Supplementary Material For: Yamaguchi, Cubizolles Et Al

Yamaguchi, Cubizolles, …..& Matthias, Page 1 of 6

Supplementary material for: Yamaguchi, Cubizolles et al.“Histone deacetylases 1 and 2 act in concert to promote the G1 to S progression“

Supplementary Figure 1: Generation of HDAC1 and HDAC2 conditional mice and MEFs

(A) At the top a schematic view of the HDAC1 protein is presented with the main functional domains indicated. The sequence of exon 6 is indicated for both HDAC1 and HDAC2 and essential residues for catalytic activity are highlighted by an asterisk. Underneath, the HDAC1 and HDAC2 gene loci are shown as well as the different modified alleles created. Modified alleles are represented as follows: T, targeted allele; F, floxed allele, , deleted allele. Black and grey boxes represent the coding exons, white rectangular boxes depict the Neomycin resistance gene cassette, white triangles symbolise the loxP sequences and S represent SacI restriction sites. (B) The recombination of HDAC1 alleles in MEFs is detected by PCR in presence or absence of Tamoxifen, as indicated.

Supplementary Figure 2: Elevated histone acetylation in MEFs lacking HDAC1 or 2

(A) Similar nuclear distribution of HDAC1 and 2. Cells were stained with an antibody against HDAC1 or 2 (green staining) and DNA was counterstained with DAPI (blue staining). (B) Microscopic analysis of the acetylation level in the different cells. Cells were stained with an antibody against acetylated H4 (green staining) and DNA was counterstained with DAPI (blue staining). The pictures of acetylated H4 stainings all have the same settings, allowing to compare the difference in signal intensity. In the case of WT cells, a longer exposed picture is also presented, allowing to see the level of acetylation.

Supplementary Figure 3: Tamoxifen does not influence cellular growth or CDKi expression

(A) HDAC1F/FMEFs were treated with Tamoxifen and monitored for proliferation. Cells were passaged every 3 or 4 days, total cell number was counted and cells were reseeded at 5x105 cells per 100-mm dish. (B) Protein extracts were prepared from WT MEF treated or not with Tamoxifen and analysed by western blot with antibodies against p21(cdkn1a), p57(cdkn1c) and actin, as indicated.

Supplementary Figure 4: HDAC1/2 double KO MEFs show the same phenotype as HDAC1/2 KO/Kd MEFs

(A) Analysis of apoptosis by annexin V staining. MEF cells from WT, HDAC1 KO and HDAC2 Kd were kept in culture and collected for apoptosis assays after 11 days of treatment with Tamoxifen. After fixation, staining with anexin V antibody, cells were analysed by flow cytometry. The bar graph represents the average of 3 independent experiments (± SD). (B) HDAC1/2F/F MEFs were infected with a retrovirus expressing Cre-ERT2 and puromycin resistance. Cells were passaged every 3 or 4 days, total cell number was counted and cells were reseeded at 5x105 cells per 100-mm dish. Each growth curve is the average (± SD) of two independent MEF cell lines. Solid black line, cells without Tamoxifen (HDAC1/2F/F cells); dashed line, cells treated with Tamoxifen (HDAC1/2-/- cells). (C) Cell cycle analysis by flow cytometry in either HDAC1/2F/F or HDAC1/2-/- cells. After a pulse of BrdU cells were collected and fixed with ethanol. Following staining with an antibody against BrdU and 7-AAD, cells were analysed by flow cytometry. Representative profiles are shown for each genotype and the percentage of cells in the different cell cycle phases are indicated beside each panel. (D) Protein extracts were prepared from WT and HDAC1/2-/- cells and analysed by western blot with antibodies against HDAC1, HDAC2, p21(cdkn1a), p57(cdkn1c) and actin, as indicated.

Supplementary Figure 5: Gene expression profiles.

(A) Gene expression profiles. Supervised hierarchical clustering was performed using comparative expression values of the gene lists indicated above each dendrogram (for gene lists, see Suppl. Table 2). In all cases, the expression value for the WT cells was used to normalize expression levels. (B) Venn diagrams based on the expression changes in each genotype.

