Independent and Complementary Functions of Caf1b and Hir1 for Chromatin Assembly in Tetrahymena thermophila

Histones and DNA associate to form the nucleosomes of eukaryotic chromatin. Chromatin assembly factor 1 (CAF-1) complex and histone regulatory protein A (HIRA) complex mediate replication-couple (RC) and replication-independent (RI) nucleosome assembly, respectively. CHAF1B and HIRA share a similar domain but play different roles in nucleosome assembly by binding to the different interactors. At present, there is limited understanding for the similarities and differences in their respective functions. Tetrahymena thermophila contains transcriptionally active polyploid macronuclei (MAC) and transcriptionally silent diploid micronuclei (MIC). Here, the distribution patterns of Caf1b and Hir1 exhibited both similarities and distinctions. Both proteins localized to the MAC and MIC during growth, and to the MIC during conjugation. However, Hir1 exhibited additional signaling on parental MAC and new MAC during sexual reproduction and displayed a punctate signal on developing anlagen. Caf1b and Hir1 only co-localized in the MIC with Pcna1 during conjugation. Knockdown of CAF1B impeded cellular growth and arrested sexual reproductive development. Loss of HIR1 led to MIC chromosome defects and aborted sexual development. Co-interference of CAF1B and HIR1 led to a more severe phenotype. Moreover, CAF1B knockdown led to the up-regulation of HIR1 expression, while knockdown of HIR1 also led to an increase in CAF1B expression. Furthermore, Caf1b and Hir1 interacted with different interactors. These results showed that CAF-1 and Hir1 have independent and complementary functions for chromatin assembly in T. thermophila.


Introduction
In eukaryotic cells, chromatin is composed of repetitive nucleosomes.The ~147 bp DNA is tightly wrapped around the histone core octamer consisting of H3, H4, H2A, and H2B subunits to form nucleosomes and is further stabilized by the H1 linker histone [1].The canonical histones and histone variants play a pivotal role in precisely modulating chromatin dynamics and regulating accessibility to specific DNA regions [2].The nucleosome conveys specific genetic information through covalently modified or incorporated histone variants.The assembly, disassembly, and reassembly of nucleosomes are precisely orchestrated processes that underlie crucial cellular functions such as DNA replication, transcription, and repair [3,4].Different histones and their variants exhibit various chromatin assembly pathways.Classic histone H3.1 is deposited onto chromatin via the replicationcouple (RC) nucleosome assembly pathway facilitated by chromatin assembly factor 1 (CAF-1) [5,6].In contrast to H3.1, the variant of histone H3 known as H3.3 follows an alternative pathway facilitated by HIRA for its incorporation into the nucleosome gap [5,7].Recently, based on BioID proximity labeling, CAF-1 not only deposits the H3.1 histone variant during the S phase but also associates with H3.3 throughout the cell cycle in mammalian cells [8].The centromere histone A (CENP-A) is deposited on the centromere with the assistance of the chaperone protein HJURP (holiday junction recognition protein) [9].
CAF-1 is ubiquitously present in all eukaryotic cells [10,11].It represents an evolutionarily conserved histone chaperone complex essential for various fundamental processes in eukaryotes, including chromatin repair following DNA synthesis, cell cycle progression, as well as the establishment and maintenance of heterochromatin [12][13][14].In most eukaryotes, CAF-1 is a heterotrimeric complex comprising the large subunit p150/CHAF1A, the medium subunit p60/CHAF1B, and the small subunit p48/CHAF1C [15][16][17].The CHAF1B contains seven WD40 repeats and a B-domain-like motif that binds to Asf1 [18,19].In vertebrate cells, the deletion of any one of the subunits in CAF-1 causes a delay in the S phase, decelerated DNA synthesis, impaired assembly of replication-coupled nucleosomes, and ultimately cell death [20].Conversely, the removal of the subunit in CAF-1 in yeast does not affect cell survival [21,22].In budding yeast, CAF-1 interacts with Cse4 and colocalizes with the Cse4 at the centromeres in the absence of Scm3 [23].Overexpressed CENP-A was mislocated in CHAF1B-depleted HeLa cells [24].CHAF1B deletion leads to abnormal hematopoietic reconstitution in bone marrow cells [25].
Tetrahymena thermophila is a ciliate protozoan exhibiting nuclear dimorphism, characterized by the transcriptionally active polyploid macronucleus MAC and the transcriptionally silent diploid germline MIC.During vegetative growth, MACs undergo amitotic division, while MICs divide through mitosis [36].During conjugation development, the MIC undergoes meiosis, followed by mitosis of pronuclei and two subsequent mitotic divisions of the postzygotic nucleus.Subsequently, the new MAC anlagen undergoes endoreplication and internal eliminated sequences (IES) deletion, while the parental MAC gradually degrades.Finally, the paired cells separate and form an exconjugant containing two MACs and one MIC.In the nutrient-sufficient environment, the exconjugant restarts proliferation [37].In Tetrahymena, there are five variants of histone H3, namely HHT1-4 and CNA1 (CENP-A-like 1).HHT1 and HHT2 encode for the canonical H3 known as H3.1, HHT3 and HHT4 encode similar H3 variants, H3.3 and H3.4.The H3.1 is deposited onto chromatin by a DNA RC pathway; in contrast, H3.3 is deposited mainly through the RI pathway and weakly through an RC pathway [38].The histones exhibit variations in their composition and post-translational modifications.In most eukaryotes, H3.1 and H3.3 differ by only four or five amino acids while Tetrahymena H3.1 and H3.3 differ at 16 residues [38,39].Furthermore, there are slow-migrating H3 S and fast-migrating H3 F unique to MIC in Tetrahymena.H3 F is generated by splitting the six amino acids at the N-terminal of H3 [40].Hence, trimethylation of H3K4 is only available in MAC.Currently, there remains a limited understanding regarding the assembly of histones with different sizes and variants into DNA, as well as the interplay between RC nucleosome assembly and RI nucleosome assembly in T. thermophila.Asf1 and Nrp1 represent pivotal upstream core proteins involved in nucleosome assembly.A decrease in Nrp1 leads to abnormal MIC mitosis and MAC amitosis [41,42].In this study, we found that Caf1b localized in both MACs and MICs during the vegetative growth stage; then, it localized in the MIC and replicated anlagen during sexual development.In contrast, Hir1 exhibited localization in both MAC and MIC throughout vegetative growth, starvation, and sexual reproduction stages.Loss of CAF1B affected cellular proliferation during asexual reproduction and led to abnormal meiosis during the sexual development stage.Impaired expression of HIR1 compromises the integrity of MIC chromatin.The co-interference of CAF1B and HIR1 led to a more serious phenotype than single-gene knockdown mutants.CAF-1 and Hir1 exhibit independent and complementary functions for chromatin assembly in Tetrahymena.

