Simplicity is the Ultimate Sophistication-Crosstalk of Post-translational Modifications on the RNA Polymerase II.
TL;DR: The highly conserved C-terminal domain (CTD) of the largest subunit of RNA polymerase II comprises a consensus heptad (Y1S2P3T4S5P6S7) repeated multiple times as mentioned in this paper.
About: This article is published in Journal of Molecular Biology.The article was published on 2021-03-04 and is currently open access. It has received 8 citations till now. The article focuses on the topics: RNA polymerase II & Eukaryotic transcription.
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TL;DR: In this paper, the authors provide an overview of the basic modes of PTM crosstalk, the proteomic methods to elucidate PTM co-stalk and approaches that can inform about the functional consequences.
65 citations
TL;DR: In this paper, a modified Intacellular Antibilinear Antibody (mintbody) was used to detect the sites of RNAP2 Ser2ph-mintbody foci.
Abstract: In eukaryotic nuclei, most genes are transcribed by RNA polymerase II (RNAP2), whose regulation is a key to understanding the genome and cell function. RNAP2 has a long heptapeptide repeat (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7), and Ser2 is phosphorylated on an elongation form. To detect RNAP2 Ser2 phosphorylation (RNAP2 Ser2ph) in living cells, we developed a genetically encoded modification-specific intracellular antibody (mintbody) probe. The RNAP2 Ser2ph-mintbody exhibited numerous foci, possibly representing transcription "factories," and foci were diminished during mitosis and in a Ser2 kinase inhibitor. An in vitro binding assay using phosphopeptides confirmed the mintbody's specificity. RNAP2 Ser2ph-mintbody foci were colocalized with proteins associated with elongating RNAP2 compared with factors involved in the initiation. These results support the view that mintbody localization represents the sites of RNAP2 Ser2ph in living cells. RNAP2 Ser2ph-mintbody foci showed constrained diffusional motion like chromatin, but they were more mobile than DNA replication domains and p300-enriched foci, suggesting that the elongating RNAP2 complexes are separated from more confined chromatin domains.
10 citations
05 Aug 2021
TL;DR: The development of various MS techniques are discussed and the pros and cons of each technique are highlighted to provide future investigators with a comprehensive overview of how MS can be used to investigate the complexities of RNAP-II mediated transcription.
Abstract: RNA polymerase II (RNAP II) is one of the primary enzymes responsible for expressing protein-encoding genes and some small nuclear RNAs. The enigmatic carboxy-terminal domain (CTD) of RNAP II and its phosphorylation state are critically important in regulating transcription in vivo. Early methods of identifying phosphorylation on the CTD heptad were plagued by issues of low specificity and ambiguous signals. However, advancements in the field of mass spectrometry (MS) have presented the opportunity to gain new insights into well-studied processes as well as explore new frontiers in transcription. By using MS, residues which are modified within the CTD heptad and across repeats are now able to be pinpointed. Likewise, identification of kinase and phosphatase specificity towards residues of the CTD has reached a new level of accuracy. Now, MS is being used to investigate the crosstalk between modified residues of the CTD and may be a critical technique for understanding how phosphorylation plays a role in the new LLPS model of transcription. Herein, we discuss the development of various MS techniques and evaluate their capabilities. By highlighting the pros and cons of each technique, we aim to provide future investigators with a comprehensive overview of how MS can be used to investigate the complexities of RNAP-II mediated transcription.
5 citations
TL;DR: In this paper, a quantitative parallel reaction monitoring (PRM) method was developed to monitor spatiotemporal changes in site-specific Ser5 phosphorylation of the carboxyl-terminal domain (CTD) by cyclin-dependent kinase 7 (CDK7) using UVPD for sequence identification, phosphosite localization, and differentiation of phosphopeptide isomers.
Abstract: The critical role of site-specific phosphorylation in eukaryotic transcription has motivated efforts to decipher the complex phosphorylation patterns exhibited by the carboxyl-terminal domain (CTD) of RNA polymerase II. Phosphorylation remains a challenging post-translational modification to characterize by mass spectrometry owing to the labile phosphate ester linkage and low stoichiometric prevalence, two features that complicate analysis by high-throughput MS/MS methods. Identifying phosphorylation sites represents one significant hurdle in decrypting the CTD phosphorylation, a problem exaggerated by a large number of potential phosphorylation sites. An even greater obstacle is decoding the dynamic phosphorylation pattern along the length of the periodic CTD sequence. Ultraviolet photodissociation (UVPD) is a high-energy ion activation method that provides ample backbone cleavages of peptides while preserving labile post-translational modifications that facilitate their confident localization. Herein, we report a quantitative parallel reaction monitoring (PRM) method developed to monitor spatiotemporal changes in site-specific Ser5 phosphorylation of the CTD by cyclin-dependent kinase 7 (CDK7) using UVPD for sequence identification, phosphosite localization, and differentiation of phosphopeptide isomers. We capitalize on the series of phospho-retaining fragment ions produced by UVPD to create unique transition lists that are pivotal for distinguishing the array of phosphopeptides generated from the CTD.
3 citations
TL;DR: In this paper, the authors developed a modification-specific Intacellular Antibody (mintbody) probe to detect the Ser2- phosphorylated, elongating form of RNA Polymerase II in living cells.
