PMC 20201221 pmc.key 4871749 NO-CC CODE no 0 0 10.1038/nchembio.2065 NIHMS769551 4871749 27089029 396 6 YEATS domain crotonylated lysine chromatin Taf14 histone PTM Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use: 398 surname:Andrews;given-names:Forest H. surname:Shinsky;given-names:Stephen A. surname:Shanle;given-names:Erin K. surname:Bridgers;given-names:Joseph B. surname:Gest;given-names:Anneliese surname:Tsun;given-names:Ian K. surname:Krajewski;given-names:Krzysztof surname:Shi;given-names:Xiaobing surname:Strahl;given-names:Brian D. surname:Kutateladze;given-names:Tatiana G. TITLE front 12 2016 0 The Taf14 YEATS domain is a reader of histone crotonylation 0.99879897 protein cleaner0 2023-07-04T16:20:21Z PR: Taf14 0.9920379 structure_element cleaner0 2023-07-04T16:20:28Z SO: YEATS domain 0.9768092 protein_type cleaner0 2023-07-04T16:21:00Z MESH: histone 0.60347056 ptm cleaner0 2023-07-04T16:18:43Z MESH: crotonylation ABSTRACT abstract 60 The discovery of new histone modifications is unfolding at startling rates, however, the identification of effectors capable of interpreting these modifications has lagged behind. Here we report the YEATS domain as an effective reader of histone lysine crotonylation – an epigenetic signature associated with active transcription. We show that the Taf14 YEATS domain engages crotonyllysine via a unique π-π-π-stacking mechanism and that other YEATS domains have crotonyllysine binding activity. protein_type MESH: cleaner0 2023-07-04T16:21:00Z histone 0.98157585 structure_element cleaner0 2023-07-04T16:20:28Z SO: YEATS domain 0.6092492 protein_type cleaner0 2023-07-04T16:21:00Z MESH: histone 0.64678025 residue_name cleaner0 2023-07-05T08:52:59Z SO: lysine 0.7005873 ptm cleaner0 2023-07-04T16:18:43Z MESH: crotonylation 0.9987513 protein cleaner0 2023-07-04T16:20:21Z PR: Taf14 0.98611736 structure_element cleaner0 2023-07-04T16:20:27Z SO: YEATS domain 0.99818015 residue_name melaniev@ebi.ac.uk 2023-07-06T15:25:20Z SO: crotonyllysine bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:20:44Z π-π-π-stacking 0.8457508 structure_element cleaner0 2023-07-05T08:49:39Z SO: YEATS domains residue_name SO: melaniev@ebi.ac.uk 2023-07-06T15:25:20Z crotonyllysine INTRO paragraph 560 Crotonylation of lysine residues (crotonyllysine, Kcr) has emerged as one of the fundamental histone post-translational modifications (PTMs) found in mammalian chromatin. This epigenetic PTM is widespread and enriched at active gene promoters and potentially enhancers. The crotonyllysine mark on histone H3K18 is produced by p300, a histone acetyltransferase also responsible for acetylation of histones. Owing to some differences in their genomic distribution, the crotonyllysine and acetyllysine (Kac) modifications have been linked to distinct functional outcomes. p300-catalyzed histone crotonylation, which is likely metabolically regulated, stimulates transcription to a greater degree than p300-catalyzed acetylation. The discovery of individual biological roles for the crotonyllysine and acetyllysine marks suggests that these PTMs can be read by distinct readers. While a number of acetyllysine readers have been identified and characterized, a specific reader of the crotonyllysine mark remains unknown (reviewed in). A recent survey of bromodomains (BDs) demonstrates that only one BD associates very weakly with a crotonylated peptide, however it binds more tightly to acetylated peptides, inferring that bromodomains do not possess physiologically relevant crotonyllysine binding activity. 0.97012514 ptm cleaner0 2023-07-04T16:18:43Z MESH: Crotonylation 0.8647513 residue_name cleaner0 2023-07-05T08:53:04Z SO: lysine 0.99726224 residue_name melaniev@ebi.ac.uk 2023-07-06T15:25:20Z SO: crotonyllysine 0.9968893 residue_name melaniev@ebi.ac.uk 2023-07-06T15:25:20Z SO: Kcr protein_type MESH: cleaner0 2023-07-04T16:21:00Z histone 0.9958947 taxonomy_domain cleaner0 2023-07-04T16:21:35Z DUMMY: mammalian 0.7653515 residue_name melaniev@ebi.ac.uk 2023-07-06T15:25:20Z SO: crotonyllysine protein_type MESH: cleaner0 2023-07-04T16:21:00Z histone protein_type MESH: cleaner0 2023-07-05T08:36:55Z H3 residue_name_number DUMMY: cleaner0 2023-07-05T08:37:14Z K18 0.98865753 protein cleaner0 2023-07-04T16:20:32Z PR: p300 0.98868376 protein_type cleaner0 2023-07-04T16:22:54Z MESH: histone acetyltransferase 0.7823207 ptm cleaner0 2023-07-04T16:18:49Z MESH: acetylation 0.9841765 residue_name melaniev@ebi.ac.uk 2023-07-06T15:25:20Z SO: crotonyllysine 0.9894874 residue_name melaniev@ebi.ac.uk 2023-07-06T15:25:20Z SO: acetyllysine 0.98921156 residue_name melaniev@ebi.ac.uk 2023-07-06T15:25:20Z SO: Kac 0.9711801 protein cleaner0 2023-07-04T16:20:33Z PR: p300 protein_type MESH: cleaner0 2023-07-04T16:21:00Z histone 0.9621592 ptm cleaner0 2023-07-04T16:18:43Z MESH: crotonylation 0.9124805 protein cleaner0 2023-07-04T16:20:33Z PR: p300 0.87436926 ptm cleaner0 2023-07-04T16:18:49Z MESH: acetylation 0.91712606 residue_name melaniev@ebi.ac.uk 2023-07-06T15:25:20Z SO: crotonyllysine 0.9561159 residue_name melaniev@ebi.ac.uk 2023-07-06T15:25:20Z SO: acetyllysine 0.8240044 residue_name melaniev@ebi.ac.uk 2023-07-06T15:25:20Z SO: acetyllysine 0.6335516 residue_name melaniev@ebi.ac.uk 2023-07-06T15:25:20Z SO: crotonyllysine 0.94788533 structure_element cleaner0 2023-07-04T16:21:48Z SO: bromodomains 0.8961469 structure_element cleaner0 2023-07-04T16:21:53Z SO: BDs 0.92061985 structure_element cleaner0 2023-07-05T08:51:22Z SO: BD 0.9774638 protein_state cleaner0 2023-07-04T16:22:06Z DUMMY: crotonylated 0.98200285 protein_state cleaner0 2023-07-04T16:22:11Z DUMMY: acetylated 0.7515075 structure_element cleaner0 2023-07-04T16:21:48Z SO: bromodomains residue_name SO: melaniev@ebi.ac.uk 2023-07-06T15:25:20Z crotonyllysine INTRO paragraph 1865 The family of acetyllysine readers has been expanded with the discovery that the YEATS (Yaf9, ENL, AF9, Taf14, Sas5) domains of human AF9 and yeast Taf14 are capable of recognizing the histone mark H3K9ac. The