Strains Culture and Mating
T. thermophila wild-type cell lines B2086 (II), CU428 (VII), and CU427 (VI) were obtained from the National Tetrahymena Stock Center (http://tetrahymena.vet.cornell.edu/,initially accessed on 10 September 2008, currently is stored in our Lab).The cells were grown in Super Proteose Peptone (SPP) medium (0.2% glucose, 0.1% yeast extract, 1% proteose peptone, and 0.003% EDTA-Fe) at 30 • C. Log-phase cells were washed with 10 mmol/L Tris-HCl (pH 7.4) and resuspended in the same buffer for 18-24 h.Equal numbers of cells from two different mating types were mixed and induced mating.

Identification of Caf1b and Hir1
The sequences of CAF1B (TTHERM_00219420) and HIR1 (TTHERM_00046490) were retrieved using BLAST search from the Tetrahymena Genome Database (http://www.ciliate.org,accessed on 9 June 2021).Protein clustering analysis was performed using MEGA11.0.11, while IBS 1.0.3 was utilized for mapping the domains of these proteins.

Phylogenetic Tree Construction
The homologous proteins of Caf1b and Hir1 were searched through sequence alignment in NCBI (https://www.ncbi.nlm.nih.gov/,accessed on 18 May 2022) or UniProt (https://www.uniprot.org/,accessed on 20 May 2022) databases, and their amino acid sequences were downloaded.The sequences of WD domains of Caf1b and Hir1 were retrieved in the Conserved Domain Database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi,accessed on 5 June 2022).Next, these amino acid sequences were subjected to ClustalW-based sequence alignment in MEGA11.0.11 to eliminate non-specific regions.Finally, phylogenetic trees were constructed using the neighbor-joining method.

Construct of HA-Tagged Caf1b and Hir1
The C-terminal region (639 bp) and 3' flanking sequence (501 bp) of the CAF1B were amplified using CAF1B-HA-5F/5R and CAF1B-HA-3F/3R (Table S1).Meanwhile, the C-terminal region (635 bp) and 3' flanking sequence (524 bp) of the HIR1 were amplified using HIR1-HA-5F/5R and HIR1-HA-3F/3R (Table S1).Subsequently, the amplified fragments were ligated to the pMD-19T vector.The C-terminal fragment was then digested with Sac I and Not I, followed by ligation into the pNeo4 vector.Similarly, the flanking sequence was digested with Xho I and Kpn I before being ligated to pHA-Neo4 using DNA ligase.Enriched fragments obtained through primer Shoot-CAF1B-HA-F/R and Shoot-HIR1-HA-F/R were introduced into T. thermophila cells via biolistic bombardment (SCIENTZ, Ningbo, China).The transformed cells were subjected to varying concentrations of paromomycin, from low to high, for better substitution.The mutants were identified by PCR using the primer Shoot-CAF1B-HA-F/R and Shoot-HIR1-HA-F/R.

Knockout of CAF1B and HIR1
The 5' (947 bp) and 3' (506 bp) flanking sequences of CAF1B were amplified using K-CAF1B-5F/5R and K-CAF1B-3F/3R primers, followed by ligation with pMD-19T.At the same time, the 5' (454 bp) and 3' (590 bp) flanking sequences of the HIR1 were amplified using K-HIR1-5F/5R and K-HIR1-3F/3R primers, followed by ligation with pMD-19T.Subsequently, the fragments of 5' flanking sequences cleaved by Sac I/Not I and the fragments of 3' flanking sequences cleaved by Xho I/Kpn I were purified and ligated to the pNeo4 vector.The resulting recombinant plasmids were digested with Sac I and Kpn I and transferred to T. thermophila via biolistic bombardment (SCIENTZ, Ningbo, China).Transformants exhibiting paromomycin resistance were selected.The mutants were identified by PCR using the primer J-CAF1B-F/R and J-HIR1-F/R.

Construction of Conditionally Induced Interference Mutants
The conserved fragment (516 bp) from CAF1B were amplified using RNAi-CAF1B-1F/1R and RNAi-CAF1B-2F/2R, which only differed in the enzymatic cleavage site.Similarly, we amplified the conserved fragment (594 bp) of HIR1 were amplified with RNAi-HIR1-1F/1R and RNAi-HIR1-2F/2R primers.The amplified fragments were ligated to pMD-19T.Fragments digested by Pst I/Sma I and BamH I/Pme I were ligated with the phpNeo5 vector cleaved using the same enzyme.Plasmids that successfully formed hairpin structures were then digested with Xho I/Not I and transformed into T. thermophila using biolistic bombardment (SCIENTZ, Ningbo, China).Transformants were selected in the presence of paromomycin, and interference efficiency was assessed by qRT-PCR using RT-CAF1B-F/R or RT-HIR1-F/R.

RT-PCR and qPCR
The cultured cells were subjected to RNA isolation using Trizol (Yeasen Biotechnology, Shanghai, China).Subsequently, 600 ng of total RNA with the gDNA removed was reversetranscribed into cDNA using a kit (Yeasen Biotechnology, Shanghai, China).The expression levels of target genes were quantified using the primers listed in Table S1.SYBR Green Master Mix (Yeasen Biotechnology, Shanghai, China) and Bio-Rad CFX Connect were employed for gene expression analysis.The internal reference used was 17SrRNA.

