Ubiquitin and ubiquitin-like modifications are central to virtually all cellular signaling pathways. They occur primarily on lysine residues of target proteins and stimulate a large number of ...downstream signals. The diversity of these signals depends on the type, location and dynamics of the modification, but the role of the exact site of modification and the selectivity for specific lysines are poorly understood. Here we review the current literature on lysine specificity in these modifications, and we highlight the known signaling mechanisms and the open questions that pose future challenges to ubiquitin research.
Histone proteins dynamically regulate chromatin structure and epigenetic signaling to maintain cell homeostasis. These processes require controlled spatial and temporal deposition and eviction of ...histones by their dedicated chaperones. With the evolution of histone variants, a network of functionally specific histone chaperones has emerged. Molecular details of the determinants of chaperone specificity for different histone variants are only slowly being resolved. A complete understanding of these processes is essential to shed light on the genuine biological roles of histone variants, their chaperones, and their impact on chromatin dynamics.
This review assesses the determinants of the specificity in histone variant‐histone chaperone interactions at the molecular and atomic level, and discusses biological roles of histone variants and chaperones.
Structure of histone-based chromatin in Archaea Mattiroli, Francesca; Bhattacharyya, Sudipta; Dyer, Pamela N. ...
Science (American Association for the Advancement of Science),
08/2017, Letnik:
357, Številka:
6351
Journal Article
Recenzirano
Odprti dostop
Small basic proteins present in most Archaea share a common ancestor with the eukaryotic core histones. We report the crystal structure of an archaeal histone-DNA complex. DNA wraps around an ...extended polymer, formed by archaeal histone homodimers, in a quasi-continuous superhelix with the same geometry as DNA in the eukaryotic nucleosome. Substitutions of a conserved glycine at the interface of adjacent protein layers destabilize archaeal chromatin, reduce growth rate, and impair transcription regulation, confirming the biological importance of the polymeric structure. Our data establish that the histone-based mechanism of DNA compaction predates the nucleosome, illuminating the origin of the nucleosome.
Complex mechanisms are in place to maintain genome stability. Ubiquitination of chromatin plays a central role in these mechanisms. The ever-growing complexity of the ubiquitin (Ub) code and of ...chromatin modifications and dynamics challenges our ability to fully understand how histone ubiquitination regulates genome stability. Here we review the current knowledge on specific, low-abundant histone ubiquitination events that are highly regulated within the cellular DNA damage response (DDR), with particular emphasis on the latest discovery of Ub phosphorylation as a novel regulator of the DDR signaling pathway. We discuss players involved and potential implications of histone (phospho)ubiquitination on chromatin structure, and we highlight exciting open questions for future research.
Histones are heavily post-translationally modified by ubiquitin. Lately, many more ubiquitin-based modifications have been mapped on histones, which are far less abundant than the canonical ones but essential to safeguard genome stability.Histones at damaged DNA can be modified by phospho-ubiquitin (i.e., pUb at Thr12), resulting in chromatin regions with new properties that impact DNA repair pathways.The discovery of new ubiquitination sites on histones and new ubiquitin modifications expands dramatically the landscape of chromatin-based signaling and crosstalk, paving the way for future studies on novel mechanisms in genome stability programs (i.e., new writers, readers, and erasers).
Nucleosome assembly in the wake of DNA replication is a key process that regulates cell identity and survival. Chromatin assembly factor 1 (CAF-1) is a H3-H4 histone chaperone that associates with ...the replisome and orchestrates chromatin assembly following DNA synthesis. Little is known about the mechanism and structure of this key complex. Here we investigate the CAF-1•H3-H4 binding mode and the mechanism of nucleosome assembly. We show that yeast CAF-1 binding to a H3-H4 dimer activates the Cac1 winged helix domain interaction with DNA. This drives the formation of a transient CAF-1•histone•DNA intermediate containing two CAF-1 complexes, each associated with one H3-H4 dimer. Here, the (H3-H4)
tetramer is formed and deposited onto DNA. Our work elucidates the molecular mechanism for histone deposition by CAF-1, a reaction that has remained elusive for other histone chaperones, and it advances our understanding of how nucleosomes and their epigenetic information are maintained through DNA replication.
