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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

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Figure S1. Phylogenetic tree of human H1 variants.The tree was constructed by amino acid sequences of H1 variants.

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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.

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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.

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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.

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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.

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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.

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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-

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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.

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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

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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

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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

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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.

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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.