SUPPLEMENTARY DATA
Linker histone variant H1T targets rDNA repeats
Ruiko Tani1,2, Koji Hayakawa1,2,, Satoshi Tanaka1 and Kunio Shiota1
1 Department of Animal Resource Sciences/Veterinary Medical Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan
2 Both authors contributed equally to this work.
Supplementary Data
Supplementary Figure S1-S7
Supplementary table S1-S3
SUPPLEMENTARY DATA
Figure S1. Phylogenetic tree of human H1 variants.
The tree was constructed by amino acid sequences of H1 variants.
Figure S2. Fluorescent immunostaining of H1C (green) and B23 (red).
Rabbit and mouse Immunoglobulin G (rIgG and mIgG) was used as a negative control. HAEC, human aortic endothelial cell. SkMC, skeletal muscle cell. Scale bars, 4 µm.
Figure S3. Ratios of multi-position reads and unique reads in H1T ChIP-seq libraries.
(A) and (B) Chromatin immunoprecipitation (ChIP)-seq data for H1 variants were mapped to the human genome (hg19) (A) or the mouse genome (mm10) (B) using Bowtie aligner (http://bowtie-bio.sourceforge.net/). The ratio of multi-position reads (aligned >1 time) and unique reads (aligned exactly 1 time) was calculated. The total mapped reads were set to 100%. mESCs, mouse embryonic stem cells.
Figure S4. ChIP-qPCR on the H1C-enriched region.
H1C-enriched regions were detected by Model-based Analysis of ChIP-Seq (MACS) (http://liulab.dfci.harvard.edu/MACS/) using ChIP-seq data of α-H1C (accession numbers GSE49345). Values are means ± SD derived from three independent qPCR reactions and were normalized to the input signal.
Figure S5. The localization of 3xFlag-tagged H1T on rDNA repeats
(A) Establishment of cell lines stably overexpressing 3xFlag-H1T. Expression of 3xFlag (Flag, control) and 3xFlag-H1T (Flag-H1T) was analyzed by RT-PCR. Expression of ACTB was used as an internal control.
(B) ChIP-qPCR in H1T-overexpressing cells. Chromatin samples of Flag and Flag-H1T overexpressing cell lines were immunoprecipitated by Flag antibody. Values are means ± SD derived from three independent qPCR reactions and were normalized to the input signal. IP, immunoprecipitation.
Figure S6. Size of the nucleolus and nucleus in 3×Flag-H1T-overexpressing and H1T knockdown (KD) cells.
(A) The area of each nucleolus in 3×Flag-H1T-overexpressing cells. In the fluorescent immunostaining, nucleoli were visualized using an antibody against B23. Area was measured with Cell Profiler. P-values were calculated by the Wilcoxon rank-sum test.
(B) The area of each nucleus in 3×Flag-H1T-overexpressing cells. Nuclei were visualized by DAPI staining and measured with Cell Profiler.
(C) Area of each nucleolus in H1T KD cells.
(D) Area of each nucleus in H1T KD cells.
Figure S7. Validation of polyclonal H1T antibody
(A) C-terminal amino acid sequence of human and mouse H1T. The C-terminal portion of recombinant human H1T was used as antigen to produce H1T antibody.
(B) Western blotting for Flag and H1T in AGS cells overexpressing 3×Flag, 3×Flag-H1A/B/C/D/E, and 3×lag-H1T using α-H1T. Expression of ACTB was used as an internal control. The arrowhead indicates endogenous H1T. α-H1T recognized over-expressed 3×Flag-H1T, but not the other 3×Flag-tagged H1 variants (H1A, H1B, H1C, H1D, or H1E) in AGS cells.
(C) Immunoprecipitation (IP) using α-H1T. IP using 3 μg of α-H1T and AGS cross-linked chromatin (equivalent to 1 μg of genomic DNA). Normal IgG (rabbit) was used as a negative control. Precipitates were analyzed by SDS-PAGE and western blotting (WB). The results showed a single band of the predicted size in the IP lane; therefore, α-H1T could be used in chromatin IP analysis.
(D) Western blotting of mouse testis by α-H1T. Insoluble nuclear protein was used. The arrowhead indicates H1t. α-H1T detected endogenously expressed H1t in mouse testis by western blotting.
