Histone Demethylation by a Family of UBA Domain-containing Proteins
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA560890 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10003804.fastq.gz | Fastqsanger.gz | |||
| SRR10003805.fastq.gz | Fastqsanger.gz | |||
| SRR10003806.fastq.gz | Fastqsanger.gz | |||
| SRR10003807.fastq.gz | Fastqsanger.gz | |||
| SRR10003808.fastq.gz | Fastqsanger.gz |
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Project description:Histone methyl groups can be removed by histone demethylases. Although LSD1 and JmjC domain-containing proteins have been identified to be histone demethylases, enzymes responsible for many histone methylation states or sites are not identified. Here, we performed a high-content cell-based screening of a cDNA library containing 2,500 nuclear proteins and identified KDM10A as a histone demethylase specific for histone H4 lysine 20 (H4K20). Ectopic expression of KDM10A reduced the global levels of H4K20me1/2/3 in 293T cells. In vitro, KDM10A specifically demethylated H4K20me1/2/3 and generated formaldehyde. The enzymatic activity required Fe(II) and α-ketoglutarate as cofactors. Domain mapping indicated that the demethylase activity required its UBA domain. We also demonstrated that KDM10B, a protein homologous to KDM10A, had H4K20 demethylase activity in vitro and in vivo. ChIP-seq reveals that KDM10A/B demethylated H4K20 methylation at specific genomic loci in vivo. We further demonstrated that KDM10A/B activated the transcription of coding genes by demethylating H4K20me1 in the gene body, and the transcription of repetitive elements by demethylating H4K20me3 in intergenic regions. NMR analyses demonstrated that an HxxxE motif in the UBA domain is crucial for iron binding, mutation of these two residues abolished iron binding and the demethylase activity. Thus, we identified a family of UBA domain-containing proteins as histone demethylases.
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Project description:RNA binding proteins (RBPs) participate in almost all steps of gene expression, underscoring their potential as key regulators of RNA homeostasis. We structurally and functionally characterized Mip6, a four-RNA Recognition Motif (RRM)-containing RBP, as a functional and physical interactor of the mRNA export factor Mex67. Mip6 directly interacts with the ubiquitin-associated (UBA) domain of Mex67 through tryptophan 442 of Mip6-RRM4, whose mutation leads to slow growth of yeast cells in vivo. Although Mip6-RRM4 binds to RNA in vitro, Mex67 UBA binding prevents Mip6-RRM4 interaction with RNA. Mip6 shuttles between the nucleus and the cytoplasm in a Mex67-dependent manner and concentrates in cytoplasmic foci under several stressors. Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) experiments showed preferential binding of Mip6 to mRNAs regulated by the stress-response Msn2/4 transcription factors. Consistent with this binding, MIP6 deletion affected their export and expression levels. These results reveal a novel role for Mip6 in the homeostasis of Msn2/4 dependent transcripts by its direct interaction with Mex67 UBA domain.
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Project description:The assembly of a specific polymeric ubiquitin (Ub) chain on a target protein is a key event in the regulation of numerous cellular processes. Yet, the mechanisms that govern the selective synthesis of particular polyubiquitin signals remain enigmatic. The ubiquitin-conjugating (E2) enzymes Ubc1 in yeast and Ube2K in mammals exclusively generate polyubiquitin linked through lysine 48 (K48). Uniquely among E2 enzymes, Ubc1 and Ube2K harbor a Ub binding UBA domain with unknown function. We found that this UBA domain preferentially interacts with Ub chains linked through lysine 63 (K63). Based on structural modeling, in vitro ubiquitination experiments and NMR studies, we propose that the UBA domain aligns Ubc1 with K63-linked polyubiquitin and facilitates the selective assembly of K48/K63-branched Ub molecules. Genetic experiments link the activity of the UBA domain and hence the formation of this unusual Ub chain topology to the maintenance of cellular proteostasis.
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Project description:by DUBs need to be tightly controlled. Here, we identify asparagine hydroxylation as a novel posttranslational modification involved in the regulation of Cezanne (OTUD7B), a DUB that controls key cellular functions and signaling pathways. We demonstrate that Cezanne is a substrate for FIH1- and oxygen-dependent asparagine hydroxylation. FIH1 modifies Asn35 within the uncharacterized N-terminal ubiquitin-associated (UBA)-like domain of Cezanne (UBACez), which lacks conserved UBA domain properties. We show for the first time that UBACez binds Lys11-, Lys48-, Lys63- and Met1-linked ubiquitin chains in vitro, and thereby establish UBACez as a functional ubiquitin-binding domain. We reveal that interaction of UBACez with ubiquitin is mediated via a non-canonical surface, and that hydroxylation of Asn35 inhibits ubiquitin binding. Recently, it has been suggested that recruitment of Cezanne to specific target proteins depends on UBACez. Our data show that UBACez can indeed fulfil this role as regulatory domain by binding differently linked ubiquitin chains and that this interaction with ubiquitin, and thus modified substrates, can be modulated by asparagine hydroxylation.
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Project description:Assembly of polymeric ubiquitin (Ub) chains is an essential posttranslational protein modification that regulates widespread intracellular processes in eukaryotic cells. Factors and mechanisms that regulate the formation of Ub chains are key to the understanding of many cellular processes. The E2 enzyme Ubc1 (Ube2K) exclusively targets K48 in Ub for chain formation and has been linked to cell-cycle progression and proteostasis. Uniquely among E2 enzymes, it harbors a Ub binding UBA domain, which has been implicated in Ub chain formation but its function remained elusive. Through in vitro binding experiments, we unexpectedly found that the UBA domain enables preferential binding of K63-linked Ub chains. Based on structural modeling, extensive in vitro ubiquitination experiments and NMR binding studies, we propose a mechanism through which Ubc1 selectively forms K48/K63 branched Ub chains – an usual chain architecture, about whose prevalence and function little is known to date. Ultimately, we link UBA dependent activity of Ubc1 to its role in proteostasis through genetic experiments.
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Project description:To systematically investigate the regulations and functions of human WW-domain containing proteins, we conducted a proteomic analysis of 26 full-length WW-domain containing proteins and 17 WW-domains only, and identified their associated protein complexes in human HEK293T cells using tandem affinity purifications followed by LC-MSMS.
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