Additional file 2 of Multilevel interrogation of H3.3 reveals a primordial role in transcription regulation

Additional file 2: Figure S1. Endogenous tagging of H3 and H3.3 in Tetrahymena. A: Comparison of Tetrahymena H3 variants and histone chaperones’ nomenclature with human gene/protein names. B: Multiple sequence alignment showing the conservation of Tetrahymena H3 and H3.3. 15 residues vary between H3 and H3.3. Conservation score key is provided. B: Schematic depiction of epitope tagging strategy for the MAC locus. C: Indirect immunofluorescence analysis of H3GFP and H3.3-FZZ in growing Tetrahymena. DAPI was used to stain the nuclei and the position of the MAC and MIC is indicated with arrows and arrowheads, respectively. Untagged wildtype Tetrahymena were used as a control. Figure S2. Aip1 shows cytoplasmic localization in growing Tetrahymena. A: Western blotting analysis using whole cell lysates prepared from vegetative Tetrahymena cells expressing Aip1FZZ. The blots were probed with the indicated antibodies. B: Indirect immunofluorescence analysis of Nrp1-GFP in growing Tetrahymena. Untagged wildtype Tetrahymena were used as a control. C: Indirect immunofluorescence analysis of Aip1-FZZ in starved Tetrahymena cells. D: Indirect immunofluorescence analysis of macronuclear Hv1-FZZ (left) and micronuclear linker histone Mlh1-FZZ (right) in growing Tetrahymena. Note: DAPI was used to stain the nuclei and the positions of the MAC and MIC are indicated with arrows and arrowheads, respectively. Figure S3. H3 (H3.3)/H4 chaperones show similar expression profiles. Heatmap representation of microarray expression values for Asf1, Hir1, Cac2, and Nrp1. Z scores were calculated across the rows for each gene to examine its differential expression across growth, starvation, and developmental stages. L1–LH: Logarithmic growth phase, S0–24: Starvation for 24 h, C: Conjugation where 0–18 are hours post mixing the different mating types. Hierarchical clustering was used to examine the expression profiles. Figure S4. Asf1Tt structure is conserved in Tetrahymena. A) AlphaFold-predicted structure of TTHERM_00442300 (Asf1Tt) B) Model alignment comparison of Asf1Tt from this study with the Human ASF1A homolog model generated by the AlphaFold Deepmind consortium (AlphaFold Database ID: Q9Y294). Asf1Tt is colored in green; Human ASF1A is colored in magenta. C) Predicted structure of Asf1Tt colored by pLDDT per residue confidence score ranging from orange (very low: pLDDT<50) to dark blue (very high: pLDDT>90). D) Model alignment comparison of Asf1Tt from this study with Human ASF1A colored by pLDDT score. Figure S5. Cac2Tt forms β-propeller-like structure. AlphaFold-predicted structure of TTHERM_00442300 (Cac2Tt) protein depicting side (A) and top (B) views respective to the βpropeller motif. The B-domain of Cac2Tt is colored in red. Figure S6. Hir1Tt structure prediction. AlphaFold-predicted structure of TTHERM_00046490 (Hir1Tt) protein depicting side (A) and top (B) views respective to the β-Propeller motif. The B-domain of Hir1Tt is colored in red. Figure S7. Protein complex prediction of Cac2Tt and Asf1Tt. Overall AlphaFold-predicted structure of TTHERM_00219420 (Cac2Tt) bound with TTHERM_00442300 (Asf1Tt). Cac2Tt is colored in cyan and Asf1Tt is colored green. The B-domain of Cac2Tt is colored in red. Figure S8. Visualization of the predicted binding interface between Cac2Tt and Asf1Tt. Cac2Tt is colored in cyan, whereas Asf1Tt is colored green and the B-domain of Cac2Tt is highlighted in red. Labeled residues (K87-G531, D89-K534, R146-D372) are predicted to form polar intermolecular contacts between Asf1Tt and Cac2Tt within 3Å, and an intramolecular π interaction (F393-K535) involving a lysine residue within the B-domain of Cac2Tt (T527-Y545). All interactions are shown as dashed yellow lines and arrows. Figure S9. Visualization of the predicted binding interface between Hir1Tt and Asf1Tt. A: AlphaFold-predicted co-structure of TTHERM_00046490 (Hir1Tt) with Asf1Tt. Hir1Tt is colored in gray, Asf1Tt is colored green. The B-domain of Hir1Tt is colored in red. No significant intermolecular interactions were detected in our predictions. B: Left, AlphaFold-predicted co-structure. Hir1Tt is colored by pLDDT per residue confidence scores ranging from