A gain-of-function allele of cbp-1, the Caenorhabditis elegans ortholog of the mammalian CBP/p300 gene, causes an increase in histone acetyltransferase activity and antagonism of activated Ras

Abstract

An RTK-Ras-mitogen-activated protein kinase (MAPK) signaling pathway plays a key role in vulval induction in Caenorhabditis elegans. We have previously carried out screens for suppressors of activated Ras to identify factors that play critical roles in the regulation of the pathway. ku258 was isolated as a semidominant allele that suppresses the Multivulva phenotype caused by activated let-60 ras. Our genetic and molecular analyses indicate that ku258 is a gain-of-function allele resulting from two point mutations in the C. elegans homolog of the transcriptional coactivator p300/CBP, cbp-1. Genetic data also suggest that cbp-1 may act downstream of the Ras signaling pathway, but not primarily downstream of the Wnt signaling pathway, to negatively regulate vulval cell fate specification. cbp-1 may function in concert with LIN-1, an Ets transcription factor family member that is one of the targets of MAPK. In vitro histone acetylation assays have revealed that together, the two point mutations cause a sevenfold increase in the histone acetyltransferase (HAT) activity of recombinant CBP-1. To our knowledge, this is the only such HAT activity mutation isolated in a CBP/p300 family protein, and this mutation may define a negative role of the HAT activity in antagonizing Ras function in a specific developmental event.

FIG. 1.

Mapping and cloning of cbp-1(ku258). (A) ku258 was mapped to 0.80 map unit between sqv-3 and ced-7 on chromosome III. (B) ku258 was then linked to unc-69, and a ku258 unc-69(e587)/CB4856; let-60(n1046) SNP mapping strain was constructed. Forty-two Unc-non-ku258 recombinant chromosomes were isolated. Using the identified SNPs, kuP7, kuP8, SNP2, and SNP1, the region in which these recombination events occurred was determined. Using these data, ku258 is predicted to lie within 7 to 12 kb on either side of kuP7. R10E11.1/cbp-1 is the only open reading frame predicted within a 7- to 12-kb interval to the left of kuP7. (C) ku258 genomic DNA was sequenced, and two mutations, R-909-H and E-1021-K, were identified within the R10E11.1/cbp-1 ORF.

FIG. 2.

Multiple-sequence alignment of the CBP-1 bromodomain and partial HAT domain from amino acid 861 to amino acid 1054. CBP-1 (C. elegans), dCBP (Drosophila), CBP (human), and p300 (human) bromodomains are underlined with a black line. The partial HAT domains (remainder of sequence) are underlined with a gray line. cbp-1(ku258) mutant residues are marked by asterisks. Gaps introduced to maximize alignment are indicated by dashes.

FIG. 3.

Immunohistochemistry of CBP-1. Larval-stage worms were stained using a polyclonal rabbit antibody specific for CBP-1. Pictured is an L2 larval-stage worm which showed CBP-1 staining in the nuclei of the vulval precursor cells situated at the ventral surface of the animal (arrows point to VPC nuclei) (B). DAPI staining served as a positive control for the identification of nuclei (A).

FIG. 4.

CBP-1 histone acetyltransferase assay. (A) GST-CBP-1 HD fusion protein constructs were made using amino acid residues 803 to 1620 (this region contains the entire bromodomain and HAT domain). Combinations of mutations corresponding to those seen in cbp-1(ku258) animals were introduced into the constructs. Fusion proteins were purified from HEK 293 cells and quantified via silver staining. WT, wild type. (B) In histone acetyltransferase assays, the fusion proteins carrying the E-1021-K mutation in the HAT domain displayed significantly higher HAT activity than did the wild-type (WT) protein or the protein with the R-909-H mutation alone. GST-CBP-1 HD carrying both mutations present in cbp-1(ku258) displayed the highest level of HAT activity, which was nearly sevenfold higher than wild-type GST-CBP-1 HD. (C) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of histone acetyltransferase reactions followed by autoradiography demonstrated that the GST-CBP-1 HD fusion proteins were specifically able to acetylate purified mammalian histones.