Genome-wide analysis of bromodomain gene family in Arabidopsis and rice

Abstract

The bromodomain-containing proteins (BRD-proteins) belongs to family of 'epigenetic mark readers', integral to epigenetic regulation. The BRD-members contain a conserved 'bromodomain' (BRD/BRD-fold: interacts with acetylated-lysine in histones), and several additional domains, making them structurally/functionally diverse. Like animals, plants also contain multiple Brd-homologs, however the extent of their diversity and impact of molecular events (genomic duplications, alternative splicing, AS) therein, is relatively less explored. The present genome-wide analysis of Brd-gene families of Arabidopsis thaliana and Oryza sativa showed extensive diversity in structure of genes/proteins, regulatory elements, expression pattern, domains/motifs, and the bromodomain (w.r.t. length, sequence, location) among the Brd-members. Orthology analysis identified thirteen ortholog groups (OGs), three paralog groups (PGs) and four singleton members (STs). While more than 40% Brd-genes were affected by genomic duplication events in both plants, AS-events affected 60% A. thaliana and 41% O. sativa genes. These molecular events affected various regions (promoters, untranslated regions, exons) of different Brd-members with potential impact on expression and/or structure-function characteristics. RNA-Seq data analysis indicated differences in tissue-specificity and stress response of Brd-members. Analysis by RT-qPCR revealed differential abundance and salt stress response of duplicate A. thaliana and O. sativa Brd-genes. Further analysis of AtBrd gene, AtBrdPG1b showed salinity-induced modulation of splicing pattern. Bromodomain (BRD)-region based phylogenetic analysis placed the A. thaliana and O. sativa homologs into clusters/sub-clusters, mostly consistent with ortholog/paralog groups. The bromodomain-region displayed several conserved signatures in key BRD-fold elements (α-helices, loops), along with variations (1-20 sites) and indels among the BRD-duplicates. Homology modeling and superposition identified structural variations in BRD-folds of divergent and duplicate BRD-members, which might affect their interaction with the chromatin histones, and associated functions. The study also showed contribution of various duplication events in Brd-gene family expansion among diverse plants, including several monocot and dicot plant species.

Keywords: Arabidopsis thaliana; Oryza sativa; alternative splicing; bromodomain; bromodomain-containing genes; homology modeling; salt-stress response; tandem and block duplication.

Figure 1

Circle-plot representation of chromosomal distribution of Brd-genes in A. thaliana (A) and O. sativa (B) genomes. The gene designations are indicated in bold font, while the locus numbers, as per TAIR (for AtBrds) and RGAP (for OsBrds) databases, are given in the parenthesis. Colored connecting lines indicate the tandem/block-duplicated Brd-genes, ‘Chr1-5 (A. thaliana)/Chr1-12 (O. sativa)’ indicate chromosome numbers, ‘BD’ and ‘TD’ indicate block and tandem duplication events. (C) Conserved ortholog groups (OGs), paralog groups (PGs), and singleton BRD-members (STs), in A. thaliana (purple circles) and O. sativa (green circles), as per orthology analysis at Orthovenn2 server (https://orthovenn2.bioinfotoolkits.net/). Blue and red colored dotted lines indicate the block-duplicated (BD, blue line) and tandem-duplicated (TD, red line) Brds, while the functional information (based on SwissProt IDs) is indicated in the parenthesis (N/A indicates ‘No Hit’).

Figure 2

Comparison of gene structure and organization of Brd-genes from A. thaliana (A) and O. sativa (B), belonging to thirteen ortholog groups (OG1-13), three paralog groups (PG1-3), and singleton category (STs). The BRD-members specific to each group are arranged side-by-side for comparison, and the designations ‘BD’ and ‘TD’ in the names indicate the block or tandem duplication. Different regions of genes are indicated by color codes (orange: upstream/downstream region including UTR, green: exons, and dashed line: intron). Scale on the top indicates the length in kilobase (kb).

Figure 3

Comparison of domain architecture of BRD-proteins of A. thaliana (A) and O. sativa (B) belonging to thirteen ortholog groups (OG1-13), three paralog groups (PG1-3), and singleton category (STs). The BRD-members specific to each group are arranged side-by-side for comparison, and the designations ‘BD’ and ‘TD’ in the names indicate the block or tandem duplication. Domains/important functional sites (CDD and PROSITE prediction) are shown by different color codes. In AtBRD1d, OsBRDPG3c, color-coded lines (above/below) indicate spread of domains to adjacent regions. Scale on the top indicates the protein length (number of amino acids).

Figure 4

Alternative splicing (AS)-mediated changes in domain architecture of BRD isoforms of A. thaliana (A) and O. sativa (B) belonging to certain ortholog groups (OGs), paralog groups (PGs), and singleton category (STs). The members specific to each group are arranged side-by-side for comparison, and the designations ‘BD’ and ‘TD’ in the names indicate the block or tandem duplication. Domains/important functional sites among different isoforms (constitutive:.1 and alternative: .2 to .6) are shown by different color codes. Scale on the top indicates the protein length (number of amino acids).

Figure 5

Diversity of cis-regulatory elements in the upstream region (-2000 bp) of A. thaliana (A) and O. sativa (B) Brd-genes, belonging to thirteen ortholog groups (OG1-13), three paralog groups (PG1-3), and singleton category (STs), as per analysis at PlantCARE database. The Brd-members specific to each group are arranged side-by-side for comparison, and the designations ‘BD’ and ‘TD’ in the names indicate the block or tandem duplication. Different functional categories of cis-motifs are shown by different color code.