Micronuclear Integrity Assay
The genomes from wild-type and HIR1KD mutants were extracted.Ten pairs of primers of micronucleus-specific chromosome breakage sequences (Cbs) were used to identify the integrity of MIC by PCR (Table S1).The PCR cycling conditions consisted of an initial denaturation step at 94 • C for 5 min, followed by 32 cycles of denaturation at 94 • C for 30 s, annealing at 56 • C for 30 s, extension at 68 • C for 60 s, and a final extension step at 68 • C for 5 min.

Co-Immunoprecipitation and Mass Spectrometry
Cells (1 × 10 7 ) were collected and resuspended in 500 µL PBS containing 100 × Inhibitor Cocktail (Thermo Fisher Scientific, Waltham, MA, USA) and 0.5 M EDTA.Following ultrasonic disruption and subsequent centrifugation, the resulting supernatant was incubated with 20 µL of packed anti-HA agarose beads (Thermo Fisher Scientific, Waltham, MA, USA) in a spin column overnight at 4 • C with continuous end-to-end mixing.The samples were then subjected to short centrifugation for 10 s followed by seven washes using TBST buffer (25 mmol/L Tris-HCl, 0.15 mol/L NaCl, pH 7.2, 0.05% Tween-20).After each wash step, the samples were briefly centrifuged again.Subsequently, the HA-tagged protein was eluted from the beads using 25 µL non-reducing sample buffer and then boiled for 5 min.An aliquot of 5.5 µL was used for Western blot analysis while 6.5 µL was used for silver staining; any remaining sample was utilized for mass spectrometry analysis (Shanghai Applied Protein Technology Shanghai, China).After reduction and alkylation, Trypsin was added to the sample (mass ratio 1:50) and enzymolized at 37 • C for 20 h.After desalting, the enzymolysis product was lyophilized, redissolved in 0.1% FA solution, and stored at −20 • C for further use.After the column was balanced with 95% liquid A (0.1% formic acid aqueous solution), the sample was loaded into the Trap column using an automatic sampler.A total of 20 fragments were collected after each full scan (MS2 Scan).The identified protein results were obtained.We denoted the ratio of Caf1b-HA or Hir1-HA to the number of WT peptides as foldchange and proteins with a ratio more than 4 were identified as interactors of Caf1b or Hir1.

Pull-Down Assay
The codon-optimized fragments of B-like 1-3 from Caf1b and B domain of Hir1 were ligated into the pGEX-6P-1, while Asf1 was ligated into the pESUMO vector.Subsequently, the recombinant plasmids were transformed into E. coli BL21 (DE3) and induced with 0.1 mM IPTG for expression at 37 • C or 16 • C.After sonication, the supernatant of GST, GST-B-like 1-3, and GST-B were added to chromatography columns with GST-sefinose resin (BBI Life Sciences, Shanghai, China) and incubated in a shaker at 4 • C for 5 h after the addition of an equal amount of the trapped protein Asf1.Following this step, centrifugation at 1500 rpm for 1 min was performed.The gel beads were washed with PBST.Finally, proteins were eluted using 100 µL of PBS supplemented with 10 mM GSH.The identification of bait proteins and trapped proteins was analyzed using Western blot.

Characterization of Caf1b and Hir1
CAF-1 and HIRA complexes mediate RC and RI histone deposition, respectively [7,43].CAF1B (TTHERM_00219420) and HIR1 (TTHERM_00046490) are homologs of p60 and HIRA in T. thermophila.Caf1b was predicted to contain the WD domain, a conserved B-domain-like motif, and two non-conservative B-domain-like motifs (Figure 1A).The sequence from the N-terminal to C-terminal was designated as B-like 1 to 3 (Supplementary Figure S1A).Hir1 comprises a WD domain, a B domain motif, and a Hira domain (Figure 1B).The B domain of Hir1 is conserved across species (Supplementary Figure S1B).
The WD domain exhibits a characteristic seven-bladed β-propeller structure.WD domains serve as crucial subunits within multiprotein complexes involved in diverse signaling pathways [44].The phylogenetic analysis of the WD domain from Caf1b and Hir1 indicated their evolutionary conservation and divergence between these two protein families (Figure 1C).The expression of CAF1B and HIR1 is low during growth and starvation but reaches its peak at 4-6 h of sexual reproduction (Figure 1D,E).
B-domain-like motif, and two non-conservative B-domain-like motifs (Figure 1A).The sequence from the N-terminal to C-terminal was designated as B-like 1 to 3 (Supplementary Figure S1A).Hir1 comprises a WD domain, a B domain motif, and a Hira domain (Figure 1B).The B domain of Hir1 is conserved across species (Supplementary Figure S1B).The WD domain exhibits a characteristic seven-bladed β-propeller structure.WD domains serve as crucial subunits within multiprotein complexes involved in diverse signaling pathways [44].The phylogenetic analysis of the WD domain from Caf1b and Hir1 indicated their evolutionary conservation and divergence between these two protein families (Figure 1C).The expression of CAF1B and HIR1 is low during growth and starvation but reaches its peak at 4-6 h of sexual reproduction (Figure 1D,E).

Dynamic Localization of Caf1b and Hir1
Deposition of H3.1 occurs within the replication region, whereas incorporation of H3.3 takes place in the highly transcribed region [45].To gain a comprehensive understanding of both the RI and RC nucleosome assembly pathways, the localization of Caf1b and Hir1 was investigated in T. thermophila.HA-tagged Hir1 and Caf1b constructs were generated (Supplementary Figure S2A,C), which were then transformed into T. thermophila via biolistic transformation, followed by selection using paromomycin (Supplementary Figure S2B,D).Indirect immunofluorescence showed that Caf1b-HA localized in the MACs and periphery of MICs in the MIC G2 phase during vegetative growth (Figure 2A(a)).It localized into the MIC and MAC in the MIC S phase and MAC amitosis period (Supplementary Figure S3A(b,c)).After starvation, the signal of Caf1b in the MAC and MIC gradually faded away.Ultimately, Caf1b-HA was localized in the cytoplasm after 24 h of starvation.(Figure 2A(b) and Supplementary Figure S3A(f)).During sexual reproduction, Caf1b-HA localized specifically to meiotic MIC but not in the parental MACs (Figure 2A(c,d)).When the selected pronuclei replicated the DNA and performed prezygotic mitosis, the Caf1b deposition in the MIC was increased (Figure 2A(e)).Caf1b signaling was also found in mitotic MIC (Figure 2A(f)).In the anlagen stage, there was a strong Caf1b signal in the new replicating MAC (Figure 2A

