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Site-specific ubiquitylation acts as a regulator of linker histone H1

ABSTRACT: Decoding the role of histone posttranslational modifications (PTMs) is key to understand the fundamental process of epigenetic regulation. While this process is well studied for core histones and many of their PTMs, this is not the case for linker histone H1 in general and its ubiquitylation in particular due to a lack of proper tools. Here, we report on the generation of site-specifically mono-ubiquitylated H1.2 via click chemistry and identify its ubiquitin-dependent interactome on a proteome-wide scale. We show that the H1 interactome is generally modulated by ubiquitylation and that site-specific ubiquitylation results in overlapping, but distinct interactomes. We further demonstrate that site-specific ubiquitylation of H1 affects the interaction with enzymes relevant for deubiquitylation and deacetylation. We finally show that site-specific ubiquitylation at position K64 impacts H1-dependent chromatosome assembly as well as H1-induced phase separation. In summary, we propose that site-specific ubiquitylation plays a general regulatory role for linker histone H1.

INSTRUMENT(S): micrOTOF II, Orbitrap Fusion, Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Florian Stengel

LAB HEAD: Florian Stengel

PROVIDER: PXD025258 | Pride | 2021-06-07

REPOSITORIES: Pride

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The functional interactome of site-specifically ubiquitylated linker histone H1

Project description:Decoding the role of histone posttranslational modifications (PTMs) is key to understand the fundamental process of epigenetic regulation. While this process is well studied for core histones and many of their PTMs, this is not the case for linker histone H1 in general and its ubiquitylation in particular due to a lack of proper tools. Here, we report on the generation of site-specifically mono-ubiquitylated H1.2 via click chemistry and identify its ubiquitin-dependent interactome on a proteome-wide scale. We show that the H1 interactome is generally modulated by ubiquitylation and that site-specific ubiquitylation results in overlapping, but distinct interactomes. We further demonstrate that site-specific ubiquitylation of H1 affects the interaction with enzymes relevant for deubiquitylation and deacetylation. We finally show that site-specific ubiquitylation at position K64 impacts H1-dependent chromatosome assembly as well as H1-induced phase separation. In summary, we demonstrate that site-specific ubiquitylation is a general functional regulator for linker histone H1.

2021-07-20 | PXD020686 | Pride

Global, site-resolved analysis of ubiquitylation occupancy and turnover rate reveals systems properties

Project description:Ubiquitylation regulates most proteins and biological processes in a eukaryotic cell. However, the site-specific occupancy (stoichiometry) and turnover rate of ubiquitylation have not been quantified. Here we present an integrated picture of the global ubiquitylation site occupancy and half-life. Ubiquitylation site occupancy spans over four orders of magnitude, but the median ubiquitylation site occupancy is three orders of magnitude lower than that of phosphorylation. The occupancy, turnover rate, and regulation of sites by proteasome inhibitors are strongly interrelated, and these attributes distinguish sites involved in proteasomal degradation and cellular signaling. Sites in structured protein regions exhibit longer half-lives and stronger upregulation by proteasome inhibitors than sites in unstructured regions. Importantly, we discovered a surveillance mechanism that rapidly and site-indiscriminately deubiquitylates all ubiquitin-specific E1 and E2 enzymes, protecting them against accumulation of bystander ubiquitylation. The work provides a systems-scale, quantitative view of ubiquitylation properties and reveals general principles of ubiquitylation-dependent governance.