acetyllysine binding function of the AF9 YEATS domain is essential for the recruitment of the histone methyltransferase DOT1L to H3K9ac-containing chromatin and for DOT1L-mediated H3K79 methylation and transcription. Similarly, activation of a subset of genes and DNA damage repair in yeast require the acetyllysine binding activity of the Taf14 YEATS domain. Consistent with its role in gene regulation, Taf14 was identified as a core component of the transcription factor complexes TFIID and TFIIF. However, Taf14 is also found in a number of chromatin-remodeling complexes (i.e., INO80, SWI/SNF and RSC) and the histone acetyltransferase complex NuA3, indicating a multifaceted role of Taf14 in transcriptional regulation and chromatin biology. In this study, we identified the Taf14 YEATS domain as a reader of crotonyllysine that binds to histone H3 crotonylated at lysine 9 (H3K9cr) via a distinctive binding mechanism. We found that H3K9cr is present in yeast and is dynamically regulated. 0.9015483 residue_name melaniev@ebi.ac.uk 2023-07-06T15:25:20Z SO: acetyllysine 0.9973253 structure_element cleaner0 2023-07-05T08:51:28Z SO: YEATS 0.99829453 protein cleaner0 2023-07-05T07:54:03Z PR: Yaf9 0.99791545 protein cleaner0 2023-07-05T07:54:08Z PR: ENL 0.9983627 protein cleaner0 2023-07-05T07:54:14Z PR: AF9 0.9985991 protein cleaner0 2023-07-04T16:20:21Z PR: Taf14 0.9987198 protein cleaner0 2023-07-05T07:54:25Z PR: Sas5 0.96409446 species cleaner0 2023-07-04T16:22:25Z MESH: human 0.99878556 protein cleaner0 2023-07-05T07:54:15Z PR: AF9 0.9893267 taxonomy_domain cleaner0 2023-07-04T16:22:20Z DUMMY: yeast 0.9988275 protein cleaner0 2023-07-04T16:20:21Z PR: Taf14 protein_type MESH: cleaner0 2023-07-04T16:21:00Z histone protein_type MESH: cleaner0 2023-07-05T07:55:54Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:53:13Z K9ac residue_name SO: melaniev@ebi.ac.uk 2023-07-06T15:25:20Z acetyllysine 0.99856865 protein cleaner0 2023-07-05T07:54:15Z PR: AF9 0.9379061 structure_element cleaner0 2023-07-04T16:20:28Z SO: YEATS domain 0.9904277 protein_type cleaner0 2023-07-04T16:22:40Z MESH: histone methyltransferase 0.99846435 protein cleaner0 2023-07-05T07:57:04Z PR: DOT1L protein_type MESH: cleaner0 2023-07-05T07:56:42Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T12:00:58Z K9ac 0.9973254 protein cleaner0 2023-07-05T07:57:04Z PR: DOT1L protein_type MESH: cleaner0 2023-07-05T07:57:26Z H3 residue_name_number DUMMY: cleaner0 2023-07-05T07:57:40Z K79 ptm MESH: cleaner0 2023-07-05T07:57:58Z methylation 0.9963744 taxonomy_domain cleaner0 2023-07-04T16:22:20Z DUMMY: yeast residue_name SO: melaniev@ebi.ac.uk 2023-07-06T15:25:20Z acetyllysine 0.9987602 protein cleaner0 2023-07-04T16:20:21Z PR: Taf14 0.94719505 structure_element cleaner0 2023-07-04T16:20:28Z SO: YEATS domain 0.9980909 protein cleaner0 2023-07-04T16:20:21Z PR: Taf14 0.84213173 complex_assembly cleaner0 2023-07-04T16:23:51Z GO: TFIID 0.6936775 complex_assembly cleaner0 2023-07-04T16:24:02Z GO: TFIIF 0.9974746 protein cleaner0 2023-07-04T16:20:21Z PR: Taf14 0.7698535 complex_assembly cleaner0 2023-07-05T07:58:48Z GO: INO80 0.67576855 complex_assembly cleaner0 2023-07-05T07:58:53Z GO: SWI/SNF 0.5300177 complex_assembly cleaner0 2023-07-05T07:59:00Z GO: RSC 0.8560897 protein_type cleaner0 2023-07-04T16:22:53Z MESH: histone acetyltransferase 0.77899855 complex_assembly cleaner0 2023-07-05T08:50:53Z GO: NuA3 0.99761933 protein cleaner0 2023-07-04T16:20:21Z PR: Taf14 0.9986816 protein cleaner0 2023-07-04T16:20:21Z PR: Taf14 0.93180734 structure_element cleaner0 2023-07-04T16:20:28Z SO: YEATS domain 0.9986914 residue_name melaniev@ebi.ac.uk 2023-07-06T15:25:20Z SO: crotonyllysine protein_type MESH: cleaner0 2023-07-04T16:21:00Z histone protein_type MESH: cleaner0 2023-07-05T07:55:54Z H3 0.6754686 protein_state cleaner0 2023-07-04T16:22:07Z DUMMY: crotonylated residue_name_number DUMMY: cleaner0 2023-07-05T07:58:27Z lysine 9 protein_type MESH: cleaner0 2023-07-05T08:00:12Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:52:22Z K9cr protein_type MESH: cleaner0 2023-07-05T08:00:12Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:52:32Z K9cr 0.9966305 taxonomy_domain cleaner0 2023-07-04T16:22:20Z DUMMY: yeast INTRO paragraph 3072 To elucidate the molecular basis for recognition of the H3K9cr mark, we obtained a crystal structure of the Taf14 YEATS domain in complex with H3K9cr5-13 (residues 5–13 of H3) peptide (Fig. 1, Supplementary Results, Supplementary Fig. 1 and Supplementary Table 1). The Taf14 YEATS domain adopts an immunoglobin-like β sandwich fold containing eight anti-parallel β strands linked by short loops that form a binding site for H3K9cr (Fig. 1b). The H3K9cr peptide lays in an extended conformation in an orientation orthogonal to the β strands and is stabilized through an extensive network of direct and water-mediated hydrogen bonds and a salt bridge (Fig. 1c). protein_type MESH: cleaner0 2023-07-05T08:00:50Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:52:40Z K9cr 0.9973307 evidence cleaner0 2023-07-05T08:56:20Z DUMMY: crystal structure 0.9986278 protein cleaner0 2023-07-04T16:20:21Z PR: Taf14 0.9957862 structure_element cleaner0 2023-07-04T16:20:28Z SO: YEATS domain 0.9311703 protein_state cleaner0 2023-07-05T08:53:17Z DUMMY: in complex with 0.97627616 chemical cleaner0 2023-07-05T08:36:15Z CHEBI: H3K9cr5-13 0.96639997 residue_range cleaner0 2023-07-05T08:49:48Z DUMMY: 5–13 0.9895627 protein_type cleaner0 2023-07-05T07:55:54Z MESH: H3 0.998461 protein cleaner0 2023-07-04T16:20:21Z PR: Taf14 0.9965588 structure_element cleaner0 2023-07-04T16:20:28Z SO: YEATS domain 0.99343634 structure_element cleaner0 2023-07-05T08:51:32Z SO: immunoglobin-like β sandwich fold 0.96191806 structure_element cleaner0 2023-07-05T08:51:36Z SO: anti-parallel β strands 0.6941337 structure_element cleaner0 2023-07-05T08:51:44Z SO: loops 0.9979013 site cleaner0 2023-07-05T08:51:58Z SO: binding site protein_type MESH: cleaner0 2023-07-05T08:01:22Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:52:54Z