Supplementary Figure 6: Specific inhibition of p21 and p57 expression leads to partial release of G1 arrest in HDAC1/2 mutant MEFs

(A) Cell cycle analysis by flow cytometry using MEFs of different backgrounds. The lentivirus pLKO.1 vectors expressing shRNA against GFP, p21 (Cdkn1a) and p57 (Cdkn1c) were obtained from Open Biosystems and virus particles were prepared. HDAC1/2F/F MEFs carrying Cre-ERT2 were infected with lentiviruses containing either shRNA vectors against GFP or 10 different set of vectors against p21 and p57. After infection, cells were cultured in the presence (lower panels) or absence (upper panels) of Tamoxifen for 6 days. Following a 2 hour pulse of BrdU cells were collected and fixed. After staining with an antibody against BrdU and 7-AAD, cells were analyzed by flow cytometry. Representative dot plots are shown for each genotype and the percentage of cells in the different cell cycle phases are indicated beside each panel. One representative experiment is presented. (B) Relative expression of the p21,p57, HDAC1 and HDAC2 genes in the MEFs used for the cell cycle analysis. RNA was extracted from the different cells, as indicated, and was used for quantitative RT-PCR assays. The expression level in wild type cells was set to 100%, after normalization for the expression of Hprt1. The average (± SD) from three independent RNA extractions obtained in two independent lentivirus infections is presented.

Supplementary Figure 7: HDAC1/2 double mutant mature B cells fail to proliferate in vitro

(A) Mature B cells lacking HDAC1 and 2 fail to proliferate in vitro. Splenic B cells were isolated from double KO or control mice, stained with CFSE and induced to proliferate in vitro by addition of anti-IgM Fab fragments. The distribution of CFSE-positive cells was analyzed by FACS after 72 hrs stimulation. (B) Apoptosis induction in stimulated cells. Cells were stained with CFSE, stimulated for 72 hrs as above and analyzed by annexin V staining.

Supplementary Figure 8: HDAC1 and HDAC2 are engaged in specific functions

(A) Immunoprecipitation of HDAC1 or HDAC2 was performed with extracts of cells of the indicated origin (WT, HDAC1 KO or HDAC2 Kd). Immunoprecipitates were analysed by western blot with antibodies against HDAC1, HDAC2, Co-Rest and Sin3A as indicated. (B) Quantification of free HDAC1 and HDAC2 in WT MEFs. HDAC1 and HDAC2 immunoprecipitation were performed under condition of near depletion. The Input and Supernatant were analyzed by western blot using the Odyssey Infrared Imaging System with antibodies against HDAC1 and HDAC2. The quantification of band intensities is presented as a table underneath and allows to estimate the amount of HDAC1 remaining after depletion of HDAC2 (and vice versa) and consequently the relative percentage of free HDAC1 or HDAC2. (C) Analysis of Histone modifications on the p21 and p57 promoters by chromatin immunoprecipitation assays. ChIP was performed for either H3K14ac or H4K8ac in WT, HDAC1 KO and HDAC2 Kd cells, as indicated. Enrichment was assessed by quantitative PCR for the p21 and p57 promoters normalized to the amplification obtained with intergenic-region and H4 or H3 signal in each sample. Results presented are one representative experiment from three independent experiments with each PCR amplification performed in triplicate.

Supplementary Table 1:

List of genes found disregulated in genome-wide expression analysis in HDAC1 KO, HDAC2 Kd and HDAC1/2 KO/Kd cells. Threshold was set up at 2 fold changes compared to WT cells with a p Value smaller or equal to 0.05.

Supplementary Table 2:

The sequence of the primers used is indicated.

Supplementary Materials and Methods

Generation of HDAC1 and 2 conditional knockout mice and animal experiment

Each exon 6 of mouse HDAC1 and 2 was flanked by a single loxP site and a cassette expressing the neomycin resistance gene and thymidine kinase gene flanked by two loxP sites. E14 ES cells (129/Ola strain) were electroporated with either the targeting vector for mouse HDAC1 or 2 and correctly targeted ES clones were identified by PCR, sequencing and Southern blot analysis (data not shown and Suppl. Fig. 1A). Mice carrying a targeted HDAC1 or HDAC2 locus were generated by standard procedures and crossed to mice expressing cre recombinase under the control of a germline specific promoter (EIIa-Cre or Meox2-cre (MORE) mice (Lakso et al. 1996; Tallquist and Soriano 2000)). Genotyping of the offspring allowed to identify mice having lost the NeoR cassette, but retaining the loxP sites flanking exon 6 (i.e. the floxed allele, +/HDAC1F or +/HDAC2F mice), as well as mice having deleted exon 6 and therefore carrying the knockout allele (+/HDAC1, +/HDAC2 mice). Intercrossing allowed to generate mice homozygous for the floxed alleles of HDAC1 or HDAC2; in both cases these mice appeared indistinguishable from their wild type (WT) littermates and were found to produce normal levels of HDAC1 or HDAC2 in several organs examined (data not shown). HDAC1 complete knockout mice were generated by intercrossing HDAC1+/-We found that HDAC1-/-mice are embryonic lethal before E10.5 (data not shown) as described earlier (Lagger et al. 2002). HDAC2 complete knockout mice were generated in the same way. HDAC2-/- mice show partial perinatal lethality (data not shown). Surviving HDAC2-/- mice were initially about 50% smaller than wild-type and heterozygote littermates but catch up with time (data not shown).

