Drosophila HP1c is regulated by an auto-regulatory feedback loop through its binding partner Woc

Abstract

HP1 is a major component of chromatin and regulates gene expression through its binding to methylated histone H3. Most eukaryotes express at least three isoforms of HP1 with similar domain architecture. However, despite the common specificity for methylated histone H3, the three HP1 isoforms bind to different regions of the genome. Most of the studies so far focused on the HP1a isoform and its role in transcriptional regulation. As HP1a requires additional factors to bind methylated chromatin in vitro, we wondered whether another isoform might also require additional targeting factors. Indeed, we found that HP1c interacts with the DNA binding factors Woc and Row and requires Woc to become targeted to chromatin in vivo. Moreover, we show that the interaction between HP1c and Woc constitutes a transcriptional feedback loop that operates to balance the concentration of HP1c within the cell. This regulation may prevent HP1c from binding to methylated heterochromatin.

Figure 1. HP1c interacts with two Zn-finger containing proteins Woc and ROW.

(A) Comassie staining of a flag affinity purification from nuclear extracts of SL2 cells (lane 1) or SL2 cells stably transfected with an expression vector for HP1c (lane 2). Major proteins are indicated in bold letters, proteins that were co-purified to a various degree in different preps are indicated in italics. Signals derived from the M2-antibody or proteins that are also present in the controlare indicated with an asterisk. (B) GST pull down assays using GST (lane 2), GST-HP1a (lane 3) or GST-HP1c (lane 4) as a bait and in vitro translated Su(var)3–9 (top panel) ACF1 (second panel) or ROW (bottom panel) as prey (2.5% of the input material is shown in lane 1). To ensure equal loading the SDS-PAA gel was stained with coomassie blue (bottom panel). (C) Specificity of the HP1c antibody. Western Blot on purified recombinant HP1 isoforms using the monoclonal HP1c antibody used in this study (top panel) and an anti-GST antibody (bottom panel). (D) Whole extract of wt or HP1c−/− mutant flies were subjected to SDS-PAGE and blotted using an anti HP1c (top panel) or an anti tubulin antibody. (E) Immunoprecipitation assays using nuclear extracts of early Drosophila embryos (0–12 h). Co-precipitated proteins were detected by Western Blotting. A mock immunoprecipitation using a non specific antibody was performed as a control (lane 2).

Figure 2. HP1c colocalizes with Woc on polytene chromosomes.

(A) Salivary gland polytene chromosomes from wild type larvae stained with α-HP1c and α-woc (upper panel). Enlargement and generation of split images allows a detailed analysis of HP1c and woc localization (lower panel). (B) Salivary gland polytene chromosomes from woc-mutant larvae stained with α-HP1c and α-HP2 as a control. (C) Salivary gland polytene chromosomes from HP1c-mutant larvae stained with α-HP1c and α-woc. In the merged images, woc is depicted in green, HP1c in red. DNA was stained with DAPI (blue).

Figure 3. Woc and HP1c coordinate HP1c expression on a transcriptional level.

(A) Western blot using whole cell extracts of either wild type or woc-mutant Drosophila 3^rd instar larvae. (B) RT-PCR analysis using total RNA isolated from 3^rd instar larvae. HP1c expression analysis was performed with wild type larvae and larvae from two fly strains carrying different heteroallelic combinations of Woc mutant alleles. Primers for the ribosomal protein RLP12 spanning an intron were used as a control. (C) Drosophila SL2 cells transfected with (right panel) or without (left panel) an expression construct for a FLAG-tagged version of HP1c driven by an actin promoter were subjected to woc RNAi. Protein levels were determined by Western Blotting with the indicated antibodies. The asterisks indicates the endogenous HP1c (D) Whole cell extracts from Drosphila SL2 cells transfected with either HP1aFLAG or HP1cFLAG were prepared. Endogenous HP1c levels were determined by immunoblotting with an HP1c specific antibody (lower panel). Expression of the FLAG-tagged HP1 isoforms was determined as a control. (E) RT-PCR analysis of total RNA using a primer pair specific for endogenous HP1c. RNA was isolated from Drosophila SL2 cells that were either non-transfected or transfected with the indicated expression plasmids. (F) Model of HP1c action to modulate its own transcription by counteracting Woc mediated transcriptional activation.