K9cr protein_type MESH: cleaner0 2023-07-05T08:01:47Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:52:47Z K9cr 0.9527997 protein_state cleaner0 2023-07-05T08:53:22Z DUMMY: extended conformation 0.99544466 structure_element cleaner0 2023-07-05T08:51:48Z SO: β strands chemical CHEBI: melaniev@ebi.ac.uk 2023-07-21T12:02:44Z water bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:20:44Z hydrogen bonds bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:20:44Z salt bridge INTRO paragraph 3741 The most striking feature of the crotonyllysine recognition mechanism is the unique coordination of crotonylated lysine residue. The fully extended side chain of K9cr transverses the narrow tunnel, crossing the β sandwich at right angle in a corkscrew-like manner (Fig. 1b and Supplementary Figure 1b). The planar crotonyl group is inserted between Trp81 and Phe62 of the protein, the aromatic rings of which are positioned strictly parallel to each other and at equal distance from the crotonyl group, yielding a novel aromatic-amide/aliphatic-aromatic π-π-π-stacking system that, to our knowledge, has not been reported previously for any protein-protein interaction (Fig. 1d and Supplementary Fig. 1c). The side chain of Trp81 appears to adopt two conformations, one of which provides maximum π-stacking with the alkene functional group while the other rotamer affords maximum π-stacking with the amide π electrons (Supplementary Fig. 1c). The dual conformation of Trp81 is likely due to the conjugated nature of the C=C and C=O π-orbitals within the crotonyl functional group. 0.9968951 residue_name melaniev@ebi.ac.uk 2023-07-06T15:25:20Z SO: crotonyllysine 0.884992 protein_state cleaner0 2023-07-04T16:22:07Z DUMMY: crotonylated 0.98747534 residue_name cleaner0 2023-07-05T08:02:11Z SO: lysine 0.99885833 ptm melaniev@ebi.ac.uk 2023-07-21T11:52:08Z MESH: K9cr 0.9932809 structure_element cleaner0 2023-07-05T08:52:02Z SO: β sandwich chemical CHEBI: melaniev@ebi.ac.uk 2023-07-21T11:51:04Z crotonyl 0.99896455 residue_name_number cleaner0 2023-07-05T08:02:27Z DUMMY: Trp81 0.99897707 residue_name_number cleaner0 2023-07-05T08:02:31Z DUMMY: Phe62 chemical CHEBI: melaniev@ebi.ac.uk 2023-07-21T11:51:04Z crotonyl bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:20:44Z π-π-π-stacking 0.99895763 residue_name_number cleaner0 2023-07-05T08:02:27Z DUMMY: Trp81 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:20:44Z π-stacking bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:20:44Z π-stacking 0.9987826 residue_name_number cleaner0 2023-07-05T08:02:27Z DUMMY: Trp81 chemical CHEBI: melaniev@ebi.ac.uk 2023-07-21T11:51:04Z crotonyl INTRO paragraph 4832 In addition to π-π-π stacking, the crotonyl group is stabilized by a set of hydrogen bonds and electrostatic interactions. The π bond conjugation of the crotonyl group gives rise to a dipole moment of the alkene moiety, resulting in a partial positive charge on the β-carbon (Cβ) and a partial negative charge on the α-carbon (Cα). This provides the capability for the alkene moiety to form electrostatic contacts, as Cα and Cβ lay within electrostatic interaction distances of the carbonyl oxygen of Gln79 and of the hydroxyl group of Thr61, respectively. The hydroxyl group of Thr61 also participates in a hydrogen bond with the amide nitrogen of the K9cr side chain (Fig. 1d). The fixed position of the Thr61 hydroxyl group, which facilitates interactions with both the amide and Cα of K9cr, is achieved through a hydrogen bond with imidazole ring of His59. Extra stabilization of K9cr is attained by a hydrogen bond formed between its carbonyl oxygen and the backbone nitrogen of Trp81, as well as a water-mediated hydrogen bond with the backbone carbonyl group of Gly82 (Fig 1d). This distinctive mechanism was corroborated through mapping the Taf14 YEATS-H3K9cr binding interface in solution using NMR chemical shift perturbation analysis (Supplementary Fig. 2a, b). bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:20:44Z π-π-π stacking chemical CHEBI: melaniev@ebi.ac.uk 2023-07-21T11:51:04Z crotonyl bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:20:44Z hydrogen bonds bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:20:44Z electrostatic interactions bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:20:44Z π bond chemical CHEBI: melaniev@ebi.ac.uk 2023-07-21T11:51:04Z crotonyl bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:20:44Z electrostatic contacts bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:20:44Z electrostatic interaction 0.9991684 residue_name_number cleaner0 2023-07-05T08:02:41Z DUMMY: Gln79 0.9991742 residue_name_number cleaner0 2023-07-05T08:02:45Z DUMMY: Thr61 0.9991697 residue_name_number cleaner0 2023-07-05T08:02:44Z DUMMY: Thr61 bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:20:44Z hydrogen bond 0.9990522 ptm melaniev@ebi.ac.uk 2023-07-21T11:52:08Z MESH: K9cr 0.99915934 residue_name_number cleaner0 2023-07-05T08:02:45Z DUMMY: Thr61 0.9990495 ptm melaniev@ebi.ac.uk 2023-07-21T11:52:08Z MESH: K9cr bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:20:44Z hydrogen bond 0.99916136 residue_name_number cleaner0 2023-07-05T08:02:52Z DUMMY: His59 0.9988856 ptm melaniev@ebi.ac.uk 2023-07-21T11:52:08Z MESH: K9cr bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:20:44Z hydrogen bond 0.9991516 residue_name_number cleaner0 2023-07-05T08:02:27Z DUMMY: Trp81 0.9917144 chemical cleaner0 2023-07-05T08:55:01Z CHEBI: water bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:20:44Z hydrogen bond 0.99916196 residue_name_number cleaner0 2023-07-05T08:02:57Z DUMMY: Gly82 0.9983511 protein cleaner0 2023-07-04T16:20:21Z PR: Taf14 0.9934926 site cleaner0 2023-07-05T08:03:03Z SO: YEATS-H3K9cr binding interface 0.9922565 experimental_method cleaner0 2023-07-05T08:03:06Z MESH: NMR chemical shift perturbation analysis INTRO paragraph 6134 Binding of the Taf14 YEATS domain to H3K9cr is robust. The dissociation constant (Kd) for the Taf14 YEATS-H3K9cr5-13 complex was found to be 9.5 μM, as measured by fluorescence spectroscopy (Supplementary Fig. 2c). This value is in the range of binding affinities exhibited by the majority of histone readers, thus attesting to the physiological relevance of the H3K9cr recognition by Taf14. 