HAT activity is essential for CBP-1-dependent transcription and differentiation in Caenorhabditis elegans

Abstract

The p300/CBP family of transcriptional coactivators possesses multiple functional domains, including a histone acetyltransferase (HAT) and several activation domains. A number of models have been proposed to account for their roles in transcriptional activation, including interactions with basal transcription machinery and chromatin remodeling. However, individual contributions of these domains to transcriptional activation and their significance in living organisms remain unclear. We addressed the importance of the HAT activity of CBP-1, the worm ortholog of p300/CBP, in Caenorhabditis elegans with three different and complementary approaches. These include allele-specific RNA-mediated interference (RNAi), genetic rescue and the use of a specific chemical inhibitor of the p300/CBP HAT. Our findings demonstrate that HAT activity is of primary importance for CBP-1 to regulate transcription and to promote differentiation during C. elegans embryogenesis.

Fig. 1. A cbp-1 deletion mutant displays an embryonic lethal phenotype. (A) Isolation of a cbp-1 deletion mutant. An internal in-frame deletion mutant of cbp-1 was isolated from a DEB-mutagenized C. elegans library by PCR. The cbp-1 gene and the predicted 227 kDa protein are shown schematically. The HAT and bromo domains and the deleted region (aa 889–1726) in the mutant are indicated. (B) The truncated CBP-1 is stably expressed in cbp-1 heterozygous mutant animals. The wild-type and the truncated CBP-1 proteins (CBP-1Δ) are labeled on the right. The genotypes of the animals analyzed are indicated at the top. (C) Animals homozygous for cbp-1Δ die at the 1.5- to 2-fold stage of C. elegans embryonic development. The Nomarski images of a wild-type (a) and homozygous mutant animal (b) are shown. The wt embryo is at the pretzel stage while the cbp-1 mutant embryo died at the ∼1.5- to 2-fold stage (b).

Fig. 2. Allele-specific RNAi. (A) Schematic diagram of the allele-specific RNAi approach. HAT dsRNA is predicted to interfere only with the expression from the wt but not the mutant cbp-1 allele. (B) CBP-1Δ lacking the HAT domain is incapable of promoting differentiation. dsRNA corresponding to the HAT domain deleted in the cbp-1Δ was injected into wt (cbp-1 +/+) and heterozygous mutant animals (cbp-1 +/Δ). Nomarski analysis shows identical RNAi phenotypes (a and c). Wild-type CBP-1 expression was abrogated in wt (b) and cbp-1 heterozygous (d) animals by HAT dsRNA, while the expression of CBP-1Δ is unaffected (compare d with b).

Fig. 3. Analysis of the effect of Lys-CoA on CBP-1 HAT activity. (A) Partial HAT domain sequence alignment between human CBP and C. elegans CBP-1. Partial HAT sequences of human CBP (top) and C. elegans CBP-1 (bottom) are taken from Martinez-Balbas et al. (1998) and Shi and Mello (1998). The F residue mutated to A is marked by an asterisk. (B) Wild-type, but not CBP-1 with a point mutation in the HAT domain, acetylates histones. Equal amounts of purified wild-type and mutant GST–CBP-1 HAT (aa 803–1620) proteins were assayed for HAT activity using purified histones as substrates. GST–CBP-1 (lane 2), but not the CBP-1 point mutant (lane 3), acetylated all four histones. (C) Lys-CoA inhibits CBP-1 HAT activity. Lys-CoA, a CBP/p300-specific HAT inhibitor, inhibits CBP-1 HAT activity at 10 µM (lane 2). A P/CAF-specific HAT inhibitor, H3-CoA-20, does not (lane 3).

Fig. 4. Phenotype and promoter activity analyses of Lys-CoA-affected embryos. (A) Lys-CoA injection results in a cbp-1(RNAi)-like phenotype. Injection of wt animals with either cbp-1 dsRNA or Lys-CoA results in identical phenotypes by Nomarski microscopy (compare a and b). In contrast, unlike the cbp-1(RNAi) embryo (c), the Lys-CoA-affected embryo continued to express CBP-1 (d). (B) Lys-CoA de-represses a neuronal promoter but inhibits gut- and muscle-specific promoters. The expression patterns of hlh-2::GFP, hlh-1::GFP and elt-2::GFP in wild-type animals are shown in the left panels (a, d and g). After interference with CBP-1 expression using RNAi (the middle panels) or inhibition of CBP-1 HAT activity by Lys-CoA (the right panels), the same surplus number of neuronal cells (hlh-2::GFP positive) was observed in either cbp-1(RNAi) or Lys-CoA-treated embryos (b and c). In contrast, the activities of the muscle- and endoderm-specific promoters, hlh-1::GFP and elt-2::GFP, are completely abrogated in cbp-1(RNAi) and Lys-CoA-treated embryos (e, f and h, i).