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Title: Methylation of RNA polymerase II non-consensus Lysine residues marks early transcription in mammalian cells}
Article:
Abstract:
Dynamic post-translational modification of RNA polymerase II( RNAPII) coordinates the co-transcriptional recruitment of enzymatic complexes that regulate chromatin states and processing of nascent RNA. Extensive phosphorylation of serine residues at the largest RNAPII subunit occurs at its structurally-disordered C-terminal domain( CTD), which is composed of multiple heptapeptide repeats with consensus sequence Y1-S2-P3-T4-S5-P6-S7. Serine-5 and Serine-7 phosphorylation mark transcription initiation, whereas Serine-2 phosphorylation coincides with productive elongation. In vertebrates, the CTD has eight non-canonical substitutions of Serine-7 into Lysine-7, which can be acetylated( K7ac). Here, we describe mono- and di-methylation of CTD Lysine-7 residues( K7me1 and K7me2). K7me1 and K7me2 are observed during the earliest transcription stages and precede or accompany Serine-5 and Serine-7 phosphorylation. In contrast, K7ac is associated with RNAPII elongation, Serine-2 phosphorylation and mRNA expression. We identify an unexpected balance between RNAPII K7 methylation and acetylation at gene promoters, which fine-tunes gene expression levels.
Introduction:
Transcription of protein-coding genes is a complex process involving a sequence of RNA processing events that occur at different stages of the transcription cycle. Co-transcriptional recruitment of chromatin modifiers and RNA processing machinery is modulated through a complex array of post-translational modifications at the C-terminal domain( CTD) of RPB1, the largest subunit of RNAPII. This unique domain constitutes a docking platform for protein complexes that cap, splice and polyadenylate newly-made RNAs( Bentley, 2014; Buratowski, 2009; Egloff et al., 2012; Eick and Geyer, 2013; Hsin and Manley, 2012). The CTD also integrates signaling cascades that, for example, coordinate the DNA damage response and chromatin remodeling with gene expression( Munoz et al., 2009; Winsor et al., 2013). The CTD is a large, structurally disordered domain composed of a tandem heptapeptide repeat structure with the canonical sequence Y1-S2-P3-T4-S5-P6-S7. Extensive remodeling of the CTD occurs during distinct steps of the transcription cycle( Buratowski, 2003, 2009). RNAPII binds to promoter regions in a hypophosphorylated state, before the CTD becomes phosphorylated at Serine-5( S5p) and Serine-7( S7p), marking the earliest stages of transcription( Akhtar et al., 2009; Chapman et al., 2007; Tietjen et al., 2010). Productive elongation is characterized by an increase in phosphorylation of Serine-2( S2p) throughout gene bodies, with the highest levels found around transcription end sites( TES). S5p is important for recruitment of the capping machinery, while S2p is involved in the recruitment of splicing and polyadenylation factors( Corden, 2013; Ghosh et al., 2011; Gu et al., 2013; Lunde et al., 2010). Although the tandem repeat structure of the CTD was acquired very early in eukaryotic evolution, and general features of serine phosphorylation are fairly conserved from yeast to mammals, the number of repeats is highly variable among different taxa( Chapman et al., 2008; Yang and Stiller, 2014). The most complex multicellular organisms, such as vertebrates, generally have longer CTDs( e. g. 52 heptad repeats in mammals), whereas Drosophila melanogaster, Caenorhabditis elegans and unicellular yeast have 44, 42 and 26–29 copies, respectively. The mammalian CTD retains a core of 21 consensus repeats, but has accumulated a diversity of non-consensus repeats, particularly at its most C-terminal region( Figure 1a). In vertebrates, non-canonical amino-acid residues occur most frequently at the seventh position of the heptapeptide repeat, and the most frequent substitution replaces the canonical S7 residue with a lysine( K7; Figure 1a). The number of non-canonical K7-containing repeats increases from zero in yeast to one, three and eight repeats in C. elegans, D. melanogaster and vertebrates, respectively( Figure 1b). Previous work has shown that non-canonical CTD-K7 residues can be acetylated, and that CTD-K7ac is associated with transcriptional pausing at epidermal growth factor( EGF)-inducible genes in mouse fibroblasts( Schroeder et al., 2013). Evolutionary analyses also suggest that CTD-K7ac played a role in the origin of complex Metazoan lineages( Simonti et al., 2015). 