Analysis of histone acetyltransferase and histone deacetylase families of Arabidopsis thaliana suggests functional diversification of chromatin modification among multicellular eukaryotes

Abstract

Sequence similarity and profile searching tools were used to analyze the genome sequences of Arabidopsis thaliana, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans and Drosophila melanogaster for genes encoding three families of histone deacetylase (HDAC) proteins and three families of histone acetyltransferase (HAT) proteins. Plants, animals and fungi were found to have a single member of each of three subfamilies of the GNAT family of HATs, suggesting conservation of these functions. However, major differences were found with respect to sizes of gene families and multi-domain protein structures within other families of HATs and HDACs, indicating substantial evolutionary diversification. Phylogenetic analysis identified a new class of HDACs within the RPD3/HDA1 family that is represented only in plants and animals. A similar analysis of the plant-specific HD2 family of HDACs suggests a duplication event early in dicot evolution, followed by further diversification in the lineage leading to Arabidopsis. Of three major classes of SIR2-type HDACs that are found in animals, fungi have representatives only in one class, whereas plants have representatives only in the other two. Plants possess five CREB-binding protein (CBP)-type HATs compared with one to two in animals and none in fungi. Domain and phylogenetic analyses of the CBP family proteins showed that this family has evolved three distinct types of CBPs in plants. The domain architecture of CBP and TAF(II)250 families of HATs show significant differences between plants and animals, most notably with respect to bromodomain occurrence and their number. Bromodomain-containing proteins in Arabidopsis differ strikingly from animal bromodomain proteins with respect to the numbers of bromodomains and the other types of domains that are present. The substantial diversification of HATs and HDACs that has occurred since the divergence of plants, animals and fungi suggests a surprising degree of evolutionary plasticity and functional diversification in these core chromatin components.

Figure 1

Alternative splicing of HDA2, HDA15 and SRT2. Sequence coordinates indicate the position of exons within the unspliced transcripts relative to the start of the ‘alt1’ RT–PCR product sequences. The approximate location of predicted protein domains and conserved amino acid motifs is marked by brackets, their Pfam accessions are listed here. HDAC, the histone deacetylase domain (PF00850); SIR2, the multidomain core [including the conserved GAG, NID and CYS motifs (89) (PF02146)]; zf-RanBP, Ran-binding protein zinc finger (PF00641); (NLS), sequence similar to the bipartite nuclear localization sequence. SRT2alt3 and alt6 completely lack exon 2, which contains the predicted translation initiation codon of SRT2alt1. The nearest downstream ATG codon for translation initiation from SRT2alt3 and alt6 is located at position 446 of the unprocessed transcript. A consequence of translation initiation at this position would be a protein lacking a putative nuclear localization signal. Alternative splicing of exon 2 in SRT2alt2 and alt5 removes 39 nt of the 5′-UTR. Alternative splicing of exon 5 in SRT2alt4, alt5, and alt6 introduces a premature nonsense codon within the conserved multi-domain SIR2 core; alternative splicing at this position is conserved between SRT2 and a putative ortholog in tomato represented by ESTs 12635152 and 12625887.

Figure 2

Phylogenetic analysis of the RPD3/HDA1 HDAC superfamily. Unrooted neighbor-joining tree of 76 RPD3/HDA1 superfamily sequences includes four double-domain sequences with each domain being analyzed separately. Confidence levels of the branching patterns are: filled circle, excellent support (>99% of bootstrap replicas); empty square, good or >70%; empty circle, majority support or >50%. Eukaryotic gene names and sequence accession numbers are listed in Table 2. The plant proteins are highlighted in bold and the three eukaryotic classes are represented in gray shaded ovals. Prokaryotic genes are represented by Acu (acetoin utilization proteins) or by Aph (acetylpolyamine aminohydrolase proteins). All the proteins have abbreviated species names as prefix. The proteins and their accession numbers are identified in Table 2. Abbreviations for species are: Aeropyrum pernix (ap), Arabidopsis thaliana (at), Archaeoglobus fulgidus (af), Aquifex aeolicus (aa), Aspergillus nidulans (an), Bacillus halodurans (bh), Bacillus subtilis (bs), Caenorhabditis elegans (ce), Deinococcus radiodurans (dr), Drosophila melanogaster (dm), Glycine max (gm), Halobacterium sp. NRC-1 (halo), Homo sapiens (hs), Leishmania major (lm), Mesembryanthemum crystallinum (mc), Methanobacterium thermoautotrophicum (mt), Methanococcus jannaschii (mj), Mus musculus (mm), Mycoplana ramose (mr), Neisseria meningitides (nm), Oryza sativa (os), Plasmodium falciparum (pf), Pseudomonas aeruginosa (ps), Pyrococcus abyssi (pa), Pyrococcus horikoshii (ph), Saccharomyces cerevisiae (sc), Schizosaccharomyces pombe (sp), Staphylococcus xylosus (sx), Streptomyces coelicolor (stco), Synechococcus PCC7002 (syp), Synechocystis PCC6803 (syn), Tetrahymena thermophila (tt), Vibrio cholerae (vc), Zea mays (zm).

