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Functional comparison of H1 histones in Xenopus reveals isoform-specific regulation by Cdk1 and RanGTP.

ABSTRACT: H1 "linker" histones bind dynamically to nucleosomes and promote their compaction into chromatin fibers. Developmental H1 isoforms are evolutionarily conserved, but their function, regulation, and posttranslational modifications are poorly understood. In Xenopus egg extracts, the embryonic linker histone H1M does not affect nuclear assembly or replication but is required for proper chromosome architecture during mitosis. We report here that somatic H1 isoforms, which are more positively charged and feature multiple Cdk1 phosphorylation sites, cannot substitute for H1M at endogenous concentrations, instead causing chromatin compaction during interphase and dissociating from chromosomes at the onset of mitosis. Mitotic Cdk1 phosphorylation is not responsible for this dissociation and instead functions to enhance H1 binding in egg extracts and embryos. Nuclear import receptors RanBP7 and importin beta bind tightly to somatic H1 but not H1M, and addition of a constitutively active Ran mutant abolishes this interaction and enhances the ability of somatic H1 to rescue mitotic chromosome architecture. Our results reveal distinct regulatory mechanisms among linker histone isoforms and a specific role for H1M to compact chromosomes during egg meiotic arrest and early embryonic divisions.

SUBMITTER: Freedman BS

PROVIDER: S-EPMC2902237 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Functional comparison of H1 histones in Xenopus reveals isoform-specific regulation by Cdk1 and RanGTP.

Freedman Benjamin S BS Heald Rebecca R

Current biology : CB 20100513 11

H1 "linker" histones bind dynamically to nucleosomes and promote their compaction into chromatin fibers. Developmental H1 isoforms are evolutionarily conserved, but their function, regulation, and posttranslational modifications are poorly understood. In Xenopus egg extracts, the embryonic linker histone H1M does not affect nuclear assembly or replication but is required for proper chromosome architecture during mitosis. We report here that somatic H1 isoforms, which are more positively charged ...[more]

PMID: 20471264

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Project description:Padavannil et al. 2019 show that Importin-9 (Imp9) transports Histones H2A-H2B from the cytoplasm to the nucleus using a non-canonical mechanism whereby binding of a GTP-bound Ran GTPase (RanGTP) fails to evict the H2A-H2B cargo. Instead, a stable complex forms, comprised of equimolar RanGTP, Imp9, and H2A-H2B. Unlike the binary Imp9•H2A-H2B complex, this RanGTP•Imp9•H2A-H2B ternary complex can release H2A-H2B to an assembling nucleosome. Here, we define the molecular basis for this RanGTP-activated nucleosome assembly by Imp9. We use hydrogen-deuterium exchange coupled with mass spectrometry and compare the dynamics and interfaces of the RanGTP•Imp9•H2A-H2B ternary complex to those in the Imp9•H2A-H2B or Imp9•RanGTP binary complexes. Our data are consistent with the Imp9•H2A-H2B structure by Padavannil et al. 2019 showing that Imp9 HEAT repeats 4-5 and 18-19 contact H2A-H2B, as well as many homologous importin•RanGTP structures showing that importin HEAT repeats 1 and 3, and the h8 loop, contact RanGTP. We show that Imp9 stabilizes H2A-H2B beyond the direct binding site, similar to other histone chaperones. Importantly, we reveal that binding of RanGTP releases H2A-H2B interaction at Imp9 HEAT repeats 4-5, but not 18-19. This exposes DNA- and histone-binding surfaces of H2A-H2B, thereby facilitating nucleosome assembly. We also reveal that RanGTP has a weaker affinity for Imp9 when H2A-H2B is bound. This may ensure that H2A-H2B is only released in high RanGTP concentrations near chromatin. We delineate the molecular link between the nuclear import of H2A-H2B and its deposition into chromatin by Imp9.SignificanceImp9 is the primary importin for shuttling H2A-H2B from the cytoplasm to the nucleus. It employs an unusual mechanism where the binding of RanGTP alone is insufficient to release H2A-H2B. The resulting stable RanGTP•Imp9•H2A-H2B complex gains nucleosome assembly activity as H2A-H2B can be deposited onto an assembling nucleosome. We show that H2A-H2B is allosterically stabilized via interactions with both N- and C-terminal portions of Imp9, reinforcing its chaperone-like behavior. RanGTP binding causes H2A-H2B release from the N-terminal portion of Imp9 only. The newly-exposed H2A-H2B surfaces can interact with DNA or H3-H4 in nucleosome assembly. Imp9 thus plays a multi-faceted role in histone import, storage, and deposition regulated by RanGTP, controlling histone supply in the nucleus and to chromatin.

Project description:Histones possess significant antimicrobial potential, yet their activity against biofilms remains underexplored. Moreover, concerns regarding adverse effects limit their clinical implementation. We investigated the antibacterial efficacy of human recombinant histone H1 subtypes against Pseudomonas aeruginosa PAO1, both planktonic and in biofilms. After the in vitro tests, toxicity and efficacy were assessed in a P. aeruginosa PAO1 infection model using Galleria mellonella larvae. Histones were also evaluated in combination with ciprofloxacin (Cpx) and gentamicin (Gm). Our results demonstrate antimicrobial activity of all three histones against P. aeruginosa PAO1, with H1.0 and H1.4 showing efficacy at lower concentrations. The bactericidal effect was associated with a mechanism of membrane disruption. In vitro studies using static and dynamic models showed that H1.4 had antibiofilm potential by reducing cell biomass. Neither H1.0 nor H1.4 showed toxicity in G. mellonella larvae, and both increased larvae survival when infected with P. aeruginosa PAO1. Although in vitro synergism was observed between ciprofloxacin and H1.0, no improvement over the antibiotic alone was noted in vivo. Differences in antibacterial and antibiofilm activity were attributed to sequence and structural variations among histone subtypes. Moreover, the efficacy of H1.0 and H1.4 was influenced by the presence and strength of the extracellular matrix. These findings suggest histones hold promise for combating acute and chronic infections caused by pathogens such as P. aeruginosa.IMPORTANCEThe constant increase of multidrug-resistant bacteria is a critical global concern. The inefficacy of current therapies to treat bacterial infections is attributed to multiple mechanisms of resistance, including the capacity to form biofilms. Therefore, the identification of novel and safe therapeutic strategies is imperative. This study confirms the antimicrobial potential of three histone H1 subtypes against both Gram-negative and Gram-positive bacteria. Furthermore, histones H1.0 and H1.4 demonstrated in vivo efficacy without associated toxicity in an acute infection model of Pseudomonas aeruginosa PAO1 in Galleria mellonella larvae. The bactericidal effect of these proteins also resulted in biomass reduction of P. aeruginosa PAO1 biofilms. Given the clinical significance of this opportunistic pathogen, our research provides a comprehensive initial evaluation of the efficacy, toxicity, and mechanism of action of a potential new therapeutic approach against acute and chronic bacterial infections.

Dataset Information

Functional comparison of H1 histones in Xenopus reveals isoform-specific regulation by Cdk1 and RanGTP.

Publications

Functional comparison of H1 histones in Xenopus reveals isoform-specific regulation by Cdk1 and RanGTP.

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