10. 7554/eLife. 11215. 003Figure 1. Structure and evolutionary conservation of the C-terminal domain of RPB1.( a) Mouse RPB1 CTD is composed of 52 heptapeptide repeats with consensus amino-acid sequence YSPTSPS, which is represented 21 times at the most proximal CTD region. Non-consensus amino acids are enriched for at the distal region. Most abundant non-consensus residues are lysines, all found at heptad position 7( K7; represented in red). Other non-consensus residues are represented in blue.( b) Amino-acid sequence alignment of the most distal part of the CTD containing K7 residues across different species: Mus musculus( M. mus); Xenopus tropicalis( X. tro); Danio rerio( D. rer); Drosophila melanogaster( D. mel); and, Caenorhabditis elegans( C. ele). Conservation of CTD K7 residues is highlighted in yellow. CTD repeat numbering was done according to the mouse CTD sequence between repeats 35 and 49, and aligned to the other species CTDs from the position of the repeat containing the first lysine in each organism. RPB1, RNA polymerase II large subunit; CTD, C-terminal domain. DOI: http://dx. doi. org/10. 7554/eLife. 11215. 003 To further explore the increasing complexity of CTD modifications over evolution, their temporal sequence, and how they interplay with each other, we have investigated the possibility of additional modification of non-canonical CTD residues. We identify mono- and di-methylation of CTD-K7 residues in both vertebrates and invertebrates. We produce new antibodies specific to CTD-K7me1 and CTD-K7me2 and show that these novel modifications precede or accompany phosphorylation of S5 and S7, upstream of S2 phosphorylation. Using biochemical and genome-wide approaches, we show that CTD-K7 methylation is present at the promoters of genes that are productively transcribed into mature RNA, but defines the earliest stages of the transcription cycle. Through detailed analysis of abundance and distribution of different CTD modifications at gene promoters in embryonic stem( ES) cells, we show that gene expression levels depend on the balance between CTD-K7 methylation and acetylation.
Discussion:
The expansion of the RPB1 CTD in repeat number and amino acid diversity has been associated with increased complexity in specific groups of organisms, including vertebrates and plants( Corden, 2013; Yang and Stiller, 2014). Larger CTDs are thought to accommodate larger protein complexes. Non-canonical CTD repeats have the potential to expand the diversity of post-translational modifications, resulting in more complex signaling through the CTD. Although there is increasing recognition that complex CTDs may have specific roles in the regulation of more complex developmental programs, gene structures or gene organization( Litingtung et al., 1999; Liu et al., 2010; Simonti et al., 2015), our understanding of CTD signaling remains extremely limited, particularly as we have not yet identified all CTD modifications. A small number of modifications to non-canonical residues have been identified, and their importance for the expression of specific subgroups of genes revealed. In mammalian cells, methylation of a single CTD-R7 is involved in expression of snRNAs and snoRNAs( Sims et al., 2011). Acetylation of CTD-K7 residues, the most common non-canonical substitution, has been associated with inducible gene expression of EGF-regulated genes( Schroeder et al., 2013). In the present study, we show that CTD-K7 residues are mono- and di-methylated in mouse and human cells, and in the invertebrates C. elegans and D. melanogaster. Using murine cell lines with K7-to-S7 substitutions, we show that several CTD-K7 residues are simultaneously mono- and di-methylated, including a single K7 residue at repeat 35 of the murine CTD. Conservation of CTD-K7 methylation, and expansion of the number of methylated CTD-K7 residues from invertebrates to mammals, suggests functional relevance of this modification across taxa. Model systems characterized by K7 expansion( e. g. Plasmodium parasites; Kishore et al., 2009) further support the need for studying the extent of K7 modifications at RNAPII CTD. We demonstrate that CTD-K7 mono- and di-methylation are associated with the earliest stages of transcription, are refractory to CDK9 inhibition using flavopiridol, and are distributed in discrete foci in the nucleoplasm of single cells. We mapped the genome-wide occupancy of K7me1 and K7me2 