Colocalization of Caf1b and Hir1 with Pcna1
PCNA plays crucial roles in both DNA replication and repair [46,47].p60 colocalizes with PCNA in HeLa cells [48].The localization of Hir1 and Caf1b on the MIC was similar but was different in MAC.To further investigate whether Caf1b and Hir1 are involved in the replication progress in T. thermophila, colocalization between Pcna1 and Caf1b, as well as between Pcna1 and Hir1, was investigated.Caf1b-HA and Pcna1 colocalized in the MACs and MICs during vegetative growth; furthermore, they co-localized in the MICs during the conjugation stage (Figure 3A(b,c)).Hir1-HA and Pcna1 also colocalized in the Hir1-HA localized in the MACs and MICs during vegetative growth (Figure 2B(a), Supplementary Figure S3B).During starvation, Hir1-HA localized in the MAC and the periphery of the MIC (Figure 2B(b), Supplementary Figure S3B).The signaling of Hir1 occurs on both the parental MAC and the new MAC, as well as in both meiotic and mitotic micronuclei (Figure 2B(c-g)).Specifically, Hir1-HA formed a ring structure around pronuclei and new MICs (Figure 2B(e,g)).Surprisingly, Hir1-HA exhibited a punctate signal within expanded postmitotic MIC (Figure 2B(f)).The signal of Hir1 disappeared in the degradable parental MAC at the late sexual development stage (Figure 2B(g)).

Colocalization of Caf1b and Hir1 with Pcna1
PCNA plays crucial roles in both DNA replication and repair [46,47].p60 colocalizes with PCNA in HeLa cells [48].The localization of Hir1 and Caf1b on the MIC was similar but was different in MAC.To further investigate whether Caf1b and Hir1 are involved in the replication progress in T. thermophila, colocalization between Pcna1 and Caf1b, as well as between Pcna1 and Hir1, was investigated.Caf1b-HA and Pcna1 colocalized in the MACs and MICs during vegetative growth; furthermore, they co-localized in the MICs during the conjugation stage (Figure 3A(b,c)).Hir1-HA and Pcna1 also colocalized in the MAC and MIC during the vegetative growth stage.They also co-localized in the selected MICs but not MAC during conjugation (Figure 3B(b,c)).

The Absence of HIR1 Resulted in the Loss of Chromatin in MIC
To further investigate the function of Hir1, the HIR1 knockout plasmid was constructed and transformed into T. thermophila (Supplementary Figure S4A).The HIR1 knockdown mutants HIR1KDB (HIR1KD B2086) and HIR1KDC (HIR1KD CU428) were created, and the expression levels of the mutants down-regulated by 98.82% and 99.70%, respectively (Supplementary Figure S4B,C).HIR1KD mutant inhibited cellular proliferation compared to that in the WT (Figure 4A).Observation of nuclear morphology revealed smaller MIC in HIR1KD (Figure 4B,C).To further validate our findings, we extracted the genomes from both the mutants and wild-type cells and subsequently amplified micronuclear-specific fragments using 10 pairs of MIC-specific primers.The loss of MIC-specific fragments was observed in HIR1KD (Figure 4D).Furthermore, expression of CAF1B was up-regulated 1.8-fold in the HIR1KD mutants (Figure 4E).These results suggest that Hir1 is involved in the chromatin integrity of MICs.

The Absence of HIR1 Resulted in the Loss of Chromatin in MIC
To further investigate the function of Hir1, the HIR1 knockout plasmid was constructed and transformed into T. thermophila (Supplementary Figure S4A).The HIR1 knockdown mutants HIR1KDB (HIR1KD B2086) and HIR1KDC (HIR1KD CU428) were created, and the expression levels of the mutants down-regulated by 98.82% and 99.70%, respectively (Supplementary Figure S4B,C).HIR1KD mutant inhibited cellular proliferation compared to that in the WT (Figure 4A).Observation of nuclear morphology revealed smaller MIC in HIR1KD (Figure 4B,C).To further validate our findings, we extracted the genomes from both the mutants and wild-type cells and subsequently amplified micronuclear-specific fragments using 10 pairs of MIC-specific primers.The loss of MIC-specific fragments was observed in HIR1KD (Figure 4D).Furthermore, expression of CAF1B was up-regulated 1.8-fold in the HIR1KD mutants (Figure 4E).These results suggest that Hir1 is involved in the chromatin integrity of MICs.

CAF1B Knockdown Affected Proliferation and Sexual Development of T. thermophila
To investigate the biological function of Caf1b in T. thermophila, the CAF1B knockout plasmid was constructed and transformed into different mating types Tetrahymena (Supplementary Figure S5A).CAF1B knockdown mutants CAF1BKDB (CAF1BKD B2086) and CAF1BKDC (CAF1BKD CU428) were created (Supplementary Figure S5B).The expression levels of CAF1BKDB and CAF1BKDC decreased by 98.86% and 93.48%, respectively (Supplementary Figure S5C).To investigate the impact of CAF1B deletion, we assessed expression levels of CAF-1's large subunit (CAF1A), small subunit (REBL1), ASF1, and HIR1.The deletion of CAF1B led to a down-regulation of expression levels of CAF1A (14.83%),REBL1 (35.36%), and ASF1 (53.23%); in contrast, HIR1 showed a 2.28-fold up-regulation (Supplementary Figure S5D-G).Since we want to know the stage-specific function of CAF1B, CAF1B conditional interference plasmids pCAF1BhpNeo5 were constructed and transformed into T. thermophila (Supplementary Figure S6A).The expression levels of CAF1B in caf1biB and caf1biC under 0.5 µg/mL Cd 2+ induction for 96 h were reduced by 86.03% and 98.26%, respectively (Supplementary Figure S6B).The proliferation rate of caf1bi mutants after being induced using Cd 2+ decreased than that of WT (Supplementary Figure S6C).To investigate the impact of CAF1B on sexual reproduction, we subjected starving cells to 0.1 µg/mL Cd 2+ treatment for 24 h, and subsequently paired cells of different mating types.After 2 h of pairing, caf1bi exhibited a similar phenotype to the wild type, as all cells were capable of reaching their maximum stretching capacity during the crescent period while maintaining intact MIC and MAC (Figure 5A(a,b,a1,b1)).In paired 4 h samples, we observed successful completion of meiosis in the wild type (Figure 5A(c)).Conversely, in the mutants, gradual DNA loss occurred in 27.06% cells during crescent retraction and resulted in a reduction in MIC (Figure 5A(c1-e1),B).After 24 h of mixing, sexual reproduction was completed by 46.42% in the wild-type cells (Figure 5A(g),B),

