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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.
SUPPLEMENTARY DATA
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.
SUPPLEMENTARY DATA
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.
SUPPLEMENTARY DATA
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.
SUPPLEMENTARY DATA
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.
SUPPLEMENTARY DATA
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.
SUPPLEMENTARY DATA
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-
SUPPLEMENTARY DATA
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 DATA
Supplementary Table S1. Primer listFor RT-PCR
Primer name Forward Reverse
HIST1H1T 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 H1TPrimer name Sequence
NheI-H1T_Foward CATATGGTGATTCCTAAATCTACCAG
BamHI-H1T_Reverse GGATCCTTACTTCTTAGATGTGGCCTTTC
For construction of overexpression vectorsPrimer name Sequence
3xFlag_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
SUPPLEMENTARY DATA
For ChIP-PCRPrimer name Forward Reverse
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
SUPPLEMENTARY DATA
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 vectorsPrimer name Sequence
miR_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 DATA
Supplementary Table S2. List of cell culture mediumCell* 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-231D-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
MBECMammary 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 DATA
Supplementary Table S3. Antibody listName Company Cat. No. Applications (Final conc.)*
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.