Evolution of lysine acetylation in the RNA polymerase II C-terminal domain

Abstract

BackgroundRPB1, the largest subunit of RNA polymerase II, contains a highly modifiable C-terminal domain (CTD) that consists of variations of a consensus heptad repeat sequence (Y1S2P3T4S5P6S7). The consensus CTD repeat motif and tandem organization represent the ancestral state of eukaryotic RPB1, but across eukaryotes CTDs show considerable diversity in repeat organization and sequence content. These differences may reflect lineage-specific CTD functions mediated by protein interactions. Mammalian CTDs contain eight non-consensus repeats with a lysine in the seventh position (K7). Posttranslational acetylation of these sites was recently shown to be required for proper polymerase pausing and regulation of two growth factor-regulated genes.ResultsTo investigate the origins and function of RPB1 CTD acetylation (acRPB1), we computationally reconstructed the evolution of the CTD repeat sequence across eukaryotes and analyzed the evolution and function of genes dysregulated when acRPB1 is disrupted. Modeling the evolutionary dynamics of CTD repeat count and sequence content across diverse eukaryotes revealed an expansion of the CTD in the ancestors of Metazoa. The new CTD repeats introduced the potential for acRPB1 due to the appearance of distal repeats with lysine at position seven. This was followed by a further increase in the number of lysine-containing repeats in developmentally complex clades like Deuterostomia. Mouse genes enriched for acRPB1 occupancy at their promoters and genes with significant expression changes when acRPB1 is disrupted are enriched for several functions, such as growth factor response, gene regulation, cellular adhesion, and vascular development. Genes occupied and regulated by acRPB1 show significant enrichment for evolutionary origins in the early history of eukaryotes through early vertebrates.ConclusionsOur combined functional and evolutionary analyses show that RPB1 CTD acetylation was possible in the early history of animals, and that the K7 content of the CTD expanded in specific developmentally complex metazoan lineages. The functional analysis of genes regulated by acRPB1 highlight functions involved in the origin of and diversification of complex Metazoa. This suggests that acRPB1 may have played a role in the success of animals.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0327-z) contains supplementary material, which is available to authorized users.

Phosphorylation Induces Sequence-Specific Conformational Switches in the RNA Polymerase II C-Terminal Domain

“…The number and complexity of these non-consensus heptads roughly correlate with developmental complexity 3 . Expression of genes involved in multicellularity were affected by mutating non-consensus heptads of mouse cells in culture 4 , despite data indicating that the non-consensus heptads are not essential for the viability of human cells grown in culture 5 . Likewise, deletion of a small region encompassing several non-consensus heptads caused severe developmental defects and growth retardation in mice 6 , demonstrating that non-consensus heptads may contribute to development and cellular differentiation.…”

“…The diversity of CTD sequences across eukaryotes has been recently acknowledged 3 , though the functional significance of many non-consensus heptads has not been widely investigated. Non-consensus CTD repeats may expand the repertoire of available PTMs, thus increasing the complexity of signalling through the CTD 4,[35][36][37][38] . In our minimal system, we observed that non-consensus motifs that contained conservative deviations from the consensus responded similarly to Dm P-TEFb and Ssu72.…”

“…For NMR experiments, expression of CTD2 0 was performed in M9 minimal media enriched with 15 N-NH 4 Cl and/or 13 C-D-Glucose (Cambridge Isotope Laboratories). Following purification, samples were buffer exchanged in Amicon Ultra-15 3,000 NMWL centrifugal filters (Merck Millipore Ltd.).…”

Crosstalk between RNA Pol II C-Terminal Domain Acetylation and Phosphorylation via RPRD Proteins

“…In addition, lysine residues at position 7 of eight heptad repeats are acetylated by the acetyltransferase p300/CBP (KAT3A/B) (K7ac), and are also mono-and di-methylated by an asyet-unknown methyltransferase (Dias et al, 2015;Schroder et al, 2013;Voss et al, 2015;Weinert et al, 2018). These lysine residues evolved in higher eukaryotes in the common ancestor of the metazoan lineage, and are highly conserved among vertebrates (Simonti et al, 2015). While lysine-7 mono-and di-methylation marks are found near promoters, K7ac is enriched in gene bodies (Dias et al, 2015).…”

“…Importantly, K7ac marks are found at ~80% of actively transcribed genes, with a peak in signal +500 bp downstream of the transcription start site (TSS), indicating that the modification could more broadly regulate the transition from transcription initiation to productive elongation (Schroder et al, 2013). In a genetic model in which all eight K7 residues were mutated to arginines (8KR), cells expressing 8KR RPB1 exhibited altered expression of genes relating to development, multicellularity and cell adhesion, underscoring a critical role of K7ac in the development of higher eukaryotes (Simonti et al, 2015).…”