CAF1B Knockdown Affected Proliferation and Sexual Development of T. thermophila
To investigate the biological function of Caf1b in T. thermophila, the CAF1B knockout plasmid was constructed and transformed into different mating types Tetrahymena (Supplementary Figure S5A).CAF1B knockdown mutants CAF1BKDB (CAF1BKD B2086) and CAF1BKDC (CAF1BKD CU428) were created (Supplementary Figure S5B).The expression levels of CAF1BKDB and CAF1BKDC decreased by 98.86% and 93.48%, respectively (Supplementary Figure S5C).To investigate the impact of CAF1B deletion, we assessed expression levels of CAF-1's large subunit (CAF1A), small subunit (REBL1), ASF1, and HIR1.The deletion of CAF1B led to a down-regulation of expression levels of CAF1A (14.83%),REBL1 (35.36%), and ASF1 (53.23%); in contrast, HIR1 showed a 2.28-fold up-regulation (Supplementary Figure S5D-G).Since we want to know the stage-specific function of CAF1B, CAF1B conditional interference plasmids pCAF1BhpNeo5 were constructed and transformed into T. thermophila (Supplementary Figure S6A).The expression levels of CAF1B in caf1biB and caf1biC under 0.5 µg/mL Cd 2+ induction for 96 h were reduced by 86.03% and 98.26%, respectively (Supplementary Figure S6B).The proliferation rate of caf1bi mutants after being induced using Cd 2+ decreased than that of WT (Supplementary Figure S6C).To investigate the impact of CAF1B on sexual reproduction, we subjected starving cells to 0.1 µg/mL Cd 2+ treatment for 24 h, and subsequently paired cells of different mating types.After 2 h of pairing, caf1bi exhibited a similar phenotype to the wild type, as all cells were capable of reaching their maximum stretching capacity during the crescent period while maintaining intact MIC and MAC (Figure 5A(a,b,a1,b1)).In paired 4 h samples, we observed successful completion of meiosis in the wild type (Figure 5A(c)).

Caf1b and Hir1 Are Functionally Complementary and Required for Sexual Reproduction
Caf1b and Hir1 played important roles in the growth and development of T. thermophila.To further elucidate the relationship between Caf1b and Hir1, HIR1 conditional interference mutants were created (Supplementary Figure S6D).Expression levels of HIR1 in hir1iB and hir1iC were reduced by 83.25% and 68.89%, respectively (Supplementary Figure S6E).During conjugation, the hir1i mutant strain exhibited aberrant pronuclei selection and subsequent mitosis during sexual reproduction (Figure 5A(d2-f2)) and 9.04% cells formed exconjugants with two MACs and one MIC (Figure 5A(g2),B).Subsequently, caf1bi and hir1i mutants were mixed and initiated sexual development.At 4 h of mixing, 35.65% cells in caf1bi×hir1i were abnormally meiotic, which is more than caf1bi (Figure 5A(c3-e3),B), and none of the cells completed sexual reproduction at 8 h of sexual reproduction (Figure 5A(g3),B).The combined interference of CAF1B and HIR1 led to a more

Caf1b and Hir1 Are Functionally Complementary and Required for Sexual Reproduction
Caf1b and Hir1 played important roles in the growth and development of T. thermophila.To further elucidate the relationship between Caf1b and Hir1, HIR1 conditional interference mutants were created (Supplementary Figure S6D).Expression levels of HIR1 in hir1iB and hir1iC were reduced by 83.25% and 68.89%, respectively (Supplementary Figure S6E).During conjugation, the hir1i mutant strain exhibited aberrant pronuclei selection and subsequent mitosis during sexual reproduction (Figure 5A(d2-f2)) and 9.04% cells formed exconjugants with two MACs and one MIC (Figure 5A(g2),B).Subsequently, caf1bi and hir1i mutants were mixed and initiated sexual development.At 4 h of mixing, 35.65% cells in caf1bi×hir1i were abnormally meiotic, which is more than caf1bi (Figure 5A(c3-e3),B), and none of the cells completed sexual reproduction at 24 h of sexual reproduction (Figure 5A(g3),B).The combined interference of CAF1B and HIR1 led to a more serious phenotype compared to individual interference, which suggests the functional complement of Caf1b and Hir1.

Caf1b and Hir1 Interacted with Different Interactors
Although Caf1b and Hir1 interacting proteins were detected previously, specific interactors for Hir1 and Caf1b under different development stages were less clear [49].To further investigate the function of Caf1b and Hir1, interactors of Caf1b and Hir1 were identified.Given that CAF1B and HIR1 exhibit peak expression during 4-6 h post-pairing, we performed Co-IP analysis using proteins extracted from Caf1b-HA, Hir1-HA, and wildtype cells at 5 h post-pairing.The eluted target proteins following Co-IP were subjected to Western blot analysis, confirming the presence of bait proteins (Figure 6A,B).Subsequent AP-MS assay revealed distinct protein interactions with Caf1b and Hir1.A total of 76 proteins interacting with Caf1b were identified, including histone chaperone Spt6 and Caf1a; proteins specific to chromatin structure Smc2/Smc4; Holliday junction ATP-dependent DNA helicase Rvb1 and Rvb2; DNA replication licensing factor Mcm2, Mcm4, Mcm5, and Mcm6; dynein family proteins; conjugation-specific proteins Giw1 (Gentleman-in-waiting 1) and Zfr3 (Zinc finger-related 3); and proteins related to degradation, transcription, cell cycle, and nuclear import (Figure 6C).serious phenotype compared to individual interference, which suggests the functional complement of Caf1b and Hir1.