0.99884135 protein cleaner0 2023-07-04T16:20:21Z PR: Taf14 0.94870484 structure_element cleaner0 2023-07-04T16:20:28Z SO: YEATS domain protein_type MESH: cleaner0 2023-07-05T08:03:24Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:54:19Z K9cr 0.995806 evidence cleaner0 2023-07-05T08:03:41Z DUMMY: dissociation constant 0.9937781 evidence cleaner0 2023-07-05T08:03:45Z DUMMY: Kd 0.9887676 complex_assembly cleaner0 2023-07-05T08:35:55Z GO: Taf14 YEATS-H3K9cr5-13 0.9937454 experimental_method cleaner0 2023-07-05T08:04:07Z MESH: fluorescence spectroscopy 0.99432206 evidence cleaner0 2023-07-05T08:04:01Z DUMMY: binding affinities protein_type MESH: cleaner0 2023-07-05T08:04:40Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:54:27Z K9cr 0.9988524 protein cleaner0 2023-07-04T16:20:21Z PR: Taf14 INTRO paragraph 6527 To determine whether H3K9cr is present in yeast, we generated whole cell extracts from logarithmically growing yeast cells and subjected them to Western blot analysis using antibodies directed towards H3K9cr, H3K9ac and H3 (Fig. 2a, b, Supplementary Fig. 3 and Supplementary Table 2). Both H3K9cr and H3K9ac were detected in yeast histones; to our knowledge, this is the first report of H3K9cr occurring in yeast. We next asked if H3K9cr is regulated by the actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Towards this end, we probed extracts derived from yeast cells in which major yeast HATs (HAT1, Gcn5, and Rtt109) or HDACs (Rpd3, Hos1, and Hos2) were deleted. As shown in Figure 2a, b and Supplementary Fig. 3e, H3K9cr levels were abolished or reduced considerably in the HAT deletion strains, whereas they were dramatically increased in the HDAC deletion strains. Furthermore, fluctuations in the H3K9cr levels were more substantial than fluctuations in the corresponding H3K9ac levels. Together, these results reveal that H3K9cr is a dynamic mark of chromatin in yeast and suggest an important role for this modification in transcription as it is regulated by HATs and HDACs. protein_type MESH: cleaner0 2023-07-05T08:08:27Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:54:36Z K9cr 0.99658173 taxonomy_domain cleaner0 2023-07-04T16:22:20Z DUMMY: yeast 0.8414014 experimental_method cleaner0 2023-07-05T08:57:36Z MESH: whole cell extracts 0.9963198 taxonomy_domain cleaner0 2023-07-04T16:22:20Z DUMMY: yeast 0.9293416 experimental_method cleaner0 2023-07-05T08:57:39Z MESH: Western blot analysis protein_type MESH: cleaner0 2023-07-05T08:09:10Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:54:53Z K9cr protein_type MESH: cleaner0 2023-07-05T08:09:38Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:55:01Z K9ac 0.7814144 protein_type cleaner0 2023-07-05T07:55:54Z MESH: H3 protein_type MESH: cleaner0 2023-07-05T08:10:08Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:54:45Z K9cr protein_type MESH: cleaner0 2023-07-05T08:10:36Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:55:10Z K9ac 0.99671257 taxonomy_domain cleaner0 2023-07-04T16:22:20Z DUMMY: yeast protein_type MESH: cleaner0 2023-07-05T08:57:52Z histones protein_type MESH: cleaner0 2023-07-05T08:38:01Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:55:18Z K9cr 0.9966935 taxonomy_domain cleaner0 2023-07-04T16:22:20Z DUMMY: yeast protein_type MESH: cleaner0 2023-07-05T08:38:30Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:55:26Z K9cr 0.99616396 protein_type cleaner0 2023-07-05T08:06:46Z MESH: histone acetyltransferases 0.9961572 protein_type cleaner0 2023-07-05T08:06:50Z MESH: HATs 0.99562824 protein_type cleaner0 2023-07-05T08:07:02Z MESH: histone deacetylases 0.9862266 protein_type cleaner0 2023-07-05T08:07:06Z MESH: HDACs 0.9961592 taxonomy_domain cleaner0 2023-07-04T16:22:20Z DUMMY: yeast 0.99671113 taxonomy_domain cleaner0 2023-07-04T16:22:20Z DUMMY: yeast 0.9969458 protein_type cleaner0 2023-07-05T08:06:51Z MESH: HATs 0.99758315 protein cleaner0 2023-07-05T08:07:12Z PR: HAT1 0.9978067 protein cleaner0 2023-07-05T08:07:16Z PR: Gcn5 0.9977192 protein cleaner0 2023-07-05T08:07:20Z PR: Rtt109 0.9930031 protein_type cleaner0 2023-07-05T08:07:06Z MESH: HDACs 0.99792385 protein cleaner0 2023-07-05T08:07:24Z PR: Rpd3 0.99804014 protein cleaner0 2023-07-05T08:07:27Z PR: Hos1 0.9978777 protein cleaner0 2023-07-05T08:07:31Z PR: Hos2 0.53316224 experimental_method cleaner0 2023-07-05T09:00:08Z MESH: deleted protein_type MESH: cleaner0 2023-07-05T08:11:11Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:55:34Z K9cr protein_type MESH: cleaner0 2023-07-05T08:58:28Z HAT 0.47926757 experimental_method cleaner0 2023-07-05T09:00:12Z MESH: deletion protein_type MESH: cleaner0 2023-07-05T08:58:22Z HDAC experimental_method MESH: cleaner0 2023-07-05T09:04:29Z deletion protein_type MESH: cleaner0 2023-07-05T08:11:41Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:55:48Z K9cr protein_type MESH: cleaner0 2023-07-05T08:12:21Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:55:41Z K9ac protein_type MESH: cleaner0 2023-07-05T08:12:47Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:55:56Z K9cr 0.9963509 taxonomy_domain cleaner0 2023-07-04T16:22:20Z DUMMY: yeast 0.9969043 protein_type cleaner0 2023-07-05T08:06:51Z MESH: HATs 0.9925954 protein_type cleaner0 2023-07-05T08:07:06Z MESH: HDACs INTRO paragraph 7741 We have previously shown that among acetylated histone marks, the Taf14 YEATS domain prefers acetylated H3K9 (also see Supplementary Fig. 3b), however it binds to H3K9cr tighter. The selectivity of Taf14 towards crotonyllysine was substantiated by 1H,15N HSQC experiments, in which either H3K9cr5-13 or H3K9ac5-13 peptide was titrated into the 15N-labeled Taf14 YEATS domain (Fig. 2c and Supplementary Fig. 