A Drosophila ESC-E(Z) protein complex is distinct from other polycomb group complexes and contains covalently modified ESC

Abstract

The extra sex combs (ESC) and Enhancer of zeste [E(Z)] proteins, members of the Polycomb group (PcG) of transcriptional repressors, interact directly and are coassociated in fly embryos. We report that these two proteins are components of a 600-kDa complex in embryos. Using gel filtration and affinity chromatography, we show that this complex is biochemically distinct from previously described complexes containing the PcG proteins Polyhomeotic, Polycomb, and Sex comb on midleg. In addition, we present evidence that ESC is phosphorylated in vivo and that this modified ESC is preferentially associated in the complex with E(Z). Modified ESC accumulates between 2 and 6 h of embryogenesis, which is the developmental time when esc function is first required. We find that mutations in E(z) reduce the ratio of modified to unmodified ESC in vivo. We have also generated germ line transformants that express ESC proteins bearing site-directed mutations that disrupt ESC-E(Z) binding in vitro. These mutant ESC proteins fail to provide esc function, show reduced levels of modification in vivo, and are still assembled into complexes. Taken together, these results suggest that ESC phosphorylation normally occurs after assembly into ESC-E(Z) complexes and that it contributes to the function or regulation of these complexes. We discuss how biochemically separable ESC-E(Z) and PC-PH complexes might work together to provide PcG repression.

FIG. 1

Expression of ESC and E(Z) during development. Detection of HA-ESC and E(Z) proteins by immunoblotting of wild-type HA-esc extracts from the indicated embryonic, larval, and pupal stages. Approximately equal amounts of total protein were loaded per lane. The two ESC forms are indicated by arrows.

FIG. 2

Gel filtration analysis of embryo extracts from HA-esc transformants. Nuclear extracts were fractionated by Superose 6 chromatography. Fraction numbers are indicated at the top. The elution positions of molecular mass standards are indicated by arrows. (Top) Detection of HA-ESC and E(Z) by immunoblotting. (Bottom) Detection of PH by immunoblotting.

FIG. 3

Tests for coenrichment of PcG proteins with HA-ESC by immunoaffinity chromatography. Immunoblots to detect the indicated PcG proteins are shown. NE, nuclear extract starting material; FT, flowthrough containing unbound material; W, final wash of affinity column; HA, material eluted with HA peptide. The HA lanes on the E(Z), PH, SCM, and PHO blots contain sixfold more material loaded than for the corresponding lane on the HA-ESC blot.

FIG. 4

Tests for ESC phosphorylation. Wild-type HA-esc extracts were treated with phosphatases and detected by immunoblotting. I, phosphatase inhibitors; E, enzyme. The arrows indicate the two ESC forms. (A) Phosphatase treatments of total embryonic extracts. The enzyme used for lanes 4 and 5 was potato acid phosphatase, and the enzyme used for lanes 9 and 10 was calf alkaline phosphatase. Lanes 1 and 6 show untreated extracts. (B) Calf alkaline phosphatase treatments of nuclear extracts.

FIG. 5

Expression of HA-ESC and E(Z) in temperature-sensitive E(z) mutant embryos. Immunoblots to detect HA-ESC and E(Z) from embryos collected at permissive (20°C) and restrictive (29°C) temperatures are shown. Embryo genotypes: wt, wild-type; E(z)²⁸ or E(z)⁶¹, homozygous for the indicated temperature-sensitive E(z) mutation. Blots were reprobed with antibodies to β-tubulin as a control for amounts of total protein loaded per lane.

FIG. 6

Expression of mutant HA-ESC proteins. Immunoblot detection of wild-type (lanes 1 to 3) and the indicated mutant (lanes 4 to 12) HA-ESC proteins from 6- to 12-h total-embryo extracts is shown. Three independent lines were used for each transgene construct. All lines were homozygous for the transgene. Approximately equal amounts of total protein were loaded per lane.

FIG. 7

Effect of ESC surface loop mutations upon ESC modification. Immunoblot detection of wild-type and mutant HA-ESC proteins from 6- to 12-h embryo extracts is shown. Arrows indicate the two ESC forms. Mutants in panel A show severe loss of esc function in vivo, and the mutant in panel B shows moderate loss of function in vivo.

FIG. 8

Gel filtration analysis of mutant HA-ESC. Nuclear extract from embryos expressing HA-ESC with the RDE216AAA DFST278AFAA mutation was fractionated by Superose 6 chromatography. Fraction numbers are indicated at the top. Elution positions of molecular mass standards are indicated by arrows. HA-ESC and E(Z) proteins were detected by immunoblotting with anti-HA and anti-E(Z) antibodies, respectively.

FIG. 9

Division of labor in the PcG. The model shows two biochemically separable PcG complexes with components based on this work and previous studies (17, 26, 35, 51, 61, 62). Members of each complex and established direct interactions between these members are indicated. Question marks indicate that there are likely additional components in these complexes to be identified. Arrows indicate that the complexes work through a common regulatory target in chromatin.