RESUMO
The HIRA histone chaperone complex is composed of the proteins HIRA, UBN1, and CABIN1 and cooperates with the histone chaperone ASF1a to specifically bind and deposit H3.3/H4 into chromatin. We recently reported that the UBN1 Hpc2-related domain (HRD) specifically binds to H3.3/H4 over H3.1/H4. However, the mechanism for HIRA complex deposition of H3.3/H4 into nucleosomes remains unclear. Here, we characterize a central region of UBN1 (UBN1 middle domain) that is evolutionarily conserved and predicted to have helical secondary structure. We report that the UBN1 middle domain has dimer formation activity and binds to H3/H4 in a manner that does not discriminate between H3.1 and H3.3. We additionally identify a nearby DNA-binding domain in UBN1, located between the UBN1 HRD and middle domain, which binds DNA through electrostatic contacts involving several conserved lysine residues. Together, these observations suggest a mechanism for HIRA-mediated H3.3/H4 deposition whereby UBN1 associates with DNA and dimerizes to mediate formation of an (H3.3/H4)2 heterotetramer prior to chromatin deposition.
Assuntos
DNA/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Cromatina/metabolismo , Dimerização , Histonas/genética , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Alinhamento de Sequência , Eletricidade Estática , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
The human enzyme histone acetyltransferase binding to ORC1 (HBO1) regulates DNA replication, cell proliferation, and development. HBO1 is part of a multiprotein histone acetyltransferase (HAT) complex that also contains inhibitor of growth family member (ING) 4/5, MYST/Esa1-associated factor (MEAF) 6, and the scaffolding proteins Jade family PHD finger (JADE) 1/2/3 or bromodomain and PHD finger-containing protein (BRPF) 2/3 to acetylate histone H4 H4K5/8/12 or H3K14, respectively. Within this four-protein complex, JADE1 determines histone H4 substrate specificity of the HBO1-HAT complex. However, the mechanism by which JADE1 controls the H4-specific acetyltransferase activity of HBO1 is unknown. Here we used recombinant proteins in vitro to dissect the specific regions and activities of HBO1 and JADE1 that mediate histone H3-H4 acetylation via the HBO1-HAT domain. We found that JADE1 increases the catalytic efficiency of HBO1 acetylation of an H3-H4 substrate by about 5-fold through an N-terminal, 21-residue HBO1- and histone-binding domain and a nearby second histone core-binding domain. We also demonstrate that HBO1 contains an N-terminal histone-binding domain (HBD) that makes additional contacts with H3-H4 independent of JADE1 interactions with histones and that the HBO1 HBD does not significantly contribute to HBO1's overall HAT activity. Experiments with JADE1 deletions in vivo recapitulated these in vitro interactions and their roles in HBO1 histone acetylation activity. Together, these results indicate that the N-terminal region of JADE1 functions as a platform that brings together the catalytic HBO1 subunit with its cognate H3-H4 substrate for histone acetylation.
Assuntos
Cromatina/metabolismo , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Acetilação , Sequência de Aminoácidos , Cromatina/genética , Replicação do DNA , Células HEK293 , Histona Acetiltransferases/genética , Histonas/genética , Proteínas de Homeodomínio/genética , Humanos , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/genética , Homologia de Sequência , Especificidade por Substrato , Proteínas Supressoras de Tumor/genéticaRESUMO
Until recently, a major limitation of hydrogen-deuterium exchange mass spectrometry (HDX-MS) was that resolution of deuterium localization was limited to the length of the peptide generated during proteolysis. However, electron transfer dissociation (ETD) has been shown to preserve deuterium label in the gas phase, enabling better resolution. To date, this technology remains mostly limited to small, already well-characterized proteins. Here, we optimize, expand, and adapt HDX-MS tandem MS (MS/MS) capabilities to accommodate histone and nucleosomal complexes on top-down HDX-MS/MS and middle-down HDX-MS/MS platforms and demonstrate that near site-specific resolution of deuterium localization can be obtained with high reproducibility. We are able to study histone tail dynamics in unprecedented detail, which have evaded analysis by traditional structural biology techniques for decades, revealing important insights into chromatin biology. Together, the results of these studies highlight the versatility, reliability, and reproducibility of ETD-based HDX-MS/MS methodology to interrogate large protein and protein/DNA complexes.