(E) Immunohistochemistry and (F) fluorescent immunostaining of mouse testis by α-H1T. a, spermatogonium; b, spermatocyte; c, spermatid. Rabbit Ig was used as a negative control. α-H1T confirmed H1t expression in spermatocytes and early spermatids, but not in spermatogonia, which verified the expression status previously reported for testis. Thus, α-H1T could recognize mouse H1t protein.
Supplementary Table S1. Primer list
For RT-PCR
Primer name / Forward / ReverseHIST1H1T / AGCAGAAGAGCCCAGTGAAG / AGAGCCTTTGGGTTCTTTCC
H1FOO / GGATCATCCAGGTCTCCTGA / TGTGCAGGATGTAGAGCTTGA
H1FNT / TCCACAAAGACCTCCCCTAA / AGTGTGACAGCCGCTCTTCT
ACTB / CCAACCGCGAGAAGATGA / CCAGAGGCGTACAGGGATAG
Hist1h1t / ACCTCGGGGTTTCTCAGTTT / CTTGAGGGCCAGCTTGATAC
Actb / TTCTACAATGAGCTGCGTGTGG / ATGGCTGGGGTGTTGAAGGT
3xFlag / CCCCTTCACCATGGACTACA / GCCCACCCTTCTACTTGTCA
3xFlag-H1T / CCCCTTCACCATGGACTACA / CTACACCAGCACTGGCAGAA
For construction of vector for expression of recombinant H1T
Primer name / SequenceNheI-H1T_Foward / CATATGGTGATTCCTAAATCTACCAG
BamHI-H1T_Reverse / GGATCCTTACTTCTTAGATGTGGCCTTTC
For construction of overexpression vectors
Primer name / Sequence3xFlag_F2 / CACCATGGACTACAAAGACCATGACGGTGATTATAAAGATCATGATATCGATTACAAGG
3xFlag_R / CTACTTGTCATCGTCATC
3xFlag-H1A_F / ATCATGATATCGATTACAAGGATGACGATGACAAGATGTCTGAAACAGTGCCTCC
3xFlag-H1A_R / TTACTTTTTCTTGGGTGCCG
3xFlag-H1B_F / ATCATGATATCGATTACAAGGATGACGATGACAAGATGTCGGAAACCGCTCCTGC
3xFlag-H1B_R / CTACTTCTTTTTGGCAGCCG
3xFlag-H1C_F / ATCATGATATCGATTACAAGGATGACGATGACAAGATGTCCGAGACTGCTCCTGC
3xFlag-H1C_R / CTATTTCTTCTTGGGCGCCG
3xFlag-H1D_F / ATCATGATATCGATTACAAGGATGACGATGACAAGATGTCGGAGACTGCTCCACTTGC
3xFlag-H1D_R / TCACTTTTTCTTCGGAGCTGC
3xFlag-H1E_F / ATCATGATATCGATTACAAGGATGACGATGACAAGATGTCCGAGACTGCGCCTGC
3xFlag-H1E_R / CTACTTTTTCTTGGCTGCCGCCTTC
3xFlag-H1T_F / ATCATGATATCGATTACAAGGATGACGATGACAAGATGTCTGAAACCGTGCCTGC
3xFlag-H1T_R / TTACTTCTTAGATGTGGCCTTTCTAAC
For ChIP-PCR
CRYGS (#27)* / TTTCAAATAGCTGGGGGTAAAA / TTTTTGCGCTTCCACTGAT
OPA1 (#47)* / GCACAAATAAATTTTCACCTTGG / GAACTTGTTCTTTAGCAACCAGAAT
