Meis1 Nature Supplementary Material ANP-Modified

SUPPLEMENTARY INFORMATION

doi:10.1038/nature12054

Materials and Methods Experimental Animals All protocols were approved by the Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center. All experiments were performed on age and sex matched mice, with equal ratio of male to female mice. The number of animals used for each experiment; n= 3-6.

Mouse breeding and genotyping The Meis1 floxed mice were kindly provided by Dr. R. Keith Humphries, University of British Columbia, Canada. Mice were genotyped as described previously1 using Meis1 sense (5’-CCAAAGTAGCCACCAATATCATGA-3’) and Meis1 antisense (5’-AGCGTCACTTGGAAAAGCAATGAT-3’) primers. The wildtype (WT) allele was identified as a 332bp-long PCR product and the mutant allele was determined by a 440bp long PCR product on a 1.2% agarose gel. Cardiac-specific deletion of Meis1 (Meis1 KO) was achieved by crossing Meis1f/f mice with αMHC-Cre mice2. αMHC-Cre mice were genotyped using αMHC-Cre sense

(5’-GATTTCCGTCTCTGGTGTAGCTGATGATCC-3)

antisense matched

and

(5’-GCCAGGTATCTCTGACCAGAGTCATCC-3) Meis1+/+;αMHC-Cre(+)

mice

were

used

as

αMHC-Cre

primers.

controls.

Age-

Inducible

cardiomyocyte-specific deletion of Meis1 (Meis1 iKO) was achieved by crossing Meis1f/f mice with Myh6-MerCreMer (tamoxifen-inducible Cre recombinase under control

of

Myh6

cardiomyocyte-specific

promoter)

mice3.

Homologous

WWW.NATURE.COM/NATURE | 1

RESEARCH SUPPLEMENTARY INFORMATION

doi:10.1038/nature12054

recombination in cardiomyocytes was achieved by administering 3 doses of Tamoxifen injections by I.P. (1mg/day/mice) to 5-6 week-old Meis1 iKO mice. In addition, 35 mg/kg of N-acetylcysteine was administered to both the iKO and control mice at the time of tamoxifen administration only to prevent the transient left ventricular systolic dysfunction that follows Cre activation, which results in transient mitochondrial dysfunction4. Hearts were harvested one week later for proliferation indices, or 6-8 weeks later for heart size and myocyte count. Myh6MerCreMer

mice

were

genotyped

using

Myh6-MerCreMer

GATTTCCGTCTCTGGTGTAGCTGATGATCC-3) antisense

and

(5’-GCCAGGTATCTCTGACCAGAGTCATCC-3)

sense

(5’-

Myh6-MerCreMer primers.

Age-

matched Meis1+/+;Myh6-MerCreMer(+) mice were used as controls and received same dose of Tamoxifen.

Immunostaining Following antigen retrieval with EDTA Buffer (1mM EDTA, 0.05% Tween 20, pH 8.0) in boiling water for 40 min, paraffin sections were permeabilized with 0.3% triton X/PBS for 10 minutes and then blocked with 10% goat serum for 30 minutes. Primary antibodies against Meis1 (Santa Cruz Biotechnology, sc-10599) and cardiac troponin T (Tnnt) (Thermoscientific, MS-295-P1, 1:100 dilution) were incubated overnight at 4oC. Sections were subsequently washed with PBS and incubated with corresponding secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 555 (Invitrogen).



doi:10.1038/nature12054

Immunostaining on paraffin embedded sections for PH3 and Aurora B were performed as described previously5. Following antigen retrieval with 10mM NaCitrate (pH6.1) in boiling water for 20 min, sections were blocked with 10% goat serum, and incubated with PH3 or Aurora B (1:100 dilution) and Tnnt (Thermoscientific, MS-295-P1, 1:100 dilution) antibodies overnight at 4oC. Sections were subsequently washed with PBS and incubated with corresponding secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 555 (Invitrogen). Z stack imaging pictures were taken from Zeiss LSM 510 confocal (Carl Zeiss, NY) equipped with a Chameleon 2 photon laser (Coherent, CA). Image

deconvolution

was

performed

with

Autoquant

X3

(http://www.mediacy.com/index.aspx?page=AutoQuant) followed by analyses on the Imaris 7.6.0 (www.bitplane.com). All staining was performed on 3-6 hearts/group, with 3 sections/heart.

