nuclear adaptor CHIP/LDB is a major developmental regulator capable of forming tissue-specific transcription complexes with various types of transcription factors and cofactors, making it a valuable model to study the intricacies of gene regulation. To date only few CHIP/LDB complexes target genes have been identified, and possible tissue-dependent crosstalk between these complexes has not been rigorously explored. SSDP proteins protect CHIP/LDB complexes from proteasome dependent degradation and are rate-limiting cofactors for these complexes. By using mutations in SSDP, we identified 189 down-stream targets of CHIP/LDB and show that these genes are enriched for the binding sites of APTEROUS (AP) and PANNIER (PNR), two well studied transcription factors associated with CHIP/LDB complexes. We performed extensive genetic screens and identified target genes that genetically interact with components of CHIP/LDB complexes in directing the development of the wings (28 genes) and thoracic bristles (23 genes). Moreover, by in vivo RNAi silencing we uncovered novel roles for two of the target genes, xbp1 and Gs-alpha, in early development of these structures. Taken together, our results suggest that loss of SSDP disrupts the normal balance between the CHIP-AP and the CHIP-PNR transcription complexes, resulting in down-regulation of CHIP-AP target genes and the concomitant up-regulation of CHIP-PNR target genes. Understanding the combinatorial nature of transcription complexes as presented here is crucial to the study of transcription regulation of gene batteries required for development.

Figure 1. Composition and function of the CHIP-AP and CHIP-PNR transcription complexes.

(A) The CHIP-AP complex is composed of a dimer of CHIP molecules bound through their dimerization domain . Each molecule of CHIP can bind one molecule of AP , through its LIM interacting domain (LID) , and one molecule of SSDP through its LDB/CHIP conserved domain (LCCD) . (B) dLMO displacement of AP from the complex blocks AP dependant expression of target genes . (C) The CHIP-PNR complex is composed of a single CHIP molecule that binds dLMO through its LID domain. PNR binds CHIP in a region that overlaps CHIP's dimerization domain thus preventing the formation of a CHIP dimmer. The b-HLH members of this complex are the AC:SC heterodimer and the DA protein . (D) Schematic representation of the wing imaginal disc (dorsal side is up). AP expression in the dorsal area (in blue) determines the dorsal compartment. The boundary between AP expressing and non-expressing cells determines the dorsal (D) ventral (V) boundary which will give rise to the adult wing margin. The wing poach will give rise to the adult wing blade. PNR expression in the dorsal most area (in brawn) of the wing imaginal disc determines thoracic identity. The SOC cells that will give rise to the thoracic macrocheata are indicated by dots.

Figure 2. Fly SSDP specifically recognizes the SSDP binding site.

(A) Labeled single stranded oligonucleotide representing the binding site of chicken SSDP was incubated with or without cell extracts from bacteria expressing GST-tagged fly SSDP in the presence of increasing concentrations of unlabeled oligonucleotide as a competitor. (B) Labeled single stranded binding site of chicken SSDP was incubated with cell extracts from bacteria expressing or not expressing GST-tagged fly SSDP. (C) Labeled single stranded binding site of chicken SSDP was incubated with purified GST-tagged fly SSDP. B and C were taken from the same gel.

Figure 3. Genetic interaction between Dlmo and ap.

(A,B) Female & male distributions of wing phenotypes. (C–F). Wings with various severities of Dlmo^Bx2 phenotypes (anterior side is up). (A) Genotype of the test group (in gray) is Dlmo^Bx2/+; ap^UGO35/+ and the control group (in black) is Dlmo^Bx2/+. (B) Genotype of the test group (in gray) is Dlmo^Bx2/Y; ap^UGO35/+ and the control group (in black) is Dlmo^Bx2/Y. Class 1 and (C), wild type wings; Class 2, one wild type wing and the other notched on the posterior side (D); Class 3, both wings are notched on the posterior side; Class 4, one wing is notched on the posterior side only and the other is notched on the anterior side as well (E); Class 5, both wings are notched on the posterior and anterior sides; Class 6 and (F), both wings are notched on the posterior and anterior sides and at list one wing also lacks dorsal to ventral adhesion. The distribution of the wing notching phenotype for the double heterozygous flies is shifted towards the more severe phenotypic groups.

Figure 4. Genetic interaction between ap^Xa and ssdp^L7.

(A,B) Wings of ap^Xa/+ flies. (A) A typical class 1 wing. (B) A typical class 2 wing. (C) Wild type wing. (D–F) Wings of ap^Xa/+; ssdp^L7/+ flies. (D) A typical class 1 wing. (E) A typical class 2 wing. (F) A typical class 3 wing. (F) Schematic representation of the wing notching phenotypes depicted in (A–E). Classes 1–3 from ap^Xa/+; ssdp^L7/+ flies (represented by doted lines) are more severe then classes 1–2 from ap^Xa/+ flies (represented by full lines).

Figure 5. Gs-alpha60A and Xbp1 are essential for normal wing and bristle formation.

(A) Wild type fly. (B,C,E) ap-Gal4/+; UAS-RNAi-Xbp1/+. (B) Acute multiplication of bristles on the wing and scutum, wings are not fully developed and lack dorsal to ventral adhesion. White doted frames indicate areas enlarged in (C,E). (C) Enlargement of the thorax from (B), multiplication of bristles can be seen on the scutum but not on the scutelleum. (D) Thorax of a pnr-Gal4/+; UAS-RNAi-Xbp1/+ fly, multiplication of bristles is limited to the midline of the scutum, the area indicated by the white frame. The anterior pair of scutellar bristles are missing, arrows point to their expected position. (E) Enlargement of the wing from (B). Wings are underdeveloped and exhibit multiplication of bristles. (F) An ap-Gal4/+; UAS-RNAi-Gs-alpha/+ fly, wings are curled. (G) Thorax of a pnr-Gal4/+; UAS-RNAi-Gs-alpha/+ fly, the posterior pair of scutellar bristles indicated by an arrow are in reversed orientation.