4a, b). Binding of H3K9cr induced resonance changes in slow exchange regime on the NMR time scale, indicative of strong interaction. In contrast, binding of H3K9ac resulted in an intermediate exchange, which is characteristic of a weaker association. Furthermore, crosspeaks of Gly80 and Trp81 of the YEATS domain were uniquely perturbed by H3K9cr and H3K9ac, indicating a different chemical environment in the respective crotonyllysine and acetyllysine binding pockets (Supplementary Fig. 4a). These differences support our model that Trp81 adopts two conformations upon complex formation with the H3K9cr mark as compared to H3K9ac (Supplementary Figs. 1c, d and 4c). One of the conformations, characterized by the π stacking involving two aromatic residues and the alkene group, is observed only in the YEATS-H3K9cr complex. protein_state DUMMY: cleaner0 2023-07-04T16:22:11Z acetylated protein_type MESH: cleaner0 2023-07-04T16:21:00Z histone 0.9988348 protein cleaner0 2023-07-04T16:20:22Z PR: Taf14 0.9854789 structure_element cleaner0 2023-07-04T16:20:28Z SO: YEATS domain 0.9957682 protein_state cleaner0 2023-07-04T16:22:11Z DUMMY: acetylated protein_type MESH: cleaner0 2023-07-05T08:13:59Z H3 residue_name_number DUMMY: cleaner0 2023-07-05T08:14:10Z K9 protein_type MESH: cleaner0 2023-07-05T08:14:26Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:56:09Z K9cr 0.99873716 protein cleaner0 2023-07-04T16:20:22Z PR: Taf14 0.9983891 residue_name melaniev@ebi.ac.uk 2023-07-06T15:25:20Z SO: crotonyllysine 0.9863785 experimental_method cleaner0 2023-07-05T08:18:57Z MESH: 1H,15N HSQC 0.990856 chemical cleaner0 2023-07-05T08:36:15Z CHEBI: H3K9cr5-13 0.9934884 chemical cleaner0 2023-07-05T08:38:55Z CHEBI: H3K9ac5-13 0.96406466 experimental_method cleaner0 2023-07-05T09:00:19Z MESH: titrated protein_state DUMMY: cleaner0 2023-07-05T08:53:55Z 15N-labeled 0.9987318 protein cleaner0 2023-07-04T16:20:22Z PR: Taf14 0.98387945 structure_element cleaner0 2023-07-04T16:20:28Z SO: YEATS domain protein_type MESH: cleaner0 2023-07-05T08:15:14Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:56:22Z K9cr evidence DUMMY: cleaner0 2023-07-05T08:59:29Z resonance changes experimental_method MESH: cleaner0 2023-07-05T09:04:29Z NMR protein_type MESH: cleaner0 2023-07-05T08:15:40Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:56:29Z K9ac 0.49922895 evidence cleaner0 2023-07-05T08:56:27Z DUMMY: crosspeaks 0.99908006 residue_name_number cleaner0 2023-07-05T08:15:58Z DUMMY: Gly80 0.9990513 residue_name_number cleaner0 2023-07-05T08:02:27Z DUMMY: Trp81 0.98532 structure_element cleaner0 2023-07-04T16:20:28Z SO: YEATS domain protein_type MESH: cleaner0 2023-07-05T08:16:15Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:56:52Z K9cr protein_type MESH: cleaner0 2023-07-05T08:16:48Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:56:37Z K9ac site SO: cleaner0 2023-07-05T08:13:40Z crotonyllysine and acetyllysine binding pockets 0.998936 residue_name_number cleaner0 2023-07-05T08:02:27Z DUMMY: Trp81 protein_type MESH: cleaner0 2023-07-05T08:17:15Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:57:01Z K9cr protein_type MESH: cleaner0 2023-07-05T08:17:45Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:56:45Z K9ac 0.99369735 complex_assembly cleaner0 2023-07-05T08:18:01Z GO: YEATS-H3K9cr INTRO paragraph 8976 To establish whether the Taf14 YEATS domain is able to recognize other recently identified acyllysine marks, we performed solution pull-down assays using H3 peptides acetylated, propionylated, butyrylated, and crotonylated at lysine 9 (residues 1–20 of H3). As shown in Figure 2d and Supplementary Fig. 5a, the Taf14 YEATS domain binds more strongly to H3K9cr1-20, as compared to other acylated histone peptides. The preference for H3K9cr over H3K9ac, H3K9pr and H3K9bu was supported by 1H,15N HSQC titration experiments. Addition of H3K9ac1-20, H3K9pr1-20, and H3K9bu1-20 peptides caused chemical shift perturbations in the Taf14 YEATS domain in intermediate exchange regime, implying that these interactions are weaker compared to the interaction with the H3K9cr1-20 peptide (Supplementary Fig. 5b). We concluded that H3K9cr is the preferred target of this domain. From comparative structural analysis of the YEATS complexes, Gly80 emerged as candidate residue potentially responsible for the preference for crotonyllysine. In attempt to generate a mutant capable of accommodating a short acetyl moiety but discriminating against a longer, planar crotonyl moiety, we mutated Gly80 to more bulky residues, however all mutants of Gly80 lost their binding activities towards either acylated peptide, suggesting that Gly80 is absolutely required for the interaction. In contrast, mutation of Val24, a residue located on another side of Trp81, had no effect on binding (Fig. 2d and Supplementary Fig. 5a, c). 0.99862266 protein cleaner0 2023-07-04T16:20:22Z PR: Taf14 0.9849653 structure_element cleaner0 2023-07-04T16:20:28Z SO: YEATS domain residue_name SO: melaniev@ebi.ac.uk 2023-07-06T15:25:20Z acyllysine 0.99509495 experimental_method cleaner0 2023-07-05T09:00:34Z MESH: solution pull-down assays 0.99270695 protein_type cleaner0 2023-07-05T07:55:54Z MESH: H3 0.9932382 protein_state cleaner0 2023-07-04T16:22:11Z DUMMY: acetylated 0.956428 protein_state cleaner0 2023-07-05T08:40:11Z DUMMY: propionylated 0.9795297 protein_state cleaner0 2023-07-05T08:40:15Z DUMMY: butyrylated 0.97335297 protein_state cleaner0 2023-07-04T16:22:07Z DUMMY: crotonylated residue_name_number DUMMY: cleaner0 2023-07-05T08:18:20Z lysine 9 0.9283707 residue_range cleaner0 2023-07-05T08:50:16Z DUMMY: 1–20 0.951781 protein_type cleaner0 2023-07-05T07:55:54Z MESH: H3 0.99854577 protein cleaner0 2023-07-04T16:20:22Z PR: Taf14 0.98509496 structure_element cleaner0 2023-07-04T16:20:28Z SO: YEATS domain chemical CHEBI: cleaner0 2023-07-05T08:33:09Z H3K9cr1-20 0.78338593 protein_state cleaner0 2023-07-05T08:54:04Z DUMMY: acylated protein_type MESH: cleaner0 