Wheat germ agglutinin (WGA) staining Following deparaffinization, slides were rinsed 3 times in PBS and then incubated for 1 hour at room temperature with a primary antibody against WGA conjugated to Alexa Fluor 488 (50 µg/ml, Invitrogen, CA). Slides were then rinsed in PBS and mounted in Vectashield containing DAPI (Vector Labs, CA). To quantify size of cells, images at 40X zoom were captured and ImageJ was used to determine the area of each cell. Quantitative analyses involved counting of multiple fields from 3-6 independent hearts per group, and 3 sections/heart (~50 cells per field assessed, total ~250 cells per sample).


doi:10.1038/nature12054


BrdU and α-actinin staining BrdU (1mg/ml, MP biomedical LLC) introduced in the drinking water of adult (28 days old) for 2 weeks before harvesting for paraffin sections. Following deparaffinization, and antigen retrieval with EDTA Buffer in boiling water for 20 min, paraffin sections were permeabilized with 0.3% triton X/PBS for 10 minutes and then blocked with 10% goat serum for 30 minutes. Primary antibodies against BrdU (Roche, 11170376001, 1:25 dilution) and α-actinin (abcam ab68167, 1:100 dilutions) were incubated overnight at 4oC. Sections were subsequently washed with PBS and incubated with corresponding secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 555 (Invitrogen). Quantitative analyses involved counting BrdU positive cardiomyocytes in multiple fields from three independent samples per group, and 3 sections/heart. The total number of myocytes counted was 2-2.5 x103 cardiomyocytes per section.

H&E and trichrome staining Hematoxylin/eosin and Masson’s trichrome staining were performed according to standard procedures at UTSW core histology facility on paraffin sections.

Meis1 knockdown Meis1 siRNA knockdown was carried out in vitro on cultured rat neonatal cardiomyocytes. Cardiomyocytes were cultured on 6-well plates at 70% confluency, 1x105 cardiomyocytes per well, 3 wells per group. 12 ul of



doi:10.1038/nature12054

lipofectamine (invitrogen) and siRNA (50nM of siRNA) were incubated in 200 ul of OPTIMEM (Invitrogen) for 20 min at RT. Silencer Select Pre-designed siRNAs (Applied Biosystems, Ambion) for Meis1 (siRNA ID# s8662) and negative control were

diluted

into

50nM

stocks:

siRNA

ID#

s8662:

5’-

GGCAUCUACUCGUUCAGGAtt-3’ and 5’-UCCUGAACGAGUAGAUGCCgt-3’. siRNA were added to cells dropwise and incubated for 6 hours. After 6 hours incubation, cardiomyocyte growth medium was added and plates were incubated under normal growth conditions for 48 hours.

Immunostaining of neonatal cardiomyocytes following Meis1 siRNA treatment Control siRNA or Meis1 siRNA transfected neonatal cardiomyocytes were fixed on coverslips with 4% PFA for 5 min at room temperature. Following permeabilization with 0.1% Triton X in PBS for 2 min at room temperature, cells were blocked with 3% normal goat serum for 30 minutes. Coverslips were incubated with PH3 (1:100) and TnnT (1:100) primary antibodies in humid chamber for one hour at room temperature. This was followed by incubation with corresponding secondary antibodies conjugated to Alexa Fluor 488 and Alexa Fluor 555, Hoechst 33342 staining and fluorescent imaging. The number of PH3+ cardiomyocytes was counted per 40x field.

Real-Time PCR: Total RNA was isolated using Qiagen`s RNeasy Mini Kit according to manufacturer’s instructions. cDNA was synthesized from 2µg of RNA using



doi:10.1038/nature12054

SuperScript II RT (Invitrogen). Predesigned primers (table 4) from NIH mouse primer depot (http://mouseprimerdepot.nci.nih.gov/) were ordered from Integrated DNA Technologies unless otherwise stated. Real time PCR was performed with SyberGreen (Applied Biosystems) on ABI Prism 7700 Sequence Detector (Applied Biosystems). GAPDH was used as a housekeeping control to normalize gene expression using the ∆∆Ct method.

