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Open accessJournal ArticleDOI: 10.1016/J.JMB.2021.166912

Simplicity is the Ultimate Sophistication-Crosstalk of Post-translational Modifications on the RNA Polymerase II.

04 Mar 2021-Journal of Molecular Biology (Academic Press)-Vol. 433, Iss: 14, pp 166912-166912
Abstract: The highly conserved C-terminal domain (CTD) of the largest subunit of RNA polymerase II comprises a consensus heptad (Y1S2P3T4S5P6S7) repeated multiple times. Despite the simplicity of its sequence, the essential CTD domain orchestrates eukaryotic transcription and co-transcriptional processes, including transcription initiation, elongation, and termination, and mRNA processing. These distinct facets of the transcription cycle rely on specific post-translational modifications (PTM) of the CTD, in which five out of the seven residues in the heptad repeat are subject to phosphorylation. A hypothesis termed the "CTD code" has been proposed in which these PTMs and their combinations generate a sophisticated landscape for spatiotemporal recruitment of transcription regulators to Pol II. In this review, we summarize the recent experimental evidence understanding the biological role of the CTD, implicating a context-dependent theme that significantly enhances the ability of accurate transcription by RNA polymerase II. Furthermore, feedback communication between the CTD and histone modifications coordinates chromatin states with RNA polymerase II-mediated transcription, ensuring the effective and accurate conversion of information into cellular responses.

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Topics: RNA polymerase II (65%), Eukaryotic transcription (62%), RNA polymerase (59%) ... show more
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8 results found


Open accessJournal ArticleDOI: 10.1039/D1CB00083G
05 Aug 2021-
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.

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Topics: RNA polymerase II (57%), Transcription (biology) (52%), CTD (51%)

2 Citations


Open accessJournal ArticleDOI: 10.1016/J.MCPRO.2021.100129
Abstract: Post-translational modification (PTM) of proteins allows cells to regulate protein functions, transduce signals and respond to perturbations. PTMs expand protein functionality and diversity, which leads to increased proteome complexity. PTM crosstalk describes the combinatorial action of multiple PTMs on the same or on different proteins for higher order regulation. Here we review how recent advances in proteomic technologies, mass spectrometry instrumentation, and bioinformatics spurred the proteome-wide identification of PTM crosstalk through measurements of PTM sites. We provide an overview of the basic modes of PTM crosstalk, the proteomic methods to elucidate PTM crosstalk, and approaches that can inform about the functional consequences of PTM crosstalk.

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Topics: Crosstalk (biology) (56%)

1 Citations


Open accessJournal ArticleDOI: 10.1016/J.JMB.2021.167037
Dylan J. Taatjes1Institutions (1)

1 Citations


Open accessPosted ContentDOI: 10.1101/2021.04.27.441582
Satoshi Uchino1, Yuma Ito1, Yuko Sato1, Tetsuya Handa1  +3 moreInstitutions (2)
27 Apr 2021-bioRxiv
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.

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Topics: RNA polymerase II (63%), Transcription (biology) (58%), Chromatin (57%) ... show more

1 Citations


Journal ArticleDOI: 10.1016/J.BIOSYSTEMS.2021.104468
Giorgio Dieci1Institutions (1)
30 Jun 2021-BioSystems
Abstract: In eukaryotes, RNA polymerase II (Pol II) is responsible for the synthesis of all mRNAs and myriads of short and long untranslated RNAs, whose fabrication involves close spatiotemporal coordination between transcription, RNA processing and chromatin modification. Crucial for such a coordination is an unusual C-terminal domain (CTD) of the Pol II largest subunit, made of tandem repetitions (26 in yeast, 52 in chordates) of the heptapeptide with the consensus sequence YSPTSPS. Although largely unstructured and with poor sequence content, the Pol II CTD derives its extraordinary functional versatility from the fact that each amino acid in the heptapeptide can be posttranslationally modified, and that different combinations of CTD covalent marks are specifically recognized by different protein binding partners. These features have led to propose the existence of a Pol II CTD code, but this expression is generally used by authors with some caution, revealed by the frequent use of quote marks for the word 'code'. Based on the theoretical framework of code biology, it is argued here that the Pol II CTD modification system meets the requirements of a true organic code, where different CTD modification states represent organic signs whose organic meanings are biological reactions contributing to the many facets of RNA biogenesis in coordination with RNA synthesis by Pol II. Importantly, the Pol II CTD code is instantiated by adaptor proteins possessing at least two distinct domains, one of which devoted to specific recognition of CTD modification profiles. Furthermore, code rules can be altered by experimental interchange of CTD recognition domains of different adaptor proteins, a fact arguing in favor of the arbitrariness, and thus bona fide character, of the Pol II CTD code. Since the growing family of CTD adaptors includes RNA binding proteins and histone modification complexes, the Pol II CTD code is by its nature integrated with other organic codes, in particular the splicing code and the histone code. These issues will be discussed taking into account fascinating developments in Pol II CTD research, like the discovery of novel modifications at non-consensus sites, the recently recognized CTD physicochemical properties favoring liquid-liquid phase separation, and the discovery that the Pol II CTD, originated before the divergence of most extant eukaryotic taxa, has expanded and diversified with developmental complexity in animals and plants.

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Topics: CTD (54%), Histone code (50%)

1 Citations


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137 results found


Open accessJournal ArticleDOI: 10.1093/NAR/11.5.1475
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).

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Topics: RNA polymerase II (64%), Transcription factor II D (63%), Sigma factor (61%) ... show more

10,734 Citations


Journal ArticleDOI: 10.1038/47412
Brian D. Strahl1, C D Allis1Institutions (1)
06 Jan 2000-Nature
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.

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Topics: Histone code (78%), Histone H4 (72%), Histone H2A (69%) ... show more

7,838 Citations


Journal ArticleDOI: 10.1038/NG1966
Nathaniel D. Heintzman1, Rhona K. Stuart1, Gary C. Hon1, Yutao Fu2  +11 moreInstitutions (4)
01 Mar 2007-Nature Genetics
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.

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Topics: ChIA-PET (67%), Enhancer RNAs (64%), Histone code (63%) ... show more

2,955 Citations


Open accessJournal ArticleDOI: 10.1016/J.CELL.2005.01.001
28 Jan 2005-Cell
Abstract: We mapped histone H3 lysine 4 di- and trimethylation and lysine 9/14 acetylation across the nonrepetitive portions of human chromosomes 21 and 22 and compared patterns of lysine 4 dimethylation for several orthologous human and mouse loci. Both chromosomes show punctate sites enriched for modified histones. Sites showing trimethylation correlate with transcription starts, while those showing mainly dimethylation occur elsewhere in the vicinity of active genes. Punctate methylation patterns are also evident at the cytokine and IL-4 receptor loci. The Hox clusters present a strikingly different picture, with broad lysine 4-methylated regions that overlay multiple active genes. We suggest these regions represent active chromatin domains required for the maintenance of Hox gene expression. 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. This 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.

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Topics: Histone H3 Lysine 4 (63%), Histone methylation (60%), Histone methyltransferase (59%) ... show more

1,461 Citations


Journal ArticleDOI: 10.1038/NATURE00883
Zu-Wen Sun1, C. David Allis1Institutions (1)
04 Jul 2002-Nature
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.

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Topics: Histone H2B ubiquitination (78%), Histone ubiquitination (76%), Histone code (72%) ... show more

1,053 Citations


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