2023-07-05T08:20:37Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:57:16Z K9cr protein_type MESH: cleaner0 2023-07-05T08:21:11Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:57:35Z K9ac protein_type MESH: cleaner0 2023-07-05T08:21:37Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:57:44Z K9pr protein_type MESH: cleaner0 2023-07-05T08:22:10Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:57:54Z K9bu 0.98849183 experimental_method cleaner0 2023-07-05T08:19:00Z MESH: 1H,15N HSQC titration experiments chemical CHEBI: cleaner0 2023-07-05T08:33:21Z H3K9ac1-20 chemical CHEBI: cleaner0 2023-07-05T08:33:34Z H3K9pr1-20 chemical CHEBI: cleaner0 2023-07-05T08:33:45Z H3K9bu1-20 evidence DUMMY: cleaner0 2023-07-05T09:01:01Z chemical shift perturbations 0.9984143 protein cleaner0 2023-07-04T16:20:22Z PR: Taf14 0.9821952 structure_element cleaner0 2023-07-04T16:20:28Z SO: YEATS domain chemical CHEBI: cleaner0 2023-07-05T08:33:09Z H3K9cr1-20 protein_type MESH: cleaner0 2023-07-05T08:22:49Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:58:04Z K9cr 0.99294585 experimental_method cleaner0 2023-07-05T09:01:12Z MESH: comparative structural analysis 0.9990103 residue_name_number cleaner0 2023-07-05T08:15:59Z DUMMY: Gly80 0.9981839 residue_name melaniev@ebi.ac.uk 2023-07-06T15:25:20Z SO: crotonyllysine chemical CHEBI: melaniev@ebi.ac.uk 2023-07-21T11:51:04Z crotonyl 0.99031365 protein_state cleaner0 2023-07-05T08:49:33Z DUMMY: mutated 0.9989976 residue_name_number cleaner0 2023-07-05T08:15:59Z DUMMY: Gly80 protein_state DUMMY: cleaner0 2023-07-05T09:01:51Z mutants of 0.9989625 residue_name_number cleaner0 2023-07-05T08:15:59Z DUMMY: Gly80 0.885494 protein_state cleaner0 2023-07-05T08:54:04Z DUMMY: acylated 0.9990196 residue_name_number cleaner0 2023-07-05T08:15:59Z DUMMY: Gly80 0.9797934 experimental_method cleaner0 2023-07-05T09:01:59Z MESH: mutation 0.99903935 residue_name_number cleaner0 2023-07-05T08:24:25Z DUMMY: Val24 0.9990657 residue_name_number cleaner0 2023-07-05T08:02:27Z DUMMY: Trp81 INTRO paragraph 10484 To determine if the binding to crotonyllysine is conserved, we tested human YEATS domains by pull-down experiments using singly and multiply acetylated, propionylated, butyrylated, and crotonylated histone peptides (Supplementary Fig. 6). We found that all YEATS domains tested are capable of binding to crotonyllysine peptides, though they display variable preferences for the acyl moieties. While YEATS2 and ENL showed selectivity for the crotonylated peptides, GAS41 and AF9 bound acylated peptides almost equally well. 0.99843556 residue_name melaniev@ebi.ac.uk 2023-07-06T15:25:20Z SO: crotonyllysine 0.984743 protein_state cleaner0 2023-07-05T08:54:12Z DUMMY: conserved 0.99253696 species cleaner0 2023-07-04T16:22:25Z MESH: human structure_element SO: cleaner0 2023-07-05T08:49:39Z YEATS domains 0.9765474 experimental_method cleaner0 2023-07-05T09:02:03Z MESH: pull-down experiments 0.9461614 protein_state cleaner0 2023-07-04T16:22:11Z DUMMY: acetylated 0.9879154 protein_state cleaner0 2023-07-05T08:40:11Z DUMMY: propionylated 0.9869178 protein_state cleaner0 2023-07-05T08:40:15Z DUMMY: butyrylated 0.9762997 protein_state cleaner0 2023-07-04T16:22:07Z DUMMY: crotonylated protein_type MESH: cleaner0 2023-07-05T09:02:17Z histone structure_element SO: cleaner0 2023-07-05T08:49:39Z YEATS domains residue_name SO: melaniev@ebi.ac.uk 2023-07-06T15:25:20Z crotonyllysine 0.9987778 protein cleaner0 2023-07-05T08:39:48Z PR: YEATS2 0.99860966 protein cleaner0 2023-07-05T07:54:09Z PR: ENL 0.9837325 protein_state cleaner0 2023-07-04T16:22:07Z DUMMY: crotonylated 0.99857676 protein cleaner0 2023-07-05T08:39:56Z PR: GAS41 0.9983223 protein cleaner0 2023-07-05T07:54:15Z PR: AF9 0.99105185 protein_state cleaner0 2023-07-05T08:54:04Z DUMMY: acylated INTRO paragraph 11007 Unlike the YEATS domain, a known acetyllysine reader, bromodomain, does not recognize crotonyllysine. We assayed a large set of BDs in pull-down experiments and found that this module is highly specific for acetyllysine and propionyllysine containing peptides (Supplementary Fig. 7). However, bromodomains did not interact (or associated very weakly) with longer acyl modifications, including crotonyllysine, as in the case of BDs of TAF1 and BRD2, supporting recent reports. These results demonstrate that the YEATS domain is currently the sole reader of crotonyllysine. 0.9855747 structure_element cleaner0 2023-07-04T16:20:28Z SO: YEATS domain 0.80789137 protein_type cleaner0 2023-07-05T08:52:52Z MESH: acetyllysine reader 0.9922646 structure_element cleaner0 2023-07-05T08:52:07Z SO: bromodomain 0.99855334 residue_name melaniev@ebi.ac.uk 2023-07-06T15:25:20Z SO: crotonyllysine 0.7588632 structure_element cleaner0 2023-07-04T16:21:53Z SO: BDs 0.9541701 experimental_method cleaner0 2023-07-05T09:02:34Z MESH: pull-down experiments 0.99803704 residue_name melaniev@ebi.ac.uk 2023-07-06T15:25:20Z SO: acetyllysine 0.9968952 residue_name melaniev@ebi.ac.uk 2023-07-06T15:25:20Z SO: propionyllysine 0.9787652 structure_element cleaner0 2023-07-04T16:21:48Z SO: bromodomains 0.99844176 residue_name melaniev@ebi.ac.uk 2023-07-06T15:25:20Z SO: crotonyllysine 0.8739639 structure_element cleaner0 2023-07-04T16:21:53Z SO: BDs 0.99858606 protein cleaner0 2023-07-05T08:51:13Z PR: TAF1 0.9986286 protein cleaner0 2023-07-05T08:51:16Z PR: BRD2 0.98622835 structure_element cleaner0 2023-07-04T16:20:28Z SO: YEATS domain 0.9985879 residue_name melaniev@ebi.ac.uk 2023-07-06T15:25:20Z SO: crotonyllysine INTRO paragraph 11579 In conclusion, we have identified the YEATS domain of Taf14 as the first reader of histone crotonylation. The unique and previously unobserved aromatic-amide/aliphatic-aromatic π-π-π-stacking mechanism facilitates the specific recognition of the crotonyl moiety. We further demonstrate that H3K9cr exists in yeast and is dynamically regulated by HATs and HDACs. As we previously showed the importance of acyllysine binding by the Taf14 YEATS domain for the DNA damage response and gene transcription, it will be essential in the future to define the physiological role of crotonyllysine recognition and to differentiate the activities of Taf14 that are due to binding to crotonyllysine and acetyllysine modifications. Furthermore, the functional significance of crotonyllysine recognition by other YEATS proteins will be of great importance to elucidate and compare. 