For all analyses, flox allele littermates of the same sex were used as controls. All experiments were done using mixed background (129/Ola and C57BL/6 mixed strain) mice that were 6 to 12 weeks old. For the generation of B cell specific KO mouse, we used mb1-cre(Hobeika et al. 2006) or cd23-cre(Kwon et al. 2008) transgenic mice. Animal experiments were carried out according to regulations effective in the Kanton of Basel-Stadt, Switzerland, and were in compliance with all internal FMI regulations.

PCR analysis of deleted HDAC1 and 2 alleles

DNA was extracted by overnight proteinase K digestion, phenol/chloroform extraction and ethanol precipitation. Primers used for the PCR detection of HDAC1 and 2 genotyping are shown in Suppl. Table 1. Amplicons from PCR reactions were separated on 1.5% agarose gel and visualized by ethidium bromide.

Cells, cell culture

Primary embryonic fibroblasts were harvested from 13.5 d.p.c. embryos and prepared as described (Zhang et al. 2008). Briefly, head, tail and fetal liver were removed and the remainder of the embryo was rinsed in PBS. Fetal tissue was treated with trypsin/EDTA and incubated for 30 min at 37°C and subsequently dissociated in medium. After removal of large tissue clamps, the remaining cells were plated on 100 mm dishes. After 48 h, confluent cultures were frozen down. These cells were considered as being passage 1. For continuous culturing, MEF cultures were grown at 37°C in Dulbecco’s-modified Eagle’s medium supplemented with 10% heat-inactivated FBS and split 1:3. For proliferation curve determinations, cells were seeded at 5 x 105 per 100mm dish. Cell numbers and viability were determined at each passage by using a Vi-Cell XR counter (Beckman coulter) and re-seeded at 5 x 105 per dish.

Affymetrix expression analysis

Cells were collected around 80% of confluency after 5 days of Tamoxifen treatment. RNA for the 4 different backgrounds (WT, HDAC1 KO, HDAC2Kd and HDAC1/2 KO/Kd) from three independent Tamoxifen inductions was extracted with RNeasy Minikit (Qiagen) according to the manufacturer’s protocol. As control for Tamoxifen effect on transcription, WT MEF cells (HDAC1+/+) were treated with Tamoxifen and process in parallel.

Biotinylated cRNA probes were generated using the SuperScript Choice cDNA sythesis kit from Stratagene and the Affymetrix IVT kit. The labeled cRNA probes were hybridized with the MOE430 v2.0 Affymetrix GeneChips® and signals were detected according to manufacturer’s instructions. All arrays yielded hybridization signals of comparable intensity and quality. The BioConductor Affymetrix package of the R software was used to import the CEL files from the Affymetrix Mouse Genome 430 2.0 Array.

Complete datasets are available at GEO database (accession number XXXXXX).

References

Lakso, M., Pichel, J.G., Gorman, J.R., Sauer, B., Okamoto, Y., Lee, E., Alt, F.W., and Westphal, H. 1996. Efficient in vivo manipulation of mouse genomic sequences at the zygote stage. Proc Natl Acad Sci U S A93(12): 5860-5865.

Tallquist, M.D. and Soriano, P. 2000. Epiblast-restricted Cre expression in MORE mice: a tool to distinguish embryonic vs. extra-embryonic gene function. Genesis26(2): 113-115.

Zhang, Y., Kwon, S., Yamaguchi, T., Cubizolles, F., Rousseaux, S., Kneissel, M., Cao, C., Li, N., Cheng, H.L., Chua, K. et al. 2008. Mice lacking histone deacetylase 6 have hyperacetylated tubulin but are viable and develop normally. Mol Cell Biol28(5): 1688-1701.