Figure 3

Schematic representation of the exon–intron and domain organization of the HDA18-HDA5-At5g61050 gene cluster on chromosome V. Coordinates indicate the position of the start and stop codons of the three genes in the P1 clone MAF19 (accession no. AB006696). The approximate location of predicted protein domains is marked by brackets. The dotted line indicates the missing HDAC domain in At5g61050. NES, nuclear export signal. Arrows indicate nucleic acid sequence repeats in HDA18.

Figure 4

Class III proteins in the RPD3/HDA1 protein superfamily have distinct motifs in the HDAC domain. Alignment of the HDAC domain of Arabidopsis HDA2 protein with human HDAC11, D.melanogaster HDA403 and C.elegans HDA308. These proteins and their accession numbers are identified in Table 2. Shading was done based on degree of identity or conservation using the Genedoc program. Also shown below the multiple sequence alignment is a second alignment of consensus motifs found in the proteins in all the three classes of HDACs identified in Figure 2. These motifs represent the most highly conserved sequence positions in the HDAC domain. The consensus motif for each class was identified by generating a logo sequence. Each class of proteins is indicated by a consensus of the sequences in that class: black boxes, positions conserved across all three classes; underlined, positions highly conserved within a class; upper case letters, 98% conserved within a class; lower case letters, 60% conserved within a class; X, variable positions. The amino acid positions in each sequence class refer to the location of these motifs in Arabidopsis HDA19 (Class I), HDA5 (Class II) and HDA2 (Class III) proteins.

Figure 5

Maximum likelihood analysis of the plant HD2 family nucleic acid sequences. This analysis is based upon a codon-by-codon alignment of the first 273 positions of the maize HD2 cDNA sequence, corresponding to the HDAC domain, with other plant cDNA and EST sequences listed in Table 2. The common name for each species is listed in parentheses and where common names are not available, the Latin name is included. The gene names and their accession numbers are identified in Table 2. Confidence levels for the tree branches that are best supported by bootstrap analysis are shown as percentages.

Figure 6

Phylogenetic analysis of plant SIR2 proteins. Unrooted neighbor-joining tree of 31 SIR2-related proteins shows the four previously identified classes of SIR2 proteins. The two plant protein clusters are highlighted in bold. Confidence levels of the branching patterns are: filled circle, excellent support (>99% of bootstrap replicas); empty square, good or >70%; empty circle, majority support or >50%. The genes and their accession numbers are identified in Table 2. Abbreviations for species are: Arabidopsis thaliana (at), Caenorhabditis elegans (ce), Drosophila melanogaster (dm), Homo sapiens (hs), Lycopersicon esculentum (le), Medicago truncatula (mt), Oryza sativa (os), Saccharomyces cerevisiae (sc), Schizosaccharomyces pombe (sp), Triticum aestivum (ta), Zea mays (zm).

Figure 7

Domain architecture of the CBP-type HAT family and phylogenetic analysis of their HAT domains. (A) Schematic representation of the domain organization of Arabidopsis and animal CBP proteins. Different domains are identified by different symbols and colors, and are shown at their approximate relative location in the protein sequence. The protein lengths are listed on the right. //, indicates position of extra sequence; /, indicates more sequence at the N- and C-terminus. The CBP-type HAT domain is conserved throughout its length between plants and animals, however, in plants a ZZ-type zinc finger domain is inserted near the C-terminus of the HAT domain. The Pfam accession number for the domain profiles is indicated in parentheses. (B) Unrooted neighbor-joining tree of 10 CBP-type HAT proteins based on the HAT domain. Distinct animals and Arabidopsis clusters are shown by two shaded ovals. Confidence levels of the branching patterns are: filled circle, excellent support (>99% of bootstrap replicas). The genes and their accession numbers are identified in Table 2. Abbreviations for species are as follows: Arabidopsis thaliana (at), Caenorhabditis elegans (ce), Drosophila melanogaster (dm), Homo sapiens (hs), Mus musculus (mm).

Figure 8

Domain architecture of the TAF_II250 proteins. A schematic representation is shown of the domain organization of Arabidopsis and animal TAF_II250 proteins aligned by the N-terminus of the HAT domain. Different domains are identified by different symbols and colors, and are shown at their approximate relative locations in the protein sequences. The protein lengths are listed on the right. Pfam accession numbers for the domain profiles are indicated in parentheses underneath the alignment. The sequences and their accession numbers are identified in Table 2. Abbreviations for species are: Arabidopsis thaliana (at), Caenorhabditis elegans (ce), Drosophila melanogaster (dm), Homo sapiens (hs), Saccharomyces cerevisiae (sc), Schizosaccharomyces pombe (sp).