2024-04-15 | PXD037009 | Pride

Variation in auxin sensing guides AUX/IAA repressor ubiquitylation and destruction

Project description:Auxin is a drug-like small molecule and morphogen that triggers the formation of SCFTIR1/AFB-AUX/IAA co-receptor complexes leading to ubiquitylation and proteasome-dependent degradation of AUX/IAA transcriptional repressors in plants. Here, we systematically dissect auxin sensing by SCFTIR1-IAA6 and SCFTIR1-IAA19 co-receptor complexes, and assess IAA6/IAA19 ubiquitylation in vitro and IAA6/IAA19 degradation in vivo. TIR1-IAA19 and TIR1-IAA6 interactions form co-receptors with different affinities, which specify ubiquitylation and turnover rate of the AUX/IAA. We demonstrate lysine ubiquitylation in IAA6/IAA19 and propose this ubiquitylation signature to be a consequence of auxin-mediated SCFTIR1-AUX/IAA interactions. We present evidence for an evolving AUX/IAA repertoire, typified by the IAA6/IAA19 ohnologs, that contributes differentially to auxin sensing. We postulate that AUX/IAAs have emerged as a versatility toolbox for auxin-modulated transcriptional control, as the gamut of AUX/IAA destruction kinetics enables fine-tuning of transcriptional auxin response and contributes to the complexity of hormone signaling.

2017-06-14 | PXD004027 | Pride

Degron flexibility enforces auxin sensing

Project description:Cullin RING-type E3 ubiquitin ligase SCFTIR1/AFB1-5 and their ubiquitylation targets, AUX/IAAs, sense auxin concentrations in the nucleus. TIR1 binds a surface- exposed degron in AUX/IAAs promoting their ubiquitylation and rapid auxin-regulated proteasomal degradation. Here, we resolved TIR1·auxin·IAA7 and TIR1·auxin·IAA12 complex topology, and show that flexible intrinsically disordered regions (IDRs) cooperatively position AUX/IAAs on TIR1. The AUX/IAA PB1 interaction domain also assists in non-native contacts, affecting AUX/IAA dynamic interaction states. Our results establish a role for IDRs in modulating auxin receptor assemblies. By securing AUX/IAAs on two opposite surfaces of TIR1, IDR diversity supports locally tailored positioning for targeted ubiquitylation, and might provide conformational flexibility for adopting a multiplicity of functional states. We postulate IDRs in distinct members of the AUX/IAA family to be an adaptive signature for protein interaction and initiation region for proteasome recruitment.

2020-05-27 | PXD015392 | Pride

Ubiquitylated-Rpn10 - Structure of ubiquitylated-Rpn10 provides insight into its autoregulation mechanism

Project description:MS/MS characterization of the proteasomal ubiquitin-receptor Rpn10 in its ubiquitylated state that together with structural and biochemical studies reveal a novel ubiquitin-binding patch on RPN10 that directs K84 ubiquitylation.

2018-10-26 | PXD004761 | Pride

The interactome of site-specifically acetylated linker histone H1

Project description:Linker histone H1 plays a key role in chromatin organization and maintenance, however, our knowledge of the regulation of H1 functions by its posttranslational modifications (PTMs) is very limited. In this study, we report on the generation of homogeneously and site-specifically mono- and di-acetylated H1 (H1 Ac) using genetic code expansion. We used these modified histones to identify and comprehensively characterize the acetylation-dependent cellular interactome for linker histone H1 and show that site-specific acetylation results in overlapping, but distinct groups of interacting partners. Intriguingly, H1 acetylation-specific interactors comprise translational initiation factors and are involved in transcriptional regulation, suggesting that acetylation of H1 may indeed act as a regulator of the linker histone H1 by modulation of protein-protein interactions.

2021-07-09 | PXD025832 | Pride

Internally Tagged Ubiquitin: a Tool to Identify Linear Polyubiquitin-modified Proteins by Mass Spectrometry

Project description:Even though ubiquitination controls a plethora of cellular processes, modifications by linear polyubiquitin have so far only been linked with acquired and innate immunity, lymphocyte development and genotoxic stress response. Until now, a single E3 ligase complex (LUBAC), one specific deubiquitinase (Otulin) and a very few substrates have been identified. The existing methods for the study of lysine-based polyubiquitination are not suitable for the detection of linear polyubiquitin-modified proteins. Here, we present a novel approach to discovering linear polyubiquitin-modified substrates by combining lysine-less internally tagged ubiquitin (INT-Ub.7KR) with SILAC-based mass spectrometry. We applied our approach in TNFα-stimulated T-Rex HEK293T cells and afterwards validated newly identified linear polyubiquitin targets. Moreover, we demonstrated that linear polyubiquitination of the novel LUBAC substrate TRAF6 is essential for the NFkappaB signalling. Overall, we have established a powerful method for the detection of linear polyubiquitin substrates.

