Evolution of the holozoan ribosome biogenesis regulon

Abstract

Background: The ribosome biogenesis (RiBi) genes encode a highly-conserved eukaryotic set of nucleolar proteins involved in rRNA transcription, assembly, processing, and export from the nucleus. While the mode of regulation of this suite of genes has been studied in the yeast, Saccharomyces cerevisiae, how this gene set is coordinately regulated in the larger and more complex metazoan genomes is not understood.

Results: Here we present genome-wide analyses indicating that a distinct mode of RiBi regulation co-evolved with the E(CG)-binding, Myc:Max bHLH heterodimer complex in a stem-holozoan, the ancestor of both Metazoa and Choanoflagellata, the protozoan group most closely related to animals. These results show that this mode of regulation, characterized by an E(CG)-bearing core-promoter, is specific to almost all of the known genes involved in ribosome biogenesis in these genomes. Interestingly, this holozoan RiBi promoter signature is absent in nematode genomes, which have not only secondarily lost Myc but are marked by invariant cell lineages typically producing small body plans of 1000 somatic cells. Furthermore, a detailed analysis of 10 fungal genomes shows that this holozoan signature in RiBi genes is not found in hemiascomycete fungi, which evolved their own unique regulatory signature for the RiBi regulon.

Conclusion: These results indicate that a Myc regulon, which is activated in proliferating cells during normal development as well as during tumor progression, has primordial roots in the evolution of an inducible growth regime in a protozoan ancestor of animals. Furthermore, by comparing divergent bHLH repertoires, we conclude that regulation by Myc but not by other bHLH genes is responsible for the evolutionary maintenance of E(CG) sites across the RiBi suite of genes.

Figure 1

E(CG) is a core promoter element of Drosophila ribosome biogenesis (RiBi) genes. (A) Fly RiBi genes (5 examples shown) generally possess three common features around the transcriptional start site (rightward pointing arrow) and upstream of the translational start (ATG). This distinct promoter architecture is characterized by a CG-core E-box (blue box), a specific E(CG) flanking motif (green box) and a coordinating cluster of sites matching the DNA Replication Element, DRE, (red boxes) spanning a distance less than 100 bp. The distance of E(CG) to the TSS for each gene is indicated above E(CG). G6375 corresponds to the pit gene, which is a known Myc target and an RiBi gene [18]. (B) This core promoter architecture identifies several functional groups of genes associated with RiBi (green circles). The number of genes is indicated for functional groups with more than 3 members. The sum of 151 genes (large circle) is the sum of all of the individual subfunctions with specific roles in Ribosome Biogenesis. The RiBi genes encode a variety of domains and protein folds including RNA-binding regions (RNP-1), C-terminal helicases, DEAD/DEAH box helicases, WD-40 repeats, ARM repeats, Histone-folds, AAA ATPases and many others [see Additional file 1]. (C) The results of genome queries in Drosophila for E(CG) type core promoters results in a highly significant enrichment of GO terms directly related to ribosome biogenesis (nucleolar, rRNA metabolism, rRNA binding, snoRNA complex, pseudouridine synthesis, ribosomal subunits, etc.)

Figure 2

Holozoan RiBi promoters are enriched in E(CG) sites. The percentage of E(CG)-bearing promoters in RiBi genes is two to four fold higher in D. melanogaster (Dm), H. sapiens (Hs), N. vectensis (Nv), and M. brevicollis (Mb) relative to negative control sequences composed of promoter regions of downstream conserved genes (C₁), the 3' regions of RiBi genes (C2), or the promoters of genes with GO mitochondrial classification (C_M). This difference between RiBi and C₁, C₂, or C_M is lacking in outgroup genomes such as S. cerevisiae (Sc), which lack Myc, as well as in the nematode genome of C. elegans (Ce), which has secondarily lost Myc (Fig. 3). Inset depicts phylogenetic relationships among these organisms.

Figure 3

The RiBi-E(CG) regulon occurs only in Myc-bearing holozoan genomes. (A) Specific amino acid residues in holozoan MAX (red), MAD (blue), MNT (pink), and MYC (green) allow identification among the MYC/MAX superfamily bHLH genes (common superfamily residues in yellow and underlined). Only three bHLH genes were found in the choanoflagellate genome of Monosiga brevicollis: Mb-MYC, MbMAX and MbMUSH, corresponding to Myc and Max orthologs, and a distant MITF/USF/SREBP homolog (not shown). No Myc and Max orthologs were found outside of Holozoa. The predicted amino acid sequences of the bHLH regions of the M. brevicollis Myc/Max family of genes are shown aligned to Drosophila, Caenorhabditis, and Nematostella orthologs. (B) The presence of Myc (green filled boxes) is correlated with multiple genomes possessing the E(CG)-RiBi signature (green filled boxes). Other bHLH genes in the Myc superfamily (Max, Mad, Mnt; gray filled boxes) are either not necessary (Mad or Mnt) or insufficient (Max) to explain the occurrence of E(CG) sites in the RiBi regulon. "X" boxes indicate absence of a gene or E(CG) signature as indicated.

Figure 4

Frequency of E(CG) in opisthokont RiBi promoters. The frequency of E(CG) in 25 opisthokont RiBi orthologs was investigated. These 25 RiBi orthologs were selected based on the presence of conserved E(CG) sites in the human, fly, sea anemone, and choanoflagellate orthologs (Table 1B). DNA sequences 500 bp upstream from the translational start sites in the RiBi orthologs of S. cerevisiae (Sc), C. glabrata (Cg), K. lactis (Kl), A. gossypii (Ag), P. stipitis (Ps), D. hansenii (Dh), C. albicans (Ca), Y. lipolytica (Yl), N. crassa (Nc), S. pombe (Sp), and M. brevicollis (Mb) were collected. For D. melanogaster (Dm), 500 bp of DNA sequence (± 250 bp) from the 5' annotated end was collected. The sequences of each of these opisthokont promoters was analyzed for the presence of E(CG) motifs. The percentage of (ECG) in each species' RiBi orthologs is depicted on the Y-axis with the number of orthologs containing E(CG) over the total ortholog number of orthologs displayed above each genome. Key nodes for latest common ancestors (LCAs) are depicted in the phylogenetic tree [59-61].

Figure 5

Evolution of shared motifs in opisthokont RiBi core promoters. The promoter gene sets for each species depicted in Figure 4 weres analyzed by MEME to identify common cis-regulatory motifs for each lineage [57,58]. Motifs found in greater than two-thirds of RiBi genes are depicted from left to right (highest scoring motifs to left). The frequency of each motif is expressed as a percentage in the upper left corner. The known fungal motifs RRPE [5,6] and PAC [5-7] are shown in shown in black and blue, respectfully. The holozoan E(CG) motif identified in this work is shown in orange.