TUNEL staining Paraffin embedded heart sections were deparaffinized and permeabilized with 0.2% Triton-PBS at room temperature for 10 minutes. Following 1 hour blocking with 1%BSA, 1%normal goat serum, 0.025%Tween-20, slides were incubated with Desmin (1/100 in PBS) or Tnnt (Thermoscientific, MS-295-P1, 1:100 dilution) overnight at 4oC. Following incubation with a corresponding secondary antibody conjugated to Alexa Fluor 488 (Invitrogen), TUNEL staining was performed according to manufacturer’s recommendations (In-Situ Cell Death Detection Kit, Fluorescein, Cat# 11684795910, Roche). All staining was performed on 3-6 hearts/group, with 3 sections/heart.

Echocardiography Left ventricular systolic function was determined echocardiographically on conscious mice using the Visual Sonics Vevo 2100, equipped with a 40 MHz mouse ultrasound probe.

Ejection fraction and fractional shortening were



doi:10.1038/nature12054

calculated based on end diastolic and end systolic dimensions obtained from Mmode ultrasound. Echocardiograms were performed on 3-6 hearts/group.

Cardiomyocyte Isolation Isolation of myocytes was performed as described recently6. Briefly, Fresh hearts were harvested and immediately fixed in 4% PFA for 2-3 hours. Samples were subsequently incubated with collagenase D (2.4 mg/mL, Roche) and B (1.8 mg/mL, Roche) for 12 hours at 37°C. The supernatant was collected and spun down (500rpm for two minutes) to yield the isolated cardiomyocytes. The hearts were minced to smaller pieces and the procedure was repeated until no more cardiomyocytes were dissociated from the tissue. The cardiomyocytes were stained with Connexin 43 antibody (IHC world, IW-PA1026, 10ul) and Hoechst for nucleation counts. For myocyte count, we averaged 3 different counts/sample and 3 hearts/group using a hemocytometer. The total number of myocytes counted

was

approximately

150-200

cardiomyocytes/aliquot

using

hemocytometer (10 ul aliquots samples using a wide-bore pipette from the total volume of myocytes obtained following digestion). For nucleation, approximately 1x103 cardiomyocytes were counted per sample, using 3 independent samples per group. Nucleation was plotted as percentage of counted cardiomyocytes.

Microarray RNA samples extracted from Meis1 MHC KO and Meis1f/f control hearts to study the dysregulation of developmental genes. Microarray analysis was performed by



doi:10.1038/nature12054

the University of Texas Southwestern Microarray Core Facility using the Illumina Mouse-6 v2 chip and data analysis performed using Genomestudio. Data presented are for those genes that showed more than 1.2 fold dysregulation.

Generation of Meis1 transgenic mice A 3-kb EcoRI-HindIII fragment containing the Meis1 gene was sub-cloned from pCMV-SPORT6-Meis1 (openbiosystems) into a pTRE-Tight DNA vector to generate the pTRE-Meis1 plasmid. pTRE-Meis1 was linearized with PvuI and microinjected into fertilized oocytes to generate transgenic mice using standard procedures. Founder (G0) mice were identified by PCR using genomic DNA isolated from the tails of transgenic mice using the following primers: sense 5′CAGTGATAGAGAACGTATGTCGAG-3′

and

antisense

5′-

TCGTCGTACCTTTGCGCCATC-3′. Mice that were positive for the pTRE-Meis1 transgene were bred to C57BL6/J mice determine to generate stable transgenic lines (4 lines were generated, and a line that overexpressed Meis1 2-2.5 fold was selected as it closely resembled the level of expression of Meis1 after P7). To induce Meis1 overexpression in cardiomyocytes, pTRE-Meis1 mice were crossed with αMHC-tTA mice to generate pTRE-Meis1; αMHC-tTA mice (Meis1 Tg). Agematched αMHC-tTA, pTRE-Meis1 and wild type mice were used as controls.

