Enhancer-promoter communication mediated by Chip during Pannier-driven proneural patterning is regulated by Osa

Abstract

The GATA factor Pannier activates proneural achaete/scute (ac/sc) expression during development of the sensory organs of Drosophila through enhancer binding. Chip bridges Pannier with the (Ac/Sc)-Daughterless heterodimers bound to the promoter and facilitates the enhancer-promoter communication required for proneural development. We show here that this communication is regulated by Osa, which is recruited by Pannier and Chip. Osa belongs to Brahma chromatin remodeling complexes and we show that Osa negatively regulates ac/sc. Consequently, Pannier and Chip also play an essential role during repression of proneural gene expression. Our study suggests that altering chromatin structure is essential for regulation of enhancer-promoter communication.

Figure 1

osa mutants genetically interact with pnr and Chip and display extra DC bristles. (A–D,F,G) Dorsal thoraces of flies. (A) Wild-type flies have four DC bristles (arrowhead). (B) Chip^E flies resemble hypomorphic pnr mutants lacking DC bristles (asterisks) and bearing a thoracic cleft (arrow). (C) Null alleles of osa enhance the cleft (arrow) but suppress the lack of DC bristles (arrowhead) in Chip^E flies (Chip^E/Chip^E; +/osa⁶¹⁶). (D,E) Hypomorphic transallelic combination of osa (osa^4H/osa¹⁴⁰⁶⁰) exhibits extra DC bristles (D, arrowhead) associated with extra DC precursors (E) as revealed by A101 lacZ staining in comparison with staining of wild-type disc. (F) osa⁶¹⁶ behaves as dominant enhancer of the excess of DC bristles associated with the pnr^D encoding constitutive activators of ac/sc (pnr^D1/osa⁶¹⁶). (G) Reducing the amount of brm also enhances the cleft (arrow) but suppresses the lack of DC bristles (arrowhead) in Chip^E flies (Chip^E/Chip^E; +/brm²). (H) Molecular characterization of the osa¹⁴⁰⁶⁰ and osa^4H alleles. osa encodes a protein carrying an ARID DNA binding domain (gray box) and an EHD (black box) conserved in human and Caenorhabditis elegans homologs. osa¹⁴⁰⁶⁰ and osa^4H encode truncated Osa protein where the EHD is lacking. The glutamine mutated to a stop codon in osa¹⁴⁰⁶⁰ (Q n°251) and osa^4H (Q n°1281) is indicated by an arrow.

Figure 2

Osa physically interacts with Pnr and Chip through its C-terminal EHD. Osa^4H consequently no longer interacts with Pnr and Chip. (A) Schematic drawing of the Osa domains used throughout the current study. They are as follows: Osa A, Meth1/Ser581; Osa B, Ser581/Ala903; Osa C, Thr897/Tyr1370; Osa D, Tyr1370/His1740; Osa E, His1733/Glu2550; Osa F, Ala2339/Ala2715. (B,C) Osa interacts with Pnr (B) and with Chip (C). In each case, an immunoblot of a representative set of transfected cells extracts is shown in the upper part of the panels. Immunoprecipitations of transfected cell extracts are shown in the lower part of the panels. The transfected expression vectors are shown at the top of the panels. The B10 and M2 mouse antibodies used to immunoprecipitate the extracts are shown at the left of the panels and the antibodies used to reveal the blots are indicated at the bottom. Pnr is recognized by the 2B8 monoclonal antibody, the B10-tagged Osa domains are detected with the B10 antibody, and the full-length flagged Chip is detected with the M2 antibody. The locations of the Osa domains are indicated by arrowheads. The locations of the flagged Chip and Pnr proteins as well as those of the immunoglobulin heavy chain [IgG(H)] are indicated at the sides. (D) Osa^4H no longer interacts with Pnr or Chip. The layout is as in B and C. The stars highlight the absence of Pnr (lane 13) and F-Chip (lane 14) coimmunoprecipitating with immunoprecipitated B10.Osa^4H.

Figure 3

The C-terminal domain of Pnr encompassing the two helical structures and the N-terminal homodimerization domain of Chip mediate interactions with Osa. (A) Structural features of the Pnr and Chip proteins used in the present study. The two zinc fingers (black boxes; ZF) and two amphipathic α-helices (hatched boxes; H1 and H2) of Pnr are shown. The mutations associated with the proteins of the alleles pnr^D1 and pnr^VX1 are localized within the first zinc finger and in the N terminus of the helices, respectively. The N-terminal homodimerization domain of Chip (N^T Chip; gray box; DD) and the C-terminal LIM-interacting domain (C^T Chip; black box; LID) are also shown. (B,C) The C-terminal domain of Pnr containing the α helices (B) and the N-terminal homodimerization domain of Chip (C) mediate the interactions with Osa. Immunoprecipitations of transfected cell extracts are shown. The layout is as described in Figure 2B–D. The B10 Chip C^T comigrates with the immunoglobulin light chain and was detected in B (lane 5) using a peroxidase-conjugated protein A-Sepharose. (D) Autoradiographs of SDS-PAGE gels from representative affinity chromatography experiments with the GST Chip beads (lane 3), the GST C^TPnr (lane 4), or the GST control beads (lane 2) and in vitro translated ³⁵S proteins are indicated on the left. One-tenth of the ³⁵S input is shown in lane 1. Luciferase is used as a negative input. Experiments were performed three times and, with all proteins except luciferase, 50-fold more protein bound to GST C^TPnr and GST Chip than to GST control.

Figure 4

Chip and Osa do not compete for binding to Pnr. The immunoprecipitation of transfected cell extracts is shown. The layout is as described in Figures 2B–D and 3B–C. The top part of the figure shows the immunoblot of a representative set of transfected cell extracts where a constant amount of Pnr and a constant amount of the tagged Osa E have been expressed in the presence of increasing amounts of F.Chip. The bottom part displays the immunoprecipitation of the protein extracts with a constant amount of immunoprecipitated Osa E and an increasing amount of coimmunoprecipitating Pnr when the amount of coimmunoprecipitating F Chip is increasing.

Figure 5

Osa regulates proneural expression through the DC-specific enhancer of the ac/sc complex. Late-third instar thoracic discs show lacZ expression in the DC area (Gomez-Skarmeta et al. 1995). In each case, the reaction was left for 1 h at 22°C. (A, ) Wild-type pattern (w¹¹¹⁸/+; DC:ac–LacZ/+) with DC expression (arrow). (b) Hypomorphic transallelic combination of osa (DC:ac–LacZ osa^4H/osa¹⁴⁰⁶⁰) promotes overexpression of LacZ. (c) Overexpressed Osa mimics the effects of Chip^E [UAS-Osa⁺/+; +/DC:ac–LacZ pnr^MD237(Gal4)]. (d) In Chip^E flies (Chip^E/Chip^E; DC:ac–LacZ/+), the lacZ expression driven by the enhancer is strongly reduced. (e) Lowering the dosage of osa⁺ suppresses the effects of Chip^E (Chip^E/Chip^E; DC:ac–LacZ/osa¹⁴⁰⁶⁰) and restores the activity of the promoter sequences (cf. a,d). (B) A model on how the proneural complex encompassing Pnr, Chip, and the (Ac/Sc)–Da heterodimer is regulated by Osa. The proneural complex activates ac/sc and is negatively regulated by interactions of Pnr and Chip with Osa. These interactions are mediated by the C-terminal domain of Pnr containing two α helices (H1H2), the N-terminal homodimerization domain (DD) of Chip, and the C-terminal EHD of Osa.