A heterochromatin protein 1 homologue in Caenorhabditis elegans acts in germline and vulval development

Abstract

Proteins of the highly conserved heterochromatin protein 1 (HP1) family have been found to function in the dynamic organization of nuclear architecture and in gene regulation throughout the eukaryotic kingdom. In addition to being key players in heterochromatin-mediated gene silencing, HP1 proteins may also contribute to the transcriptional repression of euchromatic genes via the recruitment to specific promoters. To investigate the role played by these different activities in specific developmental pathways, we identified HP1 homologues in the genome of Caenorhabditis elegans and used RNA-mediated interference to study their function. We show that one of the homologues, HPL-2, is required for the formation of a functional germline and for the development of the vulva by acting in an Rb-related pathway. We suggest that, by acting as repressors of gene expression, HP1 proteins may fulfil specific functions in both somatic and germline differentiation processes throughout development.

Fig. 1. (A) Alignment of amino-acid sequences of the HP1 proteins from human (HP1α, -β and -γ), Drosophila (HP1a, -b and –c) and C. elegans (HPL-1, -2a and -2b) by the Clustal W method followed by manual editing. The conserved CD and CSD are boxed in. Note that HPL-2B diverges from other HP1-like proteins in the C-terminal part of the CSD. Black and grey boxes indicate identical and conserved residues, respectively. Asterisks indicate the 5′ acidic stretch of residues found in Drosophila HP1a and all human homologues but absent from C. elegans proteins. Heavy-lined boxes within hinge region denote the previously described bipartite nuclear localization sequence (Smothers and Henikoff, 2001), which is missing from the C. elegans homologues. (B) Genomic structure of the C. elegans hpl-1 and hpl-2 genes. hpl-2a (K01G5.2a) and hpl-2b (K01G5.2b) arise from alternative splicing of a single transcript that is part of an operon including the upstream gene K01G5.1, predicted to encode a ring zinc finger protein of the C3HC4 type. The AUG start codon for hpl-2 is found 120 bp downstream from the stop codon of K01G5.1, and RT–PCR analysis confirmed the presence of an SL2 transpliced leader sequence on the hpl-2 transcript. The ATG start and stop codons and the SL1 and SL2 transpliced leader sequences are shown. Boxes correspond to exons, connecting lines to introns. (C) Schematic representation of the HPL-1 and HPL-2 proteins, showing CD and CSD domains and the GFP insertion site in the fusion proteins used in this study.

Fig. 2. Localization of HPL-2::GFP in the soma and germline. (A, D, G) DAPI fluorescence; (B, E, H) GFP fluorescence; (C, F, I) merged image of DAPI and GFP with DAPI in false red and GFP in green. (A–C) HPL-2::GFP is present in the germline and associated with the DNA of condensed nuclei in pachytene. (D–F) Twenty-eight-cell stage embryo with HPL-2::GFP expression in all nuclei. Nuclei in metaphase (m) and prophase (p) are indicated. (G–I) Nuclei of intestinal cells in interphase. Scale bars: 2 µm (A–C), 4 µm (D–F), 1 µm (G–I).

Fig. 3. Desilencing of a repetitive let-858::gfp transgene in the germline of F1 offspring of hpl-2(RNAi) animals. Germline is outlined by brackets. (C) In control animals, GFP fluorescence can be strongly detected in somatic nuclei but not in the germline. (D) In hpl-2(RNAi) animals, GFP fluorescence is detected in all germline nuclei and in oocytes (arrowheads). (A and B) Nomarski images of the same animals as in (C) and (D).

Fig. 4. Germline development is abnormal in hpl-2(RNAi) animals. Nomarski (DIC) images of the gonad of wild-type (A) and hpl-2(RNAi) (B) animals. Note that the proximal region of the gonad (arrowheads in A and B) in hpl-2(RNAi) animal is filled with enlarged, abnormal oocytes. (C and D) Fluorescent images of the same worms as in (A) and (B) in which DNA is visualized by histone H2B::GFP. Note the intense fluorescence (arrowheads in D) corresponding to accumulation of DNA in the enlarged nuclei seen in (B). Scale bar, 50 µm.