Caf1b and Hir1 Interacted with Different Interactors
Although Caf1b and Hir1 interacting proteins were detected previously, specific interactors for Hir1 and Caf1b under different development stages were less clear [49].To further investigate the function of Caf1b and Hir1, interactors of Caf1b and Hir1 were identified.Given that CAF1B and HIR1 exhibit peak expression during 4-6 h post-pairing, we performed Co-IP analysis using proteins extracted from Caf1b-HA, Hir1-HA, and wild-type cells at 5 h post-pairing.The eluted target proteins following Co-IP were subjected to Western blot analysis, confirming the presence of bait proteins (Figure 6A,B).Subsequent AP-MS assay revealed distinct protein interactions with Caf1b and Hir1.A total of 76 proteins interacting with Caf1b were identified, including histone chaperone Spt6 and Caf1a; proteins specific to chromatin structure Smc2/Smc4; Holliday junction ATP-dependent DNA helicase Rvb1 and Rvb2; DNA replication licensing factor Mcm2, Mcm4, Mcm5, and Mcm6; dynein family proteins; conjugation-specific proteins Giw1 (Gentleman-in-waiting 1) and Zfr3 (Zinc finger-related 3); and proteins related to degradation, transcription, cell cycle, and nuclear import (Figure 6C).
We identified 56 proteins that interacted with Hir1, mainly including pdd1 (programmed DNA degradation 1); heterochromatin protein 1 like 2 (Hpl2); transposase Lia5 (localized in macronuclear anlagen 5); class I histone deacetylase family protein (TTHERM_00663829); DNA mismatch repair protein (Msh6); transcription-related protein Ada2 (transcriptional adaptor 2), Elp1 (elongation protein 1), and TTHERM_011942820; proteins related to degradation and nuclear importin; and dynein family protein (Figure 6D).Western blotting was performed using anti-His and anti-GST.(F) The interaction between B-like 2 of Caf1b and Asf1.Western blotting was performed using anti-His and anti-GST.(G) The interaction between B-like 3 of Caf1b and Asf1.Western blotting was performed using His and GST antibodies.(H) The interaction between B domain of Hir1 and Asf1.Western blotting was performed using anti-His and anti-GST.

Caf1b and Hir1 Interacted with Asf1 In Vitro
Homologous proteins of Caf1b and Hir1 were found to interact with Asf1 in both mammals and yeast [19,27].However, the mass spectrometry results of Caf1b-HA and Hir1-HA did not reveal the presence of Asf1.To investigate the interaction between Asf1 and Caf1b/Hir1 in T. thermophila, we constructed fusion proteins by ligating the three predicted B-domain-like motifs of Caf1b and the B domain of Hir1 into the pGEX-6P-1 expression vector, while Asf1 was ligated to a SUMO vector for expression in E. coli.The interaction analysis between Caf1b/Hir1 and Asf1 was performed using a pull-down assay.Western blot and SDS-PAGE analyses revealed that each of the three B-domain-like motifs of Caf1b interacts with Asf1 (Figure 6E-G, Supplementary Figure S7A-C).These findings demonstrate that the binding between Caf1b and Asf1 is mediated by multiple sites.Similarly, Hir1 could interact directly with Asf1 in vitro (Figure 6H, Supplementary Figure S7D).The results indicate that both Caf1b and Hir1 interact directly with Asf1.