Generation of INK4b–ARF–INK4a locus (p16INK4a/p19ARF/p15INK4b) and p21 reporter vectors



doi:10.1038/nature12054

We have identified putative Meis1 regulatory sites by UCSC Genome Browser within genes that are downregulated in Meis1 KO Cardiomyocytes. We have found that only three genes had putative highly conserved Meis1 binding sites, namely p21, Ink4b-ARF-Ink4a locus and BRCA2. Meis1 binding sites within p21 and Ink4b-ARF-Ink4a locus were in the promoter region of the genes and highly conserved across the species (at least 5 different species as shown) though BRCA2 had a predicted site in the 9th intronic region. In addition, predicted Meis1 binding site in BRCA2 gene by the Genome Browser algorithm was based on the Meis1 cofactors Pbx1 and HoxA9 instead of canonical Meis1 motif (TGACAG). A detailed analysis of predicted site in BRCA2 gene showed that it included only Pbx1 binding motif (TGAT) instead of Meis1 motif (TGACAG). Thus, we excluded BRCA2 in our further studies, as it did not have a putative Meis1 binding motif. A 691 bp long DNA fragment (chr9:22,010,169-22,010,227) from the promoter region of INK4b–ARF–INK4a locus and a 987 bp long DNA fragment (chr6:36,650,803-36,650,829) from the promoter region of p21 (Cdkn1a) containing conserved Meis1 binding sites were amplified from mouse genomic DNA.

PCR primers for cloning include INK4b–ARF–INK4a-pGL2-F 5'-

CCCTTGAGCTTTGGTTGTAATCC-3' GGAATCTGACACACGTACTACC-3' luciferase reporter, p21-pGL-F p21-pGL-R

and for

INK4b–ARF–INK4a-pGL2-R5'-

generation

of

INK4b–ARF–INK4a

5'-GTATGTGTGTGGTAGTGTATGTGG-3' and

5'-GCCAGCCTGGTCTACAAAGTG-3'

for

generation

of

p21

luciferase reporter. PCR products were subsequently subcloned into the E1b-



doi:10.1038/nature12054

pGL2 vector to generate INK4b–ARF–INK4a and p21 luciferase reporter vectors. To test for Meis1 binding site specificity, the Meis1 binding site (TGAC) was mutated using iProof (Bio-rad) with the following primers: INK4b–ARF–INK4a del(NotI)-F INK4a

5'-agtgcggccgcCCCAGCACCACACCCGAGT-3' and INK4b–ARF–

del(NotI)-R

5'-agtgcggccgcTTAAAGCCCTCTCTGAACGT-3'.

p21mut

(NotI)-F 5'-agtgcggccgcAGTGGGAGGGAGGGAGGG-3' and p21mut (NotI)-R 5'agtgcggccgcCAGGTGCTCCAGGCTGGAGG-3'.

Luciferase reporter assays Transcriptional activation of INK4b–ARF–INK4a locus and p21 by Meis1 was evaluated using INK4b–ARF–INK4a-pGL2 and p21-pGL2 vectors as described previously1. 0.8 µg of INK4b–ARF–INK4a-pGL2 or p21-pGL2 was co-transfected with 50ng, 100ng, 200ng and 400ng of the Meis1 expression vector pCMVSPORT6-Meis1 (OpenBiosystems) and 0.2 µg of pCMV-LacZ (internal control) into COS cells in 6-well plates using lipofectamine transfection reagent (Invitrogen). 48 hours after transfection, the cell lysate was processed for luciferase activity using the luciferase reporter system (Promega), according to the manufacturer’s instructions. Luciferase measurements were calculated as firefly luciferase normalized to β-gal units.

Chromatin Immunoprecipitation Assay (ChIP) Chip assays were performed to evaluate the in vivo binding of Meis1 to its consensus sequence in the INK4b–ARF–INK4a and p21 promoter. The assays



doi:10.1038/nature12054

were done in whole mouse hearts using the ChIP kit (Upstate, cat#17-295) as described previously1.