2017-03-21 | PXD005878 | Pride

Expression data of sleeping, waking, and sleep deprived adult heterozygous aldh1l1 – eGFP-L10a mice

Project description:Transcriptomic studies revealed that hundreds of mRNAs show differential expression in the brains of sleeping versus awake rats, mice, flies, and sparrows. Although these results have offered clues regarding the molecular consequences of sleep and sleep loss, their functional significance thus far has been limited. This is because the previous studies pooled transcripts from all brain cells, including neurons and glia. In the following experiment, we studied the specific effects of sleep and wake conditions on glia cells of mouse cerebral cortex using the genetically targeted translating ribosome affinity purification (TRAP) methodology. We used bacterial artificial chromosome (BAC) transgenic mice expressing EGFP tagged ribosomal protein L10a in astrocytes which constitute a defined cellular population of the mouse brain. Using this approach, we could extract only the astrocytic mRNAs, and only those already committed to be translated into proteins (L10a is part of the translational machinery). Six mice for each vigilant state group (sleep (S), waking (W), and sleep deprivation (SD)) were considered. For each animal, cerebral cortex was dissected and immediately processed. Samples were immunoprecipitated to isolate astrocytes. The precipitated portion formed the bound sample (IP) containing astrocytes and the remaining part formed the unbound sample (UB) containing all the remaining cell types (neurons and other glia cells). Then, both IP and UB samples were processed and RNA was extracted. All the IP samples (n=18) were then hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 arrays, while UB samples were pooled together in two biological replicates (UB1+UB2+UB3 and UB4+UB5+UB6) for each group (n=6 in total) and then hybridized to Affymetrix GeneChip Mouse Genome 430 2.0 arrays.

2015-07-08 | E-GEOD-69079 | biostudies-arrayexpress

Sequential Peptide Immunopurification Methodology to Monitor the Crosstalk between SUMOylation and Ubiquitylation

Project description:We describe a method that permits the identification of proteins that are modified by both SUMOylation and ubiquitylation to better understand the role of this crosstalk. The procedure requires 3 days when starting from cell pellets and can yield more than 8000 SUMO sites and 3500 ubiquitin sites from 16 mg of cell extract.

2018-03-27 | PXD006545 | Pride

Expression data of sleeping, waking, and sleep deprived in adult heterozygous Cnp M-bM-^@M-^S eGFP-L10a mice

Project description:Transcriptomic studies revealed that hundreds of mRNAs show differential expression in the brains of sleeping versus awake rats, mice, flies, and sparrows. Although these results have offered clues regarding the molecular consequences of sleep and sleep loss, their functional significance thus far has been limited. This is because the previous studies pooled transcripts from all brain cells, including neurons and glia. In the following experiment, we studied the specific effects of sleep and wake conditions on glia cells of mouse cerebral cortex using the genetically targeted translating ribosome affinity purification (TRAP) methodology. We used bacterial artificial chromosome (BAC) transgenic mice expressing EGFP tagged ribosomal protein L10a in oligodendrocytes which constitute a defined cellular population of the mouse brain. Using this approach, we could extract only the oligodendrocitic mRNAs, and only those already committed to be translated into proteins (L10a is part of the translational machinery). Six mice for each vigilant state group (sleep (S), waking (W), and sleep deprivation (SD)) were considered. For each animal, one forebrain sample was immediately processed. Samples were immunoprecipitated to isolate oligodendrocytes. The precipitated portion formed the bound sample (IP) containing olidodendrocytes and the remaining part formed the unbound sample (UB) containing all the remaining cell types (neurons and other glia cells). Then, both IP and UB samples were processed and RNA was extracted.

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