0.99071395 structure_element cleaner0 2023-07-04T16:20:28Z SO: YEATS domain 0.9987459 protein cleaner0 2023-07-04T16:20:22Z PR: Taf14 0.5421235 protein_type cleaner0 2023-07-04T16:21:00Z MESH: histone 0.672082 ptm cleaner0 2023-07-04T16:18:43Z MESH: crotonylation bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:20:44Z π-π-π-stacking chemical CHEBI: melaniev@ebi.ac.uk 2023-07-21T11:51:04Z crotonyl protein_type MESH: cleaner0 2023-07-05T08:41:07Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T12:01:20Z K9cr 0.99646616 taxonomy_domain cleaner0 2023-07-04T16:22:20Z DUMMY: yeast 0.9973616 protein_type cleaner0 2023-07-05T08:06:51Z MESH: HATs 0.9948607 protein_type cleaner0 2023-07-05T08:07:06Z MESH: HDACs residue_name SO: melaniev@ebi.ac.uk 2023-07-06T15:25:20Z acyllysine 0.998776 protein cleaner0 2023-07-04T16:20:22Z PR: Taf14 0.9936651 structure_element cleaner0 2023-07-04T16:20:28Z SO: YEATS domain residue_name SO: melaniev@ebi.ac.uk 2023-07-06T15:25:20Z crotonyllysine 0.9987233 protein cleaner0 2023-07-04T16:20:22Z PR: Taf14 0.99826485 residue_name melaniev@ebi.ac.uk 2023-07-06T15:25:20Z SO: crotonyllysine 0.9959139 residue_name melaniev@ebi.ac.uk 2023-07-06T15:25:20Z SO: acetyllysine residue_name SO: melaniev@ebi.ac.uk 2023-07-06T15:25:20Z crotonyllysine 0.9981281 protein_type cleaner0 2023-07-05T08:52:37Z MESH: YEATS METHODS title_1 12449 ONLINE METHODS METHODS title_2 12464 Protein expression and purification METHODS paragraph 12500 The Taf14 YEATS constructs (residues 1–132 or 1–137) were expressed in E. coli BL21 (DE3) RIL in either Luria Broth or M19 minimal media supplemented with 15NH4Cl and purified as N-terminal GST fusion proteins. Cells were harvested by centrifugation and resuspended in 50 mM HEPES (pH 7.5) supplemented with 150 mM NaCl and 1 mM TCEP. Cells are lysed by freeze-thaw followed by sonication. Proteins were purified on glutathione Sepharose 4B beads and the GST tag was cleaved with PreScission protease. METHODS title_2 13006 X-ray data collection and structure determination METHODS paragraph 13056 Taf14 YEATS (residues 1–137) was concentrated to 9 mg/mL in 25 mM MES (pH 6.5) and incubated with 2 molar equivalence of the H3K9cr5-13 at RT for 30 mins prior to crystallization. Crystals were obtain via sitting drop diffusion method at 18°C by mixing 800 nL of protein/peptide solution with 800 nL of well solution composed of 44% PEG600 (v/v) and 0.2 M citric acid (pH 6.0). X-ray diffraction data was collected at a wavelength of 1.54 Å at 100 K from a single crystal on the UC Denver Biophysical Core home source composed of a Rigaku Micromax 007 high frequency microfocus X-ray generator with a Pilatus 200K 2D area detector. HKL3000 was used for indexing, scaling, and data reduction. Solution was solved via molecular replacement with Phaser using the Taf14 YEATS domain (PDB 5D7E) as search model with waters, ligands, and peptide removed. Phenix was used for refinement of structure and waters were manually placed by inception of difference maps in Coot. Ramachandran plot indicates good stereochemistry of the three-dimensional structure with 100% of all residues falling within the favored (98%) and allowed (2%) regions. The crystallographic statistics are shown in Supplementary Table 1. METHODS title_2 14263 NMR spectroscopy METHODS paragraph 14280 NMR spectroscopy was carried out on a Varian INOVA 600 MHz spectrometer outfitted with a cryogenic probe. Chemical shift perturbation (CSP) analysis was performed using uniformly 15N-labeled Taf14 (1–132). 1H,15N heteronuclear single quantum coherence (HSQC) spectra of the Taf14 YEATS domain were collected in the presence of increasing concentrations of either H3K9cr5-13, H3K9ac5-13, H3K9cr1-20, H3K9ac1-20 H3K9pr1-20, H3K9bu1-20 or free Kcr in PBS buffer pH 6.8, 8% D2O. METHODS title_2 14757 Fluorescence binding assays METHODS paragraph 14785 Tryptophan fluorescence measurements were performed on a Fluorolog spectrofluorometer at room temperature as described. The samples containing 2 μM of Taf14 YEATS in PBS (pH 7.4) and increasing concentrations of H3K9cr5-13 were excited at 295 nm. Emission spectra were recorded from 310 to 340 nm with a 1 nm step size and a 0.5 sec integration time. The Kd value was determined using a nonlinear least-squares analysis and the equation: where [L] is the concentration of the peptide, [P] is the concentration of the protein, ΔI is the observed change of signal intensity, and ΔImax is the difference in signal intensity of the free and bound states. The Kd values were averaged over 3 separate experiments, with error calculated as the standard deviation (SD). METHODS title_2 15558 Peptide pull-downs METHODS paragraph 15577 YEATS domains in pGEX vectors were expressed in SoluBL21 cells (Amsbio) by induction with 1 mM IPTG at 16–18°C overnight with shaking. Cells were lysed by freeze-thaw and sonication then purified over glutathione agarose (Pierce) in a buffer containing 50 mM Tris pH 8.0, 500 mM NaCl, 20% glycerol (v/v) and 1 mM dithiothreitol (DTT). Peptide pull-downs were performed essentially as described except that the assay buffer contained 50 mM Tris pH 8.0, 500 mM NaCl, and 0.1% NP-40, and 500 pmols of biotinylated histone peptides were loaded onto streptavidin coated magnetic beads before incubation with 40 pmols of protein. Bound proteins were detected with rabbit GST antibody (Sigma, G7781). Point mutants were generated by site-directed mutagenesis and purified/assayed as described above. The YEATS domains of Taf14, AF9, ENL, and GAS41 were previously described. METHODS title_2 16448 Western blotting METHODS paragraph 16465 Yeast