Functional Analysis Of The Histone Regulation Hir1 In Tetrahymena Thermophila

Nucleosome assembly involves both DNA replication-coupled nucleosome assembly and replication-independent nucleosome assembly,and relies on the help of some specific histone molecular chaperones in this process,such as the HIR complex that mediates the deposition of H3.3 and thus plays a major role in replication-independent nucleosome assembly.The HIR complex is highly conserved among species and consists of three core subunits:histone regulator A（Hira）,ubinuclein 1（Ubn1）,and calcineurin-binding protein1（Cabin1）.Hira is the scaffold protein of this complex and is involved in a variety of chromatin-regulated activities,including gene transcription,heterochromatin formation,chromatin remodeling,and more.Tetrahymena thermophila belongs to the ciliate protozoa and is typically characterized by the presence of two nuclei,the polyploid macronucleus and the diploid micronucleus,and the two nuclei have different structures and functions,with nuclear dimorphism.The unique features of Tetrahymena sexual development stage make it an important model system to study how histone molecular chaperones function in chromatin assembly.In this study,we identified for the first time the molecular chaperone Hir1 of the histone variant H3.3 of Tetrahymena,and systematically investigated the function of the histone regulatory protein Hir1（Histone Regulation 1）in nuclei development and its role in nucleosome assembly and chromatin structure maintenance.The main results obtained are as follows:1.Bioinformatics analysis of HIR1The homologous gene of human Hira,HIR1,was first identified by BLAST in the Tetrahymena genome database.HIR1（TTHERM＿00046490）is 3409 bp long,with three introns and open reading frame of 3078 bp,encoding 1025 amino acids,and the predicted protein molecular weight of Hir1 is 117 kDa.Hir1 includes WD40 repeat domain,B domain and Hira domain,and has a nuclear localization signal at its C-terminus.RT-PCR results showed that HIR1 was expressed at low levels during the growing and starvation periods,but was specifically highly expressed at 4 h of sexual reproduction.2.Hir1 localizes to the micronucleus and macronucleus via nuclear localization signalHir1-3HA localized in both micronucleus and macronucleus during all processes of the vegetative growth and sexual reproduction,and had a strong signal on micronucleus.As meiosis proceeded,the signal of Hir1-3HA was gradually enhanced on the micronucleus and localized in the functional macronucleus and micronucleus during Anlagen,while the localization signal disappeared in the parental macronucleus that were about to apoptosis.Hir1 has a predicted nuclear localization signal sequence at its C-terminus.After truncating the nuclear localization signal sequence,Hir1^{Tr NLS}-3HA localized in the cytoplasm and cell proliferation is slowed.During the period of sexual reproduction,the mutant cells were unable to form zygotic nucleus and completed the normal sexual reproduction process.Immunoprecipitation and mass spectrometry analysis showed that Hir1 interacts with the heterochromatin protein Hpl2,the elongation factor-associated protein Elp1,the DNA damage repair protein Rad51,the ubiquitin and proteasome-associated protein Uba1,and the DNA mismatch repair protein Msh6.3.Knockdown of HIR1 affects the normal developmentTo analyze the function of HIR1,a recombinant plasmid was constructed and knockdown mutant cell lines and Cd²⁺-induced conditional interference mutant cell lines were obtained.During the vegetative growth and starvation stage,HIR1 was knocked down,the micronucleus chromosomes were missing,its area was significantly smaller and some micronucleus were lost,and the cell proliferation rate was slowed down.During sexual reproduction,knockdown of HIR1 could not select micronucleus properly during nuclear selection,and abnormal cells with new macronucleus and new micronucleus loss during Anlagen period,and sexual reproduction process could not be completed normally.Knockdown of HIR1 affects the expression levels of histone molecular chaperones CAF1A,CAF1B,REBL1,ASF1 and POB3;HIR1 can repress histone gene transcription,and the H3K4me3 levels is markedly elevated after knockdown of HIR1.4.Interaction of Hir1 and Asf1The B domain of Hir1 and Asf1 were codon optimized and expressed in E.coli.GST-Hir1-B and His-Asf1 were expressed and purified by affinity chromatography.The interaction of Hir1 and Asf1 was verified by in vitro pull-down assay.The present study is the first to systematically analyze the function of the histone molecular chaperone Hir1 in T.thermophila.Hir1 localized in both micronucleus and macronucleus during vegetative growth and sexual reproduction.HIR1 mutation affects the normal development of micronucleus and the nuclear selection process during sexual reproduction,and regulate the level of H3K4me3.Hir1 mutations affects the stability of gene transcription and chromatin structure.The functional study of Hir1 provides new data to further elucidate the molecular mechanism of chromatin assembly of histones in higher eukaryotes.