in mouse ES cells and find that they are highly enriched at the promoters of active genes. CTD-K7 methylation levels are highly correlated with promoter levels of unmodified S2, S7p, and S5p, but are uncoupled from mRNA expression and S2p abundance. In contrast, we find that CTD-K7 acetylation expands downstream of promoter regions into gene bodies, and highly correlates with S2p and mRNA levels. One interesting aspect that emerges from studying CTD-K7 methylation and acetylation is that the two marks may compete for the eight available K7 residues at the CTD and thereby regulate gene expression levels. We observed that the ratio between K7me2 and K7ac at active promoters is inversely related to gene expression. Genes with similar promoter occupancy of RNAPII but which have higher levels of K7me2 relative to K7ac are significantly less expressed than genes that have higher levels of K7ac relative to K7me2. This suggests that a balance between K7 modifications fine-tunes gene expression levels. Further studies are required to explore the enzymatic activities that methylate and demethylate CTD-K7 residues, as well as the readers of methylated CTD-K7. We performed co-immunoprecipitation experiments of proteins associated with YFP-tagged RPB1 containing the wild-type CTD or the CTD bearing all K7-to-S7 mutations, followed by mass spectrometry, but this strategy failed to identify specific modifiers and readers( data not shown), suggesting that the interactions of RNAPII with CTD-K7 modifiers and/or readers may be transient and not easily captured by in vitro biochemical approaches. The identification of modified CTD-K7 residues( K7me1, K7me2, K7me3 and K7ac) expands the repertoire of CTD interactors to proteins containing domains that recognize these marks, such as PHD finger, bromo-, chromo-, tudor- and MBT-domains( Biggar and Li, 2015; Hamamoto et al., 2015). Cross-talk between K7 modification and other CTD modifications, such as phosphorylation, may also regulate the extent of factor recruitment. Future investigations of the CTD code should consider the contribution of K7 modifications to co-transcriptional gene regulation in development and disease.
Genes are sections of DNA that encode the instructions to make proteins . When a gene is switched on , the section of DNA is copied to make molecules of messenger ribonucleic acid ( RNA ) in a process called transcription . These messenger RNAs are then used as templates for protein production . In animals , plants and other eukaryotic organisms , an enzyme called RNA polymerase II is responsible for making messenger RNA molecules during transcription . This enzyme is made up of several proteins , the largest of which contains a long tail , called the carboxy-terminal domain . This domain is crucial for transcription because it serves as a landing platform for other enzymes that help to make the RNA molecules . The carboxy-terminal domain contains multiple repeats of a string of seven amino acids ( the building blocks of proteins ) . Normally , each repeat contains three amino acids called serines . However , in humans and other mammals , one of these serines is often substituted with another amino acid called lysine instead . This lysine ( referred to as Lysine-7 ) was known to be modified by the addition of a chemical group called an 'acetyl' tag , but it was not clear how this tag affected transcription . Dias , Rito , Torlai Triglia et al . carried out an in-depth study into how Lysine-7 is modified in mouse cells , and what effects these modifications have on transcription . The experiments show that Lysine-7 can also be modified by the addition of a different chemical group , called a 'methyl' tag . This new modification is also found in flies , worms and human cells , which suggests that it is generally important for transcription . Next , Dias , Rito , Torlai Triglia et al . found that in mouse stem cells , methyl tags are added to Lysine-7 during the earliest steps of transcription , before the acetyl tags are added . Further experiments show that a balance between the addition of methyl tags and acetyl tags to Lysine-7 fine-tunes the activity of RNA polymerase II . These findings add to our understanding of how cells control the activity of RNA polymerase II at different genes . Future challenges are to find out which enzymes are responsible for adding and removing these chemical tags , and how the balance between the methyl and acetyl modifications is controlled .