Meis1 antibody (Santa Cruz Biotechnology, sc-10599)

was used. Normal goat IgG (Santa Cruz Biotechnology, sc-2028) was used as a control as previously described1. The DNA isolated from input chromatin fragments and from the precipitated chromatin fragments by anti-Meis1 antibody or control IgG was subjected to PCR using primers flanking the consensus Meis1 binding sites on INK4b–ARF–INK4a and p21 promoters: p21-Chip-F 5’GCTACTGCCTCCTCCCAGG-3’

and

p21-Chip-R5’-

TAGGCAATCCTGAGAAACAGAAGC-3’,

INK4b–ARF–INK4a

-ChIP-For5’-

TCTTAGTTTGCCCTCTTACTGG-3’

INK4b–ARF–INK4a

-ChIP-Rev5’-

and

AGCAGTATGTTCCTTCGCTTGG-3’.

List of primers used for real time PCR Gene

Forward Primer

Reverse Primer

GAPDH

GAACCCTAAGGCCAACCGT

ACCGCTCGTTGCCAATAGTGAT

GAAAGAT

G

Meis1

GTTGTCCAAGCCATCACCTT ATCCACTCGTTCAGGAGGAA

p15INK4b (CDKN2B)

CAGTTGGGTTCTGCTCCGT

AGATCCCAACGCCCTGAAC

p16INK4a

GGGTTTCGCCCAACGCCCC

TGCAGCACCACCAGCGTGTCC

GA p21(CDKN1A)

ATCACCAGGATTGGACATG

CGGTGTCAGAGTCTAGGGGA

G



doi:10.1038/nature12054

p19ARF

GTTTTCTTGGTGAAGTTCGT

TCATCACCTGGTCCAGGATTC

GC p18(CDKN2C)

CTCCGGATTTCCAAGTTTCA

GGGGGACCTAGAGCAACTTAC

p27(CDKN1B)

GGGGAACCGTCTGAAACAT

AGTGTCCAGGGATGAGGAAG

T p57(CDKN1C)

TTCTCCTGCGCAGTTCTCTT

CTGAAGGACCAGCCTCTCTC

p19(CDKN2D)