Figure 6

Analysis of upstream region (-2000 bp) of duplicated Brd-genes of A. thaliana (A) and O. sativa (B) on PlantPAN3.0 database, for difference in CpG islands (grey boxes), transcription factor binding sites (TFBS, indicated with numerals 1-6 in different genes) and repetitive motifs (R). The designation (and locus number) of Brd-members, association with ortholog group (OG)/paralog group (PG)/singleton category (ST) is indicated, and the designations ‘BD’ and ‘TD’ in the names indicate block or tandem duplication. Scale on the top indicates the length of upstream region (bp), arrow towards right indicates translation start site.

Figure 7

Expression analysis of Brd-genes: Heatmap-based analysis of RNA-Seq data for tissue-specific and abiotic stress-responsive expression pattern of Brd-genes of A. thaliana (A) and O. sativa (B), belonging to thirteen ortholog groups (OG1-13), three paralog groups (PG1-3), and singleton category (STs). Different tissues and stress conditions are indicated on the top, and names of Brd-genes are shown on the sides, with designations ‘BD’ and ‘TD’ indicating block or tandem duplication event. A continuous color gradient scale is indicative of the expression level (blue: low levels; red: high). RT-qPCR analysis of transcript levels of six duplicated AtBrd-gene pairs (C) and five OsBrd-gene pairs (D) in seedling tissues (top panels), and in response to salt stress (NaCl, 150 mM, bottom panels), using reference genes (AtActin; OselF1α). Designations ‘BD’ and ‘TD’ indicate block or tandem duplication event. (E) Expression pattern of two isoforms of AtBrdPG1b (constitutive:.1; alternative:.2) in Arabidopsis seedlings (top panel), and in response to salt stress (bottom panel). The analysis was carried out in triplicate, data is represented as mean ± SD, and statistical significance is indicated by *(p <0.05), **(p <0.01), ***(p <0.001), ns (no significant difference).

Figure 8

(A) Sequence logo analysis of the bromodomain (BRD)-region of 28 AtBRD and 22 OsBRD-homologs, indicating conserved residues in the key BRD-fold elements (helices: αZ, αA, αB, αC; loops: ZA, AB). ‘*’ indicates conserved sites (red ‘*’: conserved residues also in human BRDs; blue ‘*’: conserved sites in At- and OsBRDs), and ‘**’ indicates an invariant residue, and # indicates region specific to a single OsBrd (OsBRD2). Positions of amino acid residues (as per the alignment in Supplementary Figure 5 ) are indicated on the x-axis. (B) Comparison of conserved sites in key elements of BRD-fold among the duplicated BRDs of A. thaliana (top panel) and O. sativa (bottom panel). The association of duplicate-BRDs to different OGs/PGs or ST category is indicated, designations ‘BD’ and ‘TD’ indicate type of duplication event, while the variations at key positions are indicated by rectangular boxes.

Figure 9

(A) Neighbor-joining phylogenetic tree based on the bromodomain (BRD)-regions of A. thaliana and O. sativa Brd-homologs (including the block and tandem duplicates) generated by MEGA-X software. Major clusters are indicated by Roman numerals (I-VI), while sub-clusters are shown by letters (A-E). The ortholog group (OG), paralog group (PG) or singleton category (ST) designation is also indicated. Numbers at the nodes indicate bootstrap values (in %, for 500-replicates), and taxa names (AtBRDs: bold font, OsBRDs: regular font) include Brd-designation used, locus numbers (in parenthesis), and type of duplication events (BD: block duplication; TD: tandem duplication). ‘*’ Indicate the gene duplication event in the species. (B) Radiation tree of the BRD-regions of A. thaliana (AtBRDs: bold font style), O. sativa (OsBRDs: regular font style), and some representative human BRD-homologs (BRD2-4, BRD8B, BRDT, WDR9, TAF1, BRWD3, BRPF1A, BRD7, GCN5L2, ASH1L, TRIM33A, TRIM66, MLL, SMCA2, SMCA4, TRIM28, SP140, shown in grey font style), placed into seven major groups (GI-GVII) and subgroups (A-H). Designation ‘BD’ and ‘TD’ indicated block and tandem duplication events, while numerals in parenthesis (1/2) indicate two different domains of the dual-BRD-containing homologs.

Figure 10

Homology models of bromodomain (BRD)-folds of A. thaliana and O. sativa Brd-homologs generated at SWISS-MODEL workspace. (A) AtBRD1c model showing key BRD-fold elements (α-helices: αZ, αA, αB, αC; loops: ZA, AB and BC), (B) ^BD2AtBRD3b, AtBRD1d, OsBRD3b and OsBRD8, (C) ^BD3OsBRD5a and ^BD3OsBRD5b, (D) AtBRD6a and OsBRD6, (E) AtBRD7b and OsBRD7, (F) OsBRDST2 and OsBRD3a. Structural superposition of homology-models of duplicate BRDs (shown in different colors): (G) ^BD3AtBRD2a-^BD3AtBRD2b, (H) ^BD5AtBRD1a-^BD5AtBRD1b, (I) ^BD1OsBRD4a-^BD1OsBRDST1, and (J) ^BD4OsBRD2-^BD4OsBRD13. Structural variations (due to sequence/length heterogeneity or duplication events) are indicated by arrows.

Figure 11

Comparative assessment of duplication events affecting Brd-gene copies among different plant genomes: (A) lower photosynthetic organisms, (B) monocots, and (C) dicots, as per analysis at PLAZA database (version 4.5). Types of duplication events are indicated by different grey shades and designations ND (no duplication), TD (tandem duplication), BD (block duplication), and TD + BD (combined tandem and block duplication).