RESUMO
The evolutionarily conserved HIRA/Hir histone chaperone complex and ASF1a/Asf1 co-chaperone cooperate to deposit histone (H3/H4)2 tetramers on DNA for replication-independent chromatin assembly. The molecular architecture of the HIRA/Hir complex and its mode of histone deposition have remained unknown. Here, we report the cryo-EM structure of the S. cerevisiae Hir complex with Asf1/H3/H4 at 2.9-6.8 Å resolution. We find that the Hir complex forms an arc-shaped dimer with a Hir1/Hir2/Hir3/Hpc2 stoichiometry of 2/4/2/4. The core of the complex containing two Hir1/Hir2/Hir2 trimers and N-terminal segments of Hir3 forms a central cavity containing two copies of Hpc2, with one engaged by Asf1/H3/H4, in a suitable position to accommodate a histone (H3/H4)2 tetramer, while the C-terminal segments of Hir3 harbor nucleic acid binding activity to wrap DNA around the Hpc2-assisted histone tetramer. The structure suggests a model for how the Hir/Asf1 complex promotes the formation of histone tetramers for their subsequent deposition onto DNA.
Assuntos
Proteínas de Ciclo Celular , Microscopia Crioeletrônica , Chaperonas de Histonas , Histonas , Ligação Proteica , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Histonas/metabolismo , Histonas/química , Histonas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Chaperonas de Histonas/metabolismo , Chaperonas de Histonas/química , Chaperonas de Histonas/genética , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Multimerização Proteica , Sítios de Ligação , Fatores de Transcrição/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Domínios e Motivos de Interação entre ProteínasRESUMO
In previous animal model studies, we demonstrated the potential of rAAV2-sVEGFRv-1, which encodes a truncated variant of the alternatively spliced soluble version of VEGF receptor-1 (VEGFR1), as a human gene therapy for age-related macular degeneration (AMD) and diabetic retinopathy (DR). Here, we elucidate in vitro some of the mechanisms by which rAAV2-sVEGFRv-1 exerts its therapeutic effects. Human umbilical vein endothelial cells (HUVECs) were infected with rAAV2-sVEGFRv-1 or a control virus vector in the presence of members of the VEGF family to identify potential binding partners via ELISA, which showed that VEGF-A, VEGF-B, and placental growth factor (PlGF) are all ligands of its transgene product. In order to determine the effects of rAAV2-sVEGFRv-1 on cell proliferation and permeability, processes that are important to the progression AMD and DR, HUVECs were infected with the therapeutic virus vector under the stimulation of VEGF-A, the major driver of the neovascularization that characterizes the forms of these conditions most associated with vision loss. rAAV2-sVEGFRv-1 treatment, as a result, markedly reduced the extent to which these processes occurred, with the latter determined by measuring zonula occludens 1 expression. Finally, the human microglial HMC3 cell line was used to show the effects of the therapeutic virus vector upon inflammatory processes, another major contributor to angiogenic eye disease pathophysiology, with rAAV2-sVEGFRv-1 reducing therein the secretion of pro-inflammatory cytokines interleukin (IL)-1ß and IL-6. Combined with our previously published in vivo data, the in vitro activity of the expressed transgene here further demonstrates the great promise of rAAV2-sVEGFRv-1 as a potential human gene therapeutic for addressing angiogenic ocular conditions.
Assuntos
Dependovirus , Terapia Genética , Células Endoteliais da Veia Umbilical Humana , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Humanos , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Dependovirus/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Proliferação de Células , Degeneração Macular/terapia , Degeneração Macular/genética , Degeneração Macular/metabolismo , Retinopatia Diabética/terapia , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Fator B de Crescimento do Endotélio Vascular/genética , Fator B de Crescimento do Endotélio Vascular/metabolismo , Fator de Crescimento Placentário/genética , Fator de Crescimento Placentário/metabolismoRESUMO
The eukaryotic Mediator, comprising a large Core (cMED) and a dissociable CDK8 kinase module (CKM), regulates RNA Polymerase II (Pol II)-dependent transcription. cMED recruits Pol II and promotes pre-initiation complex (PIC) formation in a manner inhibited by the CKM, which is also implicated in post-initiation control of gene expression. Herein we report cryo-electron microscopy structures of the human complete Mediator and its CKM, which explains the basis for CKM inhibition of cMED-activated transcription. The CKM binds to cMED through an intrinsically disordered region (IDR) in MED13 and HEAT repeats in MED12. The CKM inhibits transcription by allocating its MED13 IDR to occlude binding of Pol II and MED26 to cMED and further obstructing cMED-PIC assembly through steric hindrance with TFIIH and the +1 nucleosome. Notably, MED12 binds to the cMED Hook, positioning CDK8 downstream of the transcription start site, which sheds new light on its stimulatory function in post-initiation events.