GTCCTGGACATTGGGGCT

AACCGCTTCGGCAAGAC

ANP#1

CACCAAGGGCTTCTTCCTC

CGAGAGCACCTCCATCTCTC

ANP#2

CAGAATCGACTGCCTTTTCC GGGGGTAGGATTGACAGGAT

BNP

ACCCAGGCAGAGTCAGAAA

ACAAGATAGACCGGATCGGA

C

MHY7

GAGCCTTGGATTCTCAAAC

GTGGCTCCGAGAAAGGAAG

G

Meis1 Isoform primers for PCR

Meis1 #1

ATGCCCATTCCACTCATAGGTC

Meis1 #2

TGGCATACTTTGCAGCCCTGG



doi:10.1038/nature12054

Meis1 #3

GCCCGGAGAAGAATAGTGCAGC

Meis1 #4

CTCACACCCATTGGGCCACC

Meis1 #5

CCACTCGTTCAGGAGGAACC

Meis1 #6

TGGTCTATCATGGGCTGCAC

Meis1 #7

TCTTTCCCAAAGTAGCCACC

Hox and Pbx primers Gene

Forward Primer

Reverse Primer

Hoxa1

GAGCTGCTTGGTGGTGAAAT

GCAGACCTTTGACTGGATGA

Hoxa2

CCCTGGATGAAGGAGAAGAA GGTGTACGCGGTTCTCAGAC

Hoxa3

GCTGCCTGGTCATTCAAAGT

Hoxa4

GTCTCCAGTTCCAGGTGCTC GGCCTGCTTCTTCTGCATAC

CTGGACAACTTTGCCAGGAT

Hoxa5

CAGGGTCTGGTAGCGAGTGT

CTCAGCCCCAGATCTACCC

Hoxa6

GTCTGGTAGCGCGTGTAGGT

CCCTGTTTACCCCTGGATG

Hoxa7

CTTCTCCAGTTCCAGCGTCT

AAGCCAGTTTCCGCATCTAC

Hoxa9

GTTCCAGCGTCTGGTGTTTT

ACAATGCCGAGAATGAGAG

Hoxa10

CGTCTGGTGCTTCGTGTAAG

CTCCAGCCCCTTCAGAAAAC

Hoxa11

CAAGCTGAACTAGGCTTGGG

GGGTGTGGTCACTGGAGATT

Hoxa13

GCGGTGTCCATGTACTTGTC

GCTGCCCTACGGCTACTTC

Hoxb1

CACGGCTCAGGTATTTGTTG

GAAGGTCAAGAGAAACCCACC

Hoxb2

GGAACCAGACTTTGACCTGC

CCTACACCAACACGCAACTG

Hoxb3

GGTCATGGAGTGTAAGGCGT

CAGAACCGTCGCATGAAGTA



doi:10.1038/nature12054

Hoxb4

GCGTCAGGTAGCGGTTGTAG

GTCGTCTACCCCTGGATGC

Hoxb5

GGGTCAGGTAGCGATTGAAG

CTCCACAGATATTCCCCTGG

Hoxb6

CAGGGTCTGGTAGCGTGTG

GAGACCGAGGAGCAGAAGTG

Hoxb7

GTCTGGTAGCGCGTGTAGGT

GAGCAGAGGGACTCGGACTT

Hoxb8

CGCTTGCGAGTCAGATAGG

CACAGCTCTTTCCCTGGATG

Hoxb9

TCCAGCGTCTGGTATTTGGT

GAAGCGAGGACAAAGAGAGG

Hoxb13

CCCCTTGCTATAGGGAATGC

AGGTGAACAGAACCCACCAG

Hoxd1

GCTCTGTCAGTTGCTTGGTG

CAGCACTTTCGAGTGGATGA

Hoxd3

ACCAGCTGAGCACTCGTGTA

AGAACAGCTGTGCCACTTCA

Hoxd4

CTCCCTGGGCTGAGACTGT

CCCTGGGAACCACTGTTCT

Hoxd9

GCGTCTGGTATTTGGTGTAGG GCTGAAGGAGGAGGAGAAGC

Hoxd10

TTCTGCCACTCTTTGCAGTG

GGCCTTCCAGAAGACAGGAG

Hoxd11

GGTTGGAGGAGTAGGGGAAA

TGAACGACTTTGACGAGTGC

Hoxd12

GACCAGGAATTCGTTCTCCA

GGTAAACAGTGCCCATGCTC

Hoxd13

CTGCAGTTTGGTGTAAGGCA

AGGATCAGCCACAGGGTTC

Hoxc4

GGGTCAGGTAGCGGTTGTAA

AAAAATTCACGTTAGCACGG

Hoxc5

TTCTCGAGTTCCAGGGTCTG

ATTTACCCGTGGATGACCAA

Hoxc6

CTTCTCCAGTTCCAGGGTCT

TCCAGATTTACCCCTGGATG

Hoxc8

CAAGGTCTGATACCGGCTGT

ATCAGAACTCGTCTCCCAGC

Hoxc9

AATCTGTCTCTGTCGGCTCC

AGTCTGGGCTCCAAAGTCAC

Hoxc10

CTTTCTCCAATTCCAGCGTC

GACACCTCGGATAACGAAGC

Hoxc12

CAACTTCGAATACGGCTTGC

TCAACGAGGGCAATAAGAGC

Hoxc13

CTGGCTGCGTACTCCTTCTC

CTCTGGAAGTCTCCCTTCCC



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Pbx1

GGCTTCATTCTGTGGCAGTT

AGGACATCGGGGACATTTTA

Pbx2

GCCAGAAGCATGTTGTCCAG

TCAAGGAGAAAACTGGCCTT

Pbx3

CAGGACCTGAAACCCCTTCT

GCTCTTCAGCGTCTTGTGTG

Pbx4

CAGTCTTCCCCTTGATCTCG

CATCACCGACCAGAGCCT

Statistical Analysis Results are expressed as mean ± SEM. An unpaired Student’s t test was used to determine statistical significance of all samples. *P