ABCA6 (#88)* / GCTAACCGTGGAAAGAGACAA / GCGTGTATCAGCAAACCAAA
SLC16A7 (#87)* / CTTGCTTATTGATACTTACCTGTACCA / TCACAGACAGTCACCACATAAAAA
LINC00578 (#90)* / TGCTTCTTTCGTGGACCATA / TGTGGAAACAATGGATCAGAAT
POU6F2 (#18)* / AAAGACAACATATGATGGATTGCTA / GCTGACTCCATACCTGACCAA
GAS2 (#6)* / GGGAGTAATTTTGAATGATTATGCTT / TGCTAAATACAGTTTGGGGAAAA
OR8B3 (#26)* / AAAGCCGTGACTTTGACATCTT / TGCACTGATATTTTTCATTATCATTTT
EGFEM1P (#6)* / GGTGTTGATGTATGTTGTCATCC / GGCCTTCAGAATTCTTGTTGAG
KCNT2 (#89)* / GGCAGTGTTCAACTAACCAAAA / TGTGGTTGCCCTCATTTTTC
PYDC2 (#46)* / TCTTCACCAGCCACTCCTG / TGCGTTTGATTCATCTTTTCA
UCE** / ACTCACGGTTTCGCTTTCG / CCGAGAGCACGATCTCAAAG
UCE2** / CTCCCGCTCTGGAGACAC / GGACACCTGTCCCCAAAAAC
UCE3** / ATCCTTTCTGGCGAGTCC / GAGCCGGAAGCATTTTCG
H42.9** / CCCGGGGGAGGTATATCTTT / CCAACCTCTCCAGCGAC
5’ETS (pre-rRNA)**,*** / AAAGCCTTCTCTAGCGATCTGAG / CTACCATAACGGAGGCAGAGAC
H4-** / GGATGCGTGCATTTATCAGA / GATCGGCCCGAGGTTATCTA
H4** / CGACGACCCATTCGAACGTCT / CTCTCCGGAATCGAACCCTGA
H8** / AGTCGGGTTGCTTGGGAATGC / CCCTTACGGTACTTGTTGACT
H13** / ACCTGGCGCTAAACCATTCGT / GGACAAACCCTTGTGTCGAGG
H18** / GTTGACGTACAGGGTGGACTG / GGAAGTTGTCTTCACGCCTG
H27** / CCTTCCACGAGAGTGAGAAGCG / CTCGACCTCCCGAAATCGTACA
H32** / GGAGTGCGATGGTGTGATCT / TAAAGATTAGCTGGGCGTGG
GAPDH** / CCCCTTCATACCCTCACGTA / GACAAGCTTCCCGTTCTCAG
ACTB** / GTGGACATCTCTTGGGCACT / TCTGCAGGAGCGTACAGAAC
mouse_rDNA-promoter / ACCTATCTCCAGGTCCAATA / CCCAGGTATGACTTCCAGG
mouse_5’ETS / GTCTGCCCGTATCAGTAACTGTC / ACCACATCGATCTAAGAGTGAGC
mouse_18S / AAACGGCTACCACATCCAAG / CCTCCAATGGATCCTCGTTA
mouse_ITS1 / GGTCCATCTGTTCTCCTCTCTCT / GTATCGGTATTTCGGGTGTGAG
mouse_ITS2 / GCTGCCTCACCAGTCTTTCT / CAACCTCGACCAGAGCAGAT
mouse_28S / CCCAGTGCTCTGAATGTCAA / ATGACGAGGCATTTGGCTAC
mouse_3’ETS / AACGACCGTGTGGTGGTTG / GACGGAAGGGGAAAGAGAAAC
mouse_IGS1 / CATGGAGCTGCTCACACAGT / CCGGCTGCTCAAGTAAGTTC
mouse_IGS2 / AGGTCCTGGGACATATGCAG / CCAGGAGTGGTGTTTGTGTG
mouse_IGS3 / AGGTCCTGGGACATATGCAG / CCAGGAGTGGTGTTTGTGTG
Hist1h3b / GAAGAGAGGCGGAGAGTGTG / TATCCGCTATTGGACCGAAG
Hist1h3g / GGCAACGTTTTTCCTTTCCT / TTGAGATGGAAAAGCCCAAG
*, # indicates Universal Probe ID.