orange (very low: pLDDT<50) to dark blue (very high: pLDDT>90). Asf1Tt is colored in green. Right: AlphaFold-predicted by-residue pLDDT confidence score plot for the 5 highest-confidence Hir1Tt models. All models display low predictive confidence for residues of the Hir1Tt B-Domain (res. 453:476) and directly up and downstream of the B-domain. Figure S10. Endogenous tagging of Tetrahymena Hat1. A: Western blotting analysis using whole cell lysates prepared from vegetative Tetrahymena cells expressing Hat1-FZZ. The blots were probed with the indicated antibodies. B: Indirect immunofluorescence analysis of Hat1-FZZ in growing Tetrahymena. DAPI was used to stain the nuclei, and the positions of the MAC and MIC are indicated with arrows and arrowheads, respectively. Untagged wildtype Tetrahymena were used as a control. Figure S11. Strategy to confirm the correct integration of NEO cassette. Top, Schematic representation of the confirmation of the accurate integration of the NEO cassette at the locus of interest. Positions of PCR primers are indicated. The reverse primer is designed complementary to sequence within the NEO cassette, whereas the forward primer is specific to sequence upstream of the gene of interest. A PCR product will be observed only if the NEO cassette is integrated into the desired locus. WT cells will not show PCR products. Primers designed to amplify DNA from the promoter regions of each target gene were used as loading controls. Bottom, Agarose gel electrophoresis using genomic DNA extracted either from KO or WT Tetrahymena cells. Figure S12. KO analysis of CAC2Tt and HIR1Tt in Tetrahymena. A: Left, Fluorescence (DAPI) of vegetative and starved ∆CAC2 and wildtype Tetrahymena cells. Right, Bar plots showing the quantification of mean diameter of MACs in ∆CAC2Tt compared to wildtype Tetrahymena. Diameters were measured in micrometers for 40 individual Tetrahymena cells. Images used were taken at 40X magnification in a 1360x1024 frame. Field of view at 40X was 360 micrometers. B: Fluorescence (DAPI) analysis of conjugating wildtype, ΔHIR1Tt, and ΔCac2Tt Tetrahymena cells. Hours post mixing the Tetrahymena cells of different mating types are indicated on the left. Note: DAPI was used to stain the nuclei. Figure S13. Indirect immunofluorescence analysis in starved Tetrahymena. Indirect immunofluorescence analysis in starved Tetrahymena. A: Expression profile of Hir1 during growth and starvation in Tetrahymena. For growing cells, L-l corresponds to ~1X105 cells/mL. For starvation, ~2X105 cells/mL were collected at 0, 3, 6, 9, 12, 15, and 24 hours referred to as S-0, S-3, S-6, S-9, S-12, S-15, and S-24. Microarray data was acquired from http://tfgd.ihb.ac.cn/search/detail/gene/TTHERM_00046490 (last accessed January 20, 2023) B: Indirect immunofluorescence analysis of H3.3-FZZ in starved Tetrahymena. H3.3 is found in the MAC only. C: Indirect immunofluorescence analysis of RebL1-FZZ in starved Tetrahymena. RebL1 is found in the MAC only. DAPI was used to stain the nuclei. The positions of the MAC and MIC are indicated with arrows and arrowheads, respectively. Figure S14. H3.3 ChIP-seq replicates correlate with each other. A: Principal component analysis (PCA) of two H3.3 ChIP-seq replicates and their corresponding inputs. B: Fingerprint plot to examine the quality of H3.3 ChIP signal in comparison with inputs. ChIP-seq is enriched as more reads are found in smaller number of bins for ChIPs compared to the input. Figure S15. GO enrichment analysis of H3.3 ChIP-seq targets. A: Bar plot depicts the % overlap of upregulated and downregulated genes in H3.3 KO cells with those genes classified as highly expressed during Tetrahymena vegetative growth. B: KEGG pathway enrichment analysis of H3.3 bound genes. Number of genes for each term is indicated beside each bar. C: Dot plot representation of pfam domain enrichment analysis in H3.3-target genes (Q< 0.05). Figure legend is provided. Figure S16. Predicted CKII sites on Cac2Tt and Cac1Tt proteins. The red box shows the conserved sequence, whereas star indicates the serine residue predicted to be phosphorylated by CKII. The prediction was performed using Netphos web server https://services.healthtech.dtu.dk/services/NetPhos-3.1/ .

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