Plant orthologs of p300/CBP: conservation of a core domain in metazoan p300/CBP acetyltransferase-related proteins

Abstract

p300 and CBP participate as transcriptional coregulators in the execution of a wide spectrum of cellular gene expression programs controlling cell differentiation, growth and homeostasis. Both proteins act together with sequence-specific transcription factors to modify chromatin structure of target genes via their intrinsic acetyltransferase activity directed towards core histones and some transcription factors. So far, p300-related proteins have been described in animals ranging from Drosophila and Caenorhabditis elegans to humans. In this report, we describe p300/CBP-like polypeptides in the plant Arabidopsis thaliana. Interestingly, homology between animal and plant p300/CBP is largely restricted to a C-terminal segment, about 600 amino acids in length, which encompasses acetyltransferase and E1A-binding domains. We have examined whether this conservation in sequence is paralleled by a conservation in function. The same amino acid residues critical for acetyltransferase activity in human p300 are also critical for the function of one of the plant orthologs. Remarkably, plant proteins bind to the adenovirus E1A protein in a manner recapitulating the binding specificity of mammalian p300/CBP. The striking conservation of an extended segment of p300/CBP suggests that it may constitute a functional entity fulfilling functions that may be essential for all metazoan organisms.

Figure 1

Identification of p300/CBP-like molecules in the plant A.thaliana. (A) Schematic representations of human p300 (CBP) and PCAT1 and 2 are shown. PCAT3 and 4 are not depicted because they structurally resemble PCAT2. Regions of highest homology are highlighted as black boxes and the amino acids located at the borders of the C/H regions are numbered. Note the absence of the CREB binding region (marked CREB) and a bromodomain (marked bromo) in the plant PCAT proteins. (B) Alignment of the segments encompassing C/H2 and C/H3 regions of human p300, CBP and the two plant proteins PCAT1 and 2. The shading of the multiple sequence alignment was done using the standard settings of the Boxshade 3.21 program. Amino acids are shaded when proteins exhibit similar (green) or identical (red) residues at the respective position according to the rules specified in the documentation to the algorithm of the software: http://www.ch.embnet.org/software/BOX_doc.html. Asterisks above the aligned sequences indicate AT domain residues that were mutated in p300 and PCAT2 in Figure 4. (C) A sequence alignment of the C/H1 regions of hp300 and AthPCAT2, 3 and 4 is shown.

Figure 2

Ubiquitous expression of PCAT1–4 mRNAs. (A) Northern blot using total RNA isolated from A.thaliana seedling, stem, rosette or flower tissue. The blot was probed with a PCAT2 fragment and shows two hybridizing bands of ~5.5 and 7.3 kb. Both mRNAs are of sufficient length to harbor the complete PCAT2 protein coding region, which is 4.7 kb in length. The 5.5 kb band is marked by an arrow because it is likely to represent the mature form of the PCAT2 transcript. The panel at the bottom shows the hybridization signal of the 25S ribosomal RNA serving as a loading control. (B) PCAT1, 2, 3 and 4 transcripts are expressed in early stages of development (seedlings) and in the three major tissues (rosette, stem and flowers) of the adult plant. Fragments of each of the four PCAT transcripts (see Table 1 for location of amplified fragments) were amplified by RT–PCR from total RNA isolated from the indicated tissue. Amplification was stopped while it was still in the linear range. Signals should not be taken to reflect the precise relative or absolute levels of mRNA abundance.

Figure 3

PCAT2 exhibits HAT activity. GST-fusion proteins containing the AT domain of AthPCAT1 (amino acids 1055–1624) and AthPCAT2 (amino acids 985–1530) were incubated with calf thymus histones and [¹⁴C]acetyl-CoA and the proteins were analyzed by SDS–PAGE followed by Coomassie blue staining [shown in (A)] and viewed following autoradiography of the gel (B). For a comparison of relative activity, the AT domain of human p300 was expressed in parallel [lane 1 in (A) and (B)]. Of the two plant proteins, only the AT domain of PCAT2 exhibited enzymatic activity, suggesting that at least PCAT2 is indeed a bona fide HAT. Even after prolonged exposure, no histone acetylation could be detected in the PCAT1 lane.

Figure 4

Conserved residues which are essential for the AT activity of human p300 and CBP are also essential for the enzymatic activity of Arabidopsis PCAT2 and for stimulation of transcription by the respective AT domains. (A) Specific point mutations were introduced into the GST–AT domain fusion proteins of both species (see Fig. 1B and Materials and Methods for exact location of mutations) and HAT assays were performed. The top panel shows an autoradiograph of the SDS–PAGE gel demonstrating that mutation of FPY or WY residues abolished AT activity of both human p300 and plant PCAT2. By contrast, mutation of the residues ML did not abolish AT activity of hp300 and reduced activity of the plant protein by a factor of about 3. The bottom panel shows a Coomassie blue stained section of the same protein gel revealing expression levels of the hp300 and PCAT2 GST–AT fusion proteins. (B) U2OS cells were transiently transfected with a GAL4-dependent reporter gene and GAL4–AT expression plasmids. GAL4–CBP AT strongly activated transcription of the reporter plasmid in a manner dependent on AT activity since GAL4–CBP (WY) was inactive. In comparison, GAL4–PCAT2 AT also activated transcription in an AT-dependent manner, but less efficently than the CBP AT domain.

Figure 5

The C/H3 domain of PCAT1 and 2 specifically binds to E1A and discriminates between mutant E1A proteins in a manner recapitulating the binding pattern of human p300. (A) A schematic representation of the different adenovirus mutants used in the E1A binding assay is shown. The wild-type virus expresses wild-type 12S E1A, dl1107 expresses E1A carrying a mutation in conserved region 2 (CR2) of E1A (constituting part of the RB protein family binding site), dl1101 expresses a mutant form of E1A lacking amino acids 5–24 (constituting part of the p300 binding site) and dl312 does not express E1A at all due to a deletion in the early region removing the E1A gene. (B and C) Shown on the right are schematic representations of hp300 and PCAT1, 2 and the GST-C/H3 fusion proteins used for the E1A interaction assays. Purified GST proteins containing the C/H3 domain of hp300 [(B) and (C) lanes 1–4], of PCAT1 [(B) lanes 9–12], PCAT2 [(C) lanes 9–12] or GST alone [(B) and (C) lanes 5–8] were incubated with cell lysates prepared from U2OS cells infected with the indicated wild-type or mutant adenovirus. Bound E1A proteins were detected by western blot. The asterisk in (B) marks a non-specific background band visible in lanes 9–13. This band is due to binding of the secondary antibody to an E.coli protein copurifying with the GST–AthPCAT1–C/H3 fusion protein.