**, also used for nuclease sensitive assay.
***, also used for RT-PCR.
For construction of knockdown vectors
Primer name / SequencemiR_LacZ_top / TGCTGAAATCGCTGATTTGTGTAGTCGTTTTGGCCACTGACTGACGACTACACATCAGCGATTT
miR_LacZ_bottom / CCTGAAATCGCTGATGTGTAGTCGTCAGTCAGTGGCCAAAACGACTACACAAATCAGCGATTTC
miR_H1T-#1_top / TGCTGTCAACTTGGACACAGAGAGGTGTTTTGGCCACTGACTGACACCTCTCTGTCCAAGTTGA
miR_H1T-#1_bottom / CCTGTCAACTTGGACAGAGAGGTGTCAGTCAGTGGCCAAAACACCTCTCTGTGTCCAAGTTGAC
miR_H1T-#2_top / TGCTGTTCTCTACGTCGTAGCCAGCAGTTTTGGCCACTGACTGACTGCTGGCTGACGTAGAGAA
miR_H1T-#2_bottom / CCTGTTCTCTACGTCAGCCAGCAGTCAGTCAGTGGCCAAAACTGCTGGCTACGACGTAGAGAAC
Supplementary Table S2. List of cell culture medium
AGS / RPMI-1640 (SIGMA, R8758-500ML), 2 mM L-Glutamine (073-05391),
10% Fetal Bovine Serum (FBS) (Cell Culture Bioscience, 171012-500ML),
105 U/L Penicillin-100 mg/L Streptomycin (168-23191)
HSC-39
HSC-57
KATOIII
YMB-1
HuTu80 / D-MEM (High Glucose) (045-30285),
10% MEM (life technologies, 11095-080),
2 mM L-Glutamine, 10% FBS,
105 U/L Penicillin-100 mg/L Streptomycin
MDA-MB-231 / D-MEM, 2 mM L-Glutamine, 10% FBS,
105 U/L Penicillin-100 mg/L Streptomycin
MCF-7 / E-MEM (051-07615), 0.1 mg/mL Insulin (093-06351), 1 mM Non-Essential Amino Acid (139-15651),
1 mM Na-Pyruvate (190-14881), 105 U/L Penicillin-100 mg/L Streptomycin
MBEC / Mammary Epithelial Cell Culture Medium (ZEN-BIO, BBMEG1)
MLEC
hRPTEC / REGM BulletKit (Lonza, CC-3190)
HAEC / EGM-2 BulletKit (Lonza, CC-3162)
SkMC / SKGM BulletKit (Lonza, CC-3160)
mESC (J1 line) / D-MEM (High Glucose),
15% Knockout SR (life technologies, 10828-028), 5% FBS,
2 mM L-Glutamine, 105 U/L Penicillin-100 mg/L Streptomycin,
MEM Non-essential Amino Acids Solution (x1) (139-15651),
1 mM Sodium Pyruvate Solution,
100 µM 2-Mercaptoethanol Solution (198-15781)
1500 U/ml leukemia inhibitory factor (Millipore, ESG1107) (to keep undifferentiated state)
*All cells except HSC-39 and mESC were cultured on TC dishes, HSC-39 in Petri dishes, and mESC on a gelatin-coated dish (Sigma-Aldrich). All cells were cultured at 37°C in 5% CO2.
MBEC, mammary basal epithelial cell. MLEC, mammary luminal epithelial cell. hRPTEC, human renal proximal convoluted tubule epithelial cell. HAEC, human aortic endothelial cell. SkMC, skeletal muscle cell. mESC, mouse embryonic stem celll.
Supplementary Table S3. Antibody list
Monoclonal-ß-Actin / SIGMA-ALDRICH / A1978 / WB (1 μg/ml), IF (1 μg/ml)
Monoclonal ANTI-FLAG M2 antibody produced in mouse / SIGMA-ALDRICH / F1804 / WB (1 μg/ml), ChIP (10 μg/ml)
Rabbit Immunoglobulin Fraction (Solid-Phase Absorbed) / Dako / X 0936 / IHC (10 μg/ml)
Anti-Rabbit Ig Biotin / Dako / E0432 / IHC (1:600)
Peroxidase-AffiniPure Goat Anti-Rabbit IgG (H+L) / Jackson ImmunoResearch / 111-035-003 / WB (1:5000)
Peroxidase-AffiniPure Goat Anti-Mouse IgG (H+L) / Jackson ImmunoResearch / 115-035-003 / WB (1:5000)
pAb anti-Histone H1.2 / Novus Biologicals / NBP2-16845 / ChIP (10 μg/ml)
Anti-Nucleophosmin antibody (B23) / Abcam / ab10530 / IF (1 μg/ml)
Rabbit polyclonal IgG / Abcam / ab27478 / IF (1 μg/ml)
Rabbit IgG Wholemolecule Chrom Pure / Jackson ImmunoResearch / 011-000-003 / ChIP (10 μg/ml)
Mouse Control IgG2a [MOPC-173] - ChIP Grade / Abcam / ab18413 / ChIP (10 μg/ml)
Alexa Fluor 488 goat anti-rabbit IgG (H+L) / Invitrogen / A11034 / IF (1:1000)
Alexa Fluor 594 goat anti-mouse IgG (H+L) / Invitrogen / A11032 / IF (1:1000)
SCP-3 Antibody (D-1) / Santa Cruz / sc-74569 / IF (2μg/ml)
*IF, immunofluorescence assay; IHC, immunohistochemistry assay; WB, western blotting; ChIP, chromatin immuno precipitation assay.