Discussion
The precise wrapping of histones by DNA is crucial for maintaining genome stability and ensuring the accurate transmission of genetic information.CAF-1 and Hir1 play pivotal roles as conserved histone chaperones in eukaryotes, facilitating the deposition of histone variants H3.1 and H3.3 onto DNA, respectively.This process not only enables the efficient compaction of genomic DNA within the nuclear space but also actively participates in gene expression regulation.The functional conservation and diversity of CAF-1 and Hir1 have been observed across different eukaryotes [50][51][52].In this study, we demonstrated that Caf1b and Hir1 exhibit similar MAC and MIC localization patterns during vegetative growth.Hir1 localized in the parental MACs; however, the Caf1b signal disappeared in the parental MACs during sexual reproduction.Following CAF1B knockdown, HIR1 expression up-regulated.Similarly, after HIR1 knockdown, there was an up-regulated expression of CAF1B, which suggests a complementary relationship between Caf1b and Hir1.In addition, both Caf1b and Hir1 play important roles in cell proliferation and sexual reproduction.CAF1B and HIR1 had low expression levels during growth and starvation but showed a significant increase in expression at 4-6 h of conjugation.In budding yeast, CAF-1 is localized to centromeres throughout the cell cycle [23].In mammalian cells, p60 binds to the nucleus during interphase, dissociates from chromatin during mitosis, and concentrates in the nucleus at sites of DNA replication during the S-phase [53].During vegetative growth, Caf1b-HA localized to both the MACs and MICs during growth; however, the nuclear signal disappeared and a cytoplasmic signal increased at starving 24 h in T. thermophila (Figure 2A(a,b)).Caf1b-HA only localized in the MICs during meiosis and mitosis, the periphery of MICs during post-meiotic nuclear selection, and the new MACs during the anlagen stage (Figure 2A(c-g)).Additionally, Caf1b-HA colocalized with Pcna1 during vegetative growth and conjugation (Figure 3A(b,c)).During vegetative growth, MAC undergoes amitosis, while MIC undergoes mitosis during asexual reproduction, indicating the involvement of Caf1b in DNA replication in both MACs and MICs.Prior to micronuclear stretching, DNA double-strand breaks occur [54].Caf1b was localized to meiotic MIC, which suggests it might be involved in post-DSB repair.One selected meiotic product proceeds through mitosis while the remaining nuclei degenerate [55].The selected gamete nuclei undergo DNA replication and chromatin remodeling [56].The strong signal of Caf1b-HA at the selected MICs suggests it may involve in DNA replication and gamete remodeling.The zygotic nuclei undergo two mitosis and two of the postzygotic mitotic products develop into new MACs [37,57].Caf1b-HA localized in the mitotic zygotic nucleus and the anlagen but disappeared in the parental MAC.The different localization implied that Caf1b might be involved in the replication and remodeling of MICs and anlagen.Furthermore, Caf1b also colocalized with Pcna1 in a different development stage, which suggests that CAF-1 participates in the chromatin replication in T. thermophila.Heterochromatin was predominantly localized in the perinuclear region [58].Hir1 and Caf1b also localized to the nuclear periphery of the MIC during the G2 phase.Hir1 also localized to the periphery of pronuclei and new MICs.They might be stored in the nucleoplasm.Another possibility is that Hir1 and Caf1b associate with heterochromatin regions and participate in chromatin remodeling processes.The exact mechanisms and functions of Hir1 and Caf1b in these contexts require further investigation.
In human cells, HIRA localizes in the nucleus [59].In Mesenchymal stem cells, HIRA colocalized with H3.3 at promyelocytic leukemia (PML) bodies [60].In T. thermophila, Hir1 localized to the MACs and MICs during asexual reproduction and starvation (Figure 2B(a,b), Supplementary Figure S3B).In sexual reproduction, Hir1 was found in transcriptionally active parental MACs at an early conjugation stage (Figure 2B(c-f)), providing direct evidence for its association with transcription progress.Surprisingly, Hir1 was also observed to localize to meiotic and mitotic MICs during sexual reproduction (Figure 2B(c-f)), and co-localization with Pcna1 on the selected pronuclei (Figure 3B(c)).The results imply that Hir1 is involved in replication-related processes or chromatin remodeling.Interestingly, punctate signals were observed on the enlarged postmitotic MICs at early anlagen stage.The localization of Hir1-HA is similar to that of Cna1 on the expanded MICs [61,62].In mammalian cells, H3.3 serves as a placeholder for CENP-A during the assembly of centromeric nucleosomes [63].In T. thermophila, the formation of new MACs involves IES elimination after the replication of MICs from 4C to 8C, with no presence of Cna1 in MACs [62].Therefore, we speculate that Hir1 might be involved in Cna1 replacement and centromeric deletion.
It is reported that the deposition of H3.1 occurs within the replication region, whereas the incorporation of H3.3 takes place in the highly transcribed region [45].GFP-H3.1 is localized to the MAC and MIC in T. thermophila, whereas GFP-H3.3 is localized only to the persistently transcriptionally active MAC.The parental MAC is only transcribed and not replicated during sexual reproduction.Combining the localization of GFP-H3.3 and GFP-H3.1 with that of Caf1b-HA and Hir1-HA, we hypothesized that CAF-1 could be involved in RC-nucleosome-assembly-mediated deposition of H3.1, whereas Hir1 mediates the RI-nucleosome-assembly-mediated deposition of H3.3.

Caf1b and Hir1 Are Required for Proliferation and Sexual Reproduction of T. thermophila
In human cells, silencing of Caf1b/p60 leads to cell death and loss of chromatin assembly [64].Deletion of caf-1b in zebrafish led to S-phase arrest and impaired differentiation [65].In the chicken DT40 B-cell line, depletion of Caf1b led to delayed S-phase progression with retarded DNA synthesis and defects in a rapid nucleosome formation of newly replicated DNA, and cell death [20].However, the deletion Cac2/Caf1b in Saccharomyces cerevisiae exhibits a minimal effect on growth.Knockdown of CAF1B in T. thermophila inhibited cellular proliferation during vegetative growth (Supplementary Figure S6C) and disturbed meiotic progress (Figure 5A(c1-e1)).There were Caf1b signals on the meiotic MIC.Moreover, proteins interacted with Caf1b including Dyh1, Dyh3, Dyh4, and Dyh5 interactors associated with microtubule movement, chromosome movement, and distribution.Chromosome loss during retraction after stretching of the MIC to its maximum length upon deletion of CAF1B may be due to abnormal microtubule movement during MIC retraction, which leads to chromosome breakage and degradation or the abnormal repair of DSBs.
HIRA is indispensable for H3.3 deposition in mouse zygotes, and its deletion hinders the de novo assembly of nucleosomes [66].It was surprising to us that the absence of HIR1, a key element in RI nucleosome assembly, resulted in compromised MIC integrity.In T. thermophila, H3.3 weakly signals on the transcriptionally silent MIC via the inefficient RC pathway [38].Impairment of CAF-1-mediated deposition of H3.1 in HeLa cells promotes the deposition of H3.3 on DNA with the assistance of HIRA [5].In T. thermophila, we observed an up-regulation of HIR1 in the CAF1BKD mutants, and up-regulation of CAF1B was also observed in HIR1KD mutants (Supplementary Figure S5G, Figure 4E).Therefore, there is a complementary function between Caf1b and Hir1.Removal of Hira leads to the rapid decline of hematopoietic stem cells [67].Simultaneous deletion of CHAF1B and HIRA renders cells more susceptible to DNA damage in leukemic cells [25].In yeast, the cac1 and hir1 double-mutant cells exhibit a broad distribution of centromere histone Cse4 outside the centromere [68].In Tetrahymena, co-interference of HIR1 and CAF1B resulted in a more serious aberrant meiotic phenotype than individual mutants (Figure 5A,B).