Abstract: In eukaryotic nuclei, most genes are transcribed by RNA polymerase II (RNAP2). How RNAP2 transcription is regulated in the nucleus is a key to understanding the genome and cell function. The largest subunit of RNAP2 has a long heptapeptide repeat (Tyr1-Ser2-Pro3-Thr4-Ser5- Pro6-Ser7) at the C-terminal domain and Ser2 is phosphorylated on an elongation form of RNAP2. To detect RNAP2 Ser2 phosphorylation (RNAP2 Ser2ph) in living cells, we developed a genetically encoded modification-specific intracellular antibody (mintbody) probe. The RNAP2 Ser2ph-mintbody probe exhibited numerous foci, possibly representing transcription “factories” in living HeLa cells, and foci were diminished when cells were treated with triptolide to induce RNAP2 degradation and with flavopiridol to inhibit Ser2ph. An in vitro binding assay using phospho-peptides confirmed the Ser2ph-specific binding of the mintbody. These results support the view that mintbody localization represents the sites of RNAP2 Ser2ph in living cells. RNAP2 Ser2ph-mintbody foci were colocalized with proteins associated with elongating RNAP2, such as the CDK12 and Paf1 complex component, compared to factors involved in transcription activation around the transcription start sites, such as CDK9 and BRD4. Tracking analysis revealed that RNAP2 Ser2ph-mintbody foci showed constrained diffusional motion like chromatin, but was more mobile compared to euchromatin domains, suggesting that the elongating RNAP2 complexes are separated from the more confined initiating clusters. Summary The authors developed a genetically encoded probe to specifically detect the Ser2- phosphorylated, elongating form of RNA Polymerase II in living cells. The motion of Ser2- phosphorylated polymerase foci was more dynamic than chromatin domains, suggesting that the elongating complexes are separated from the more confined initiating clusters.
2 citations
References
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TL;DR: A procedure for preparing extracts from nuclei of human tissue culture cells that directs accurate transcription initiation in vitro from class II promoters, including tRNA and Ad 2 VA, is developed.
Abstract: We have developed a procedure for preparing extracts from nuclei of human tissue culture cells that directs accurate transcription initiation in vitro from class II promoters. Conditions of extraction and assay have been optimized for maximum activity using the major late promoter of adenovirus 2. The extract also directs accurate transcription initiation from other adenovirus promoters and cellular promoters. The extract also directs accurate transcription initiation from class III promoters (tRNA and Ad 2 VA).
10,800 citations
TL;DR: It is proposed that distinct histone modifications, on one or more tails, act sequentially or in combination to form a ‘histone code’ that is, read by other proteins to bring about distinct downstream events.
Abstract: Histone proteins and the nucleosomes they form with DNA are the fundamental building blocks of eukaryotic chromatin. A diverse array of post-translational modifications that often occur on tail domains of these proteins has been well documented. Although the function of these highly conserved modifications has remained elusive, converging biochemical and genetic evidence suggests functions in several chromatin-based processes. We propose that distinct histone modifications, on one or more tails, act sequentially or in combination to form a 'histone code' that is, read by other proteins to bring about distinct downstream events.
8,265 citations
TL;DR: Insight is given into the connections between chromatin modifications and transcriptional regulatory activity and a novel functional enhancer for the carnitine transporter SLC22A5 (OCTN2) is uncovered, providing a new tool for the functional annotation of the human genome.
Abstract: Eukaryotic gene transcription is accompanied by acetylation and methylation of nucleosomes near promoters, but the locations and roles of histone modifications elsewhere in the genome remain unclear. We determined the chromatin modification states in high resolution along 30 Mb of the human genome and found that active promoters are marked by trimethylation of Lys4 of histone H3 (H3K4), whereas enhancers are marked by monomethylation, but not trimethylation, of H3K4. We developed computational algorithms using these distinct chromatin signatures to identify new regulatory elements, predicting over 200 promoters and 400 enhancers within the 30-Mb region. This approach accurately predicted the location and function of independently identified regulatory elements with high sensitivity and specificity and uncovered a novel functional enhancer for the carnitine transporter SLC22A5 (OCTN2). Our results give insight into the connections between chromatin modifications and transcriptional regulatory activity and provide a new tool for the functional annotation of the human genome.
3,215 citations
TL;DR: Methylation patterns at orthologous loci are strongly conserved between human and mouse even though many methylated sites do not show sequence conservation notably higher than background, which suggests that the DNA elements that direct the methylation represent only a small fraction of the region or lie at some distance from the site.
1,536 citations
TL;DR: It is shown that the ubiquitin-conjugating enzyme Rad6 (Ubc2) mediates methylation of histone H3 at lysine 4 (Lys 4) through ubiquitination of H2B at Lys 123 in yeast (Saccharomyces cerevisiae) to reveal a pathway leading to gene regulation through concerted histone modifications on distinct histone tails.
Abstract: In eukaryotes, the DNA of the genome is packaged with histone proteins to form nucleosomal filaments, which are, in turn, folded into a series of less well understood chromatin structures. Post-translational modifications of histone tail domains modulate chromatin structure and gene expression. Of these, histone ubiquitination is poorly understood. Here we show that the ubiquitin-conjugating enzyme Rad6 (Ubc2) mediates methylation of histone H3 at lysine 4 (Lys 4) through ubiquitination of H2B at Lys 123 in yeast (Saccharomyces cerevisiae). Moreover, H3 (Lys 4) methylation is abolished in the H2B-K123R mutant, whereas H3-K4R retains H2B (Lys 123) ubiquitination. These data indicate a unidirectional regulatory pathway in which ubiquitination of H2B (Lys 123) is a prerequisite for H3 (Lys 4) methylation. We also show that an H2B-K123R mutation perturbs silencing at the telomere, providing functional links between Rad6-mediated H2B (Lys 123) ubiquitination, Set1-mediated H3 (Lys 4) methylation, and transcriptional silencing. Thus, these data reveal a pathway leading to gene regulation through concerted histone modifications on distinct histone tails. We refer to this as 'trans-tail' regulation of histone modification, a stated prediction of the histone code hypothesis.
1,096 citations