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Site-specific methylation and acetylation of lysine residues in the C-terminal domain (CTD) of RNA polymerase II

ABSTRACT: Dynamic modification of the carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII) regulates transcription-coupled processes in eukaryotes. The CTD in mammals is composed of 52 heptad-repeats with the consensus sequence Y1-S2-P3-T4-S5-P6-S7. Repeats in the distal part of CTD deviate from the consensus sequence and have frequently replaced serine at position 7 by lysine residues (K7). Mass spectrometry analysis revealed modification of K7 residues in heptad-repeats 39, 42, and 47/49 by acetylation and in heptad-repeats 38, 39, 40, 42, and 47/49 by mono-, di-, or trimethylation. Notably, acetylated as well as di- and tri-methylated K7 residues were found exclusively in phosphorylated CTD peptides, while mono-methylated K7 residues occurred also in non-phosphorylated CTD peptides. Methylation of K7 residues was further studied with the monoclonal antibody (mAb) 1F5, which recognizes mono- and di-methylated K7 in CTD. ChIP experiments revealed high levels of K7 methylation at the transcriptional start site of genes. The low levels of K7 methylation in the body of genes results from impairment of epitope recognition in hyper-phosphorylated CTD by mAb 1F5. In agreement with this notion, phosphatase treatment of hyper-phosphorylated CTD or treatment of cells with kinase inhibitor flavopiridol restored the reactivity of mAb 1F5 towards methylated K7 residues in CTD. We conclude that methylation and acetylation of K7 residues further expand the mammalian CTD code and potentially contribute to regulation of gene expression.

ORGANISM(S): Homo sapiens

PROVIDER: GSE72698 | GEO | 2015/12/01

SECONDARY ACCESSION(S): PRJNA294685

REPOSITORIES: GEO

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Project description:RPB1, 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. To 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. Our 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. We used a HA-tagged mouse RPB1 construct in which all K7 residues were substituted with arginines (8KR). This mutation resembles unacetylated lysines by conserving the positive charge at these positions, but preventing acetylation. To examine the potential functions of K7 acetylation in regulating gene expression, we stably expressed wildtype or 8KR HA-RPB1 in murine NIH/3T3 fibroblasts and cultured these cells in media containing α-amanitin. Both were expressed at equivalent levels, but acetylation was present only in wildtype, and not mutant HA-RPB1. We then performed gene expression profiling using the Affymetrix Mouse Gene 1.0 ST microarray with three biological replicates.

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Site-specific methylation and acetylation of lysine residues in the C-terminal domain (CTD) of RNA polymerase II

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