Caf1b and Hir1 Interacted with Different Proteins
The interaction between Asf1 and Caf1b has been observed in Drosophila, Arabidopsis, and yeast [69].Caf1b contains B-domain-like motifs that bind to Asf1 [70].Caf1b contains two tandem B-domain-like motifs at its C-terminal in mammalian cells, whereas only one B domain is present in D. melanogaster and S. cerevisiae [19].However, Caf1b contained three B-domain-like motifs in Tetrahymena.The three B-domain-like motifs of Caf1b directly interacted with Asf1 in vitro.Furthermore, distinct B-domain-like motifs also indicate subtle structural variations between Caf1b and its homologous counterparts.In T. thermophila, the interaction between Caf1b and Caf1a was identified but not RebL1, indicating a more intensive interaction with Caf1a compared to RebL1, which may be attributed to the fact that the intermediate subunit interacts with the small subunit through the large subunit.In U2OS and HeLa cells, the chromodomain Y-like protein (CDYL) mediates the interaction between CAF-1 and MCM complexes, facilitating the transfer and deposition of histones during DNA replication [71].In HeLa S3 cells, the enrichment of CAF-1 complexes comprising all three subunits (p150, p60, and p48) was observed in eH3.1 and eH3.2 immunoprecipitations (IPs), while MCM2, 4, 6, and 7 exhibited interactions with all H3 variants [72].In T. thermophila, we identified direct or indirect interactions between Caf1b and members of the MCM protein family (Figure 6C).Caf1b exhibitsed co-localization with Pcna1 and interacted with a diverse range of proteins involved in replication, cell cycle regulation, and ubiquitination-mediated protein degradation, indicating its role as a replication-associated protein regulated via the cell cycle.
In mammals, UBN1, CABIN1, and HIRA form a stable HIR complex.In yeast, the complex is composed of Hir1, Hir2, Hir3, and Hpc2 [27,32].However, we did not find a similar subunit of HIR complexes that interact with Hir1 in Tetrahymena.We also failed to identify homologs of UBN1 and CABIN1 using a bioinformatics method in Tetrahymena.We hypothesized that the Hir1 complex could form a different assembly or have a different composition compared to other organisms.Further experimental investigation is required to gain a better understanding of the specific composition and functions of the Hir1 complex.Normal expression of Hira in the mouse is essential for transcriptional regulation [35].In Plasmodium falciparum, HIR is located in the heterochromatin region [73].Hir1 interacted with the chromodomain proteins Hpl2 and Pdd1, and formed punctate structure in the early anlagen stage (Figures 2B(f) and 6D), which implies that Hir1 is involved in heterochromatin formation in T. thermophila.The interaction of Hir1 and Caf1b with distinct proteins and their formation of different complexes suggests their functional diversity and complexity.

Conclusions
Caf1b and Hir1 contain conservative WD40 domains and B domains and have similar expression profiles.Both played important roles in the sexual and asexual reproduction of T. thermophila.Although Caf1b and Hir1 formed different complexes, they exhibited functional complementarity for nucleosome assembly.These findings in Tetrahymena deepen the understanding of RC and RI nucleosome assembly mechanisms.

Cells 2023 , 19 Figure 4 .
Figure 4. HIR1 knockdown affected chromatin integrity of MIC.(A) Proliferation of HIR1KD mutants.Initial cell concentration was 0.125 × 10 5 cells/mL and cellular proliferation was analyzed every 8 h.(B) The nuclear morphology of HIR1KD.Scale bar, 10 µm.(C) The diameter of MICs in HIR1KD.A total of 100 cells were randomly photographed and micronuclear diameters were measured using ImageJ 1.46r.(D) Integrity assay of MICs of HIR1KD.Gel electrophoresis was performed to detect specific fragments of MICs from WT and HIR1KD, which were amplified using 10 pairs of primers.JMJ1 was utilized as an internal control.M, marker; C, control; I-X, 10 pairs of MIC-specific primers.(E) Expression analysis of CAF1B was performed in HIR1KD mutants and using qRT-PCR.t-test was applied for significance analysis, and the expression level of CAF1B was significantly upregulated in HIR1KD mutant.(** p < 0.01; **** p < 0.0001).

Figure 4 .
Figure 4. HIR1 knockdown affected chromatin integrity of MIC.(A) Proliferation of HIR1KD mutants.Initial cell concentration was 0.125 × 10 5 cells/mL and cellular proliferation was analyzed every 8 h.(B) The nuclear morphology of HIR1KD.Scale bar, 10 µm.(C) The diameter of MICs in HIR1KD.A total of 100 cells were randomly photographed and micronuclear diameters were measured using ImageJ 1.46r.(D) Integrity assay of MICs of HIR1KD.Gel electrophoresis was performed to detect specific fragments of MICs from WT and HIR1KD, which were amplified using 10 pairs of primers.JMJ1 was utilized as an internal control.M, marker; C, control; I-X, 10 pairs of MIC-specific primers.(E) Expression analysis of CAF1B was performed in HIR1KD mutants and using qRT-PCR.t-test was applied for significance analysis, and the expression level of CAF1B was significantly upregulated in HIR1KD mutant.(** p < 0.01; **** p < 0.0001).
Conversely, in the mutants, gradual DNA loss occurred in 27.06% cells during crescent retraction and resulted in a reduction in MIC (Figure 5A(c1-e1),B).After 24 h of mixing, sexual reproduction was completed by 46.42% in the wild-type cells (Figure 5A(g),B), whereas only 1.49% of caf1bi cells were capable of forming two MACs and one MIC, and 61.01% of the cells developed abnormal single cells (Figure 5A(g1),B).Cells 2023, 12, x FOR PEER REVIEW 11 of 19 whereas only 1.49% of caf1bi cells were capable of forming two MACs and one MIC, and 61.01% of the cells developed abnormal single cells (Figure 5A(g1),B).

Figure 6 .Figure 6 .
Figure 6.Caf1b and Hir1 interacted with different proteins.(A) Western blotting analysis of Caf1b-HA from the eluate.(B) Western blotting analysis of Hir1-HA from the eluate.(C) Caf1b-HA interaction network.(D) Hir1-HA interaction network.(E) The interaction between B-like 1 of Caf1b and Asf1.Western blotting was performed using anti-His and anti-GST.(F) The interaction between B-Figure 6. Caf1b and Hir1 interacted with different proteins.(A) Western blotting analysis of Caf1b-HA from the eluate.(B) Western blotting analysis of Hir1-HA from the eluate.(C) Caf1b-HA interaction network.(D) Hir1-HA interaction network.(E) The interaction between B-like 1 of Caf1b and Asf1.Western blotting was performed using anti-His and anti-GST.(F) The interaction between B-like 2 of Caf1b and Asf1.Western blotting was performed using anti-His and anti-GST.(G) The interaction between B-like 3 of Caf1b and Asf1.Western blotting was performed using His and GST antibodies.(H) The interaction between B domain of Hir1 and Asf1.Western blotting was performed using anti-His and anti-GST.

4. 1 .
Similar and Different Dynamic Distribution Patterns of Caf1b and Hir1 in T. thermophila