During DNA damage response, the RING E3 ligase RNF168 ubiquitinates nucleosomal H2A at K13-15. Here we show that the ubiquitination reaction is regulated by its substrate. We define a region on the ...RING domain important for target recognition and identify the H2A/H2B dimer as the minimal substrate to confer lysine specificity to the RNF168 reaction. Importantly, we find an active role for the substrate in the reaction. H2A/H2B dimers and nucleosomes enhance the E3-mediated discharge of ubiquitin from the E2 and redirect the reaction towards the relevant target, in a process that depends on an intact acidic patch. This active contribution of a region distal from the target lysine provides regulation of the specific K13-15 ubiquitination reaction during the complex signalling process at DNA damage sites.
All vertebrate genomes encode for three large histone H2A variants that have an additional metabolite-binding globular macrodomain module, macroH2A. MacroH2A variants impact heterochromatin ...organization and transcription regulation and establish a barrier for cellular reprogramming. However, the mechanisms of how macroH2A is incorporated into chromatin and the identity of any chaperones required for histone deposition remain elusive. Here, we develop a split-GFP-based assay for chromatin incorporation and use it to conduct a genome-wide mutagenesis screen in haploid human cells to identify proteins that regulate macroH2A dynamics. We show that the histone chaperone ANP32B is a regulator of macroH2A deposition. ANP32B associates with macroH2A in cells and in vitro binds to histones with low nanomolar affinity. In vitro nucleosome assembly assays show that ANP32B stimulates deposition of macroH2A-H2B and not of H2A-H2B onto tetrasomes. In cells, depletion of ANP32B strongly affects global macroH2A chromatin incorporation, revealing ANP32B as a macroH2A histone chaperone.
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•Split-GFP can be used as a high-throughput readout for chromatin incorporation•Genetic screen identified macroH2A regulators•ANP32B binds macroH2A-H2B dimers and deposits them onto tetrasomes in vitro•Loss of ANP32B affects macroH2A chromatin localization on a genome wide scale
Mandemaker et al. establish a split-GFP based assay to measure chromatin incorporation and use it to perform a genetic screen to identify regulators of the histone variant macroH2A. They show that ANP32B can directly bind to macroH2A and affect its deposition both in vitro and in cells.
Prostate cancer (PCa) is a major lethal malignancy in men, but the molecular events and their interplay underlying prostate carcinogenesis remain poorly understood. Epigenetic events and the ...upregulation of polycomb group silencing proteins including Bmi1 have been described to occur during PCa progression. Here, we found that conditional overexpression of Bmi1 in mice induced prostatic intraepithelial neoplasia, and elicited invasive adenocarcinoma when combined with PTEN haploinsufficiency. In addition, Bmi1 and the PI3K/Akt pathway were coactivated in a substantial fraction of human high-grade tumors. We found that Akt mediated Bmi1 phosphorylation, enhancing its oncogenic potential in an Ink4a/Arf-independent manner. This process also modulated the DNA damage response and affected genomic stability. Together, our findings demonstrate the etiological role of Bmi1 in PCa, unravel an oncogenic collaboration between Bmi1 and the PI3K/Akt pathway, and provide mechanistic insights into the modulation of Bmi1 function by phosphorylation during prostate carcinogenesis.
For a complete understanding of biochemical reactions, information on complex stoichiometry is essential. However, measuring stoichiometry is experimentally challenging. Our lab has developed a ...FRET-based assay to study protein complex stoichiometry
. This assay, also known as Job plot, is set up as a continuous variation of the molar ratio between the two species, kept at constant total concentration. The FRET (Fluorescence Resonance Energy Transfer) between the two fluorescently-labeled proteins is measured and the stoichiometry is inferred from the sample with highest FRET signal. This approach allows us to assess complex stoichiometry in solution.
Nucleosomes organize the eukaryotic genome into chromatin. In cells, nucleosome assembly relies on the activity of histone chaperones, proteins with high binding affinity to histones. At least a ...subset of histone chaperones promotes histone deposition
. However, it has been challenging to characterize this activity, due to the lack of quantitative assays. Here we developed a quantitative nucleosome assembly (NAQ) assay to measure the amount of nucleosome formation
. This assay relies on a Micrococcal nuclease (MNase) digestion step that yields DNA fragments protected by the deposited histone proteins. A subsequent run on the Bioanalyzer machine allows the accurate quantification of the fragments (length and amount), relative to a loading control. This allows us to measure nucleosome formation by following the signature DNA length of ~150 bp. This assay finally enables the characterization of the nucleosome assembly activity of different histone chaperones, a step forward in the understanding of the functional roles of these proteins
.
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