cultures were grown in YPD media at 30°C to mid-log phase and extracts were prepared as previously described. Proteins from cell lysates were separated by SDS-PAGE and transferred to a PVDF membrane. Anti-H3K9ac (Millipore, 07-352) and anti-H3K9cr (PTM Biolabs, PTM-516) were diluted to 1:2000 and 1:1000, respectively, in 1x Superblock (ThermoScientific). An HRP-conjugated anti-rabbit (GE Healthcare) was used for detection. Bands were quantified using the ImageJ program. METHODS title_2 16947 Dot blotting METHODS paragraph 16960 Increasing concentrations of biotinylated histone peptides (0.06–1.5 μg) were spotted onto a PVDF membrane then probed with the anti-H3K9ac (Millipore, 07-352) or H3K9cr (PTM Biolabs, PTM-516) at 1:2000 in a 5% non-fat milk solution and detected with an HRP-conjugated anti-rabbit by enhanced chemiluminesence (ECL). METHODS title_2 17280 Bromodomains pull-downs METHODS paragraph 17304 cDNAs of GST-fused bromodomains were obtained either from EpiCypher Inc. or as a kind gift from Katrin Chua (Stanford University). GST fusions were expressed as described above except that the preparation buffer contained 50 mM Tris (pH 7.5), 150 mM NaCl, 10% glycerol (v/v), and 1 mM DTT. Pull-down assays were preformed as described above except that the assay buffer contained 50 mM Tris (pH 8.0), 300 mM NaCl, and 0.1% NP-40. SUPPL title_1 17734 Supplementary Material SUPPL footnote 17757 Accession codes. Coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 5IOK. SUPPL footnote 17881 Author contributions SUPPL footnote 17902 F.H.A., S.A.S., E.K.S., J.B.B., A.G., I.K.T and K.K. performed experiments and together with X.S., B.D.S and T.G.K. analyzed the data. F.H.A., S.A.S., B.D.S. and T.G.K. wrote the manuscript with input from all authors. SUPPL footnote 18121 Competing Financial Interest SUPPL footnote 18150 The authors declare no competing financial interests. 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H3K9cr protein_type MESH: cleaner0 2023-07-05T08:46:18Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:59:06Z K9cr nihms769551f1.jpg F1 FIG fig_caption 20565 (a) Chemical structure of crotonyllysine. (b) The crystal structure of the Taf14 YEATS domain (wheat) in complex with the H3K9cr5-13 peptide (green). (c) H3K9cr is stabilized via an extensive network of intermolecular electrostatic and polar interactions with the Taf14 YEATS domain. (d) The π-π-π stacking mechanism involving the alkene moiety of crotonyllysine. 0.99838877 residue_name melaniev@ebi.ac.uk 2023-07-06T15:25:20Z SO: crotonyllysine 0.99736893 evidence cleaner0 2023-07-05T08:56:31Z DUMMY: crystal structure 0.99835616 protein cleaner0 2023-07-04T16:20:22Z PR: Taf14 0.97807664 structure_element cleaner0 2023-07-04T16:20:28Z SO: YEATS domain protein_state DUMMY: cleaner0 2023-07-05T08:54:40Z in complex with 0.80304146 chemical cleaner0 2023-07-05T08:36:15Z CHEBI: H3K9cr5-13 protein_type MESH: cleaner0 2023-07-05T08:46:58Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:58:57Z K9cr bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:20:44Z electrostatic and polar interactions 0.99865216 protein cleaner0 2023-07-04T16:20:22Z PR: Taf14 0.9790957 structure_element cleaner0 2023-07-04T16:20:28Z SO: YEATS domain bond_interaction MESH: melaniev@ebi.ac.uk 2023-07-28T14:20:44Z π-π-π stacking 0.9983652 residue_name melaniev@ebi.ac.uk 2023-07-06T15:25:20Z SO: crotonyllysine nihms769551f2.jpg F2 FIG fig_title_caption 20932 H3K9cr is a selective target of the Taf14 YEATS domain protein_type MESH: cleaner0 2023-07-05T08:47:40Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:59:33Z K9cr 0.9988475 protein cleaner0 2023-07-04T16:20:22Z PR: Taf14 0.9892417 structure_element cleaner0 2023-07-04T16:20:28Z SO: YEATS domain nihms769551f2.jpg F2 FIG fig_caption 20987 (a, b) Western blot analysis comparing the levels of H3K9cr and H3K9ac in wild type (WT), HAT deletion, or HDAC deletion yeast strains. Total H3 was used as a loading control. (c) Superimposed 1H,15N HSQC spectra of Taf14 YEATS recorded as H3K9cr5-13 and H3K9ac5-13 peptides were titrated in. Spectra are color coded according to the protein:peptide molar ratio. (d) Western blot analyses of peptide pull-down assays using wild-type and mutated Taf14 YEATS domains and indicated peptides. 0.98307806 experimental_method cleaner0 2023-07-05T09:03:32Z MESH: Western blot protein_type MESH: cleaner0 2023-07-05T08:48:14Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T11:59:41Z K9cr protein_type MESH: cleaner0 2023-07-05T08:48:42Z H3 ptm MESH: melaniev@ebi.ac.uk 2023-07-21T12:00:02Z K9ac 0.9924296 protein_state cleaner0 2023-07-05T08:49:13Z DUMMY: wild type 0.98962665 protein_state cleaner0 2023-07-05T08:49:18Z DUMMY: WT protein_type MESH: melaniev@ebi.ac.uk 2023-07-21T12:00:29Z HAT protein_type MESH: cleaner0 2023-07-05T09:03:46Z HDAC experimental_method MESH: cleaner0 2023-07-05T09:04:00Z deletion 0.99134904 taxonomy_domain cleaner0 2023-07-04T16:22:20Z DUMMY: yeast 0.9896226 protein_type cleaner0 2023-07-05T07:55:54Z MESH: H3 0.90694416 experimental_method cleaner0 2023-07-05T09:03:35Z MESH: 1H,15N HSQC 0.65750146 evidence cleaner0 2023-07-05T08:57:05Z DUMMY: spectra 0.9981918 protein cleaner0 2023-07-04T16:20:22Z PR: Taf14 0.99271107 structure_element cleaner0 2023-07-05T08:52:13Z SO: YEATS 0.7894704 chemical cleaner0 2023-07-05T08:36:15Z CHEBI: H3K9cr5-13 chemical CHEBI: cleaner0 2023-07-05T08:38:55Z H3K9ac5-13 experimental_method MESH: cleaner0 2023-07-05T09:04:10Z titrated 0.6348235 evidence cleaner0 2023-07-05T08:57:07Z DUMMY: Spectra 0.98869205 experimental_method cleaner0 2023-07-05T08:49:22Z MESH: Western blot 0.99291915 experimental_method cleaner0 2023-07-05T08:49:24Z MESH: peptide pull-down assays 0.99699277 protein_state cleaner0 2023-07-05T08:49:30Z DUMMY: wild-type 0.959159 protein_state cleaner0 2023-07-05T08:49:33Z DUMMY: mutated 0.9981369 protein cleaner0 2023-07-04T16:20:22Z PR: Taf14 0.981699 structure_element cleaner0 2023-07-05T08:49:39Z SO: YEATS domains