Coactivator-associated arginine methyltransferase 1 (CARM1) is a positive regulator of the Cyclin E1 gene -- El Messaoudi et al. 103 (36): 13351 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

Messaoudi et al. 10.1073/pnas.0605692103.

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Supporting Figure 6
Supporting Figure 7
Supporting Figure 8
Supporting Figure 9
Supporting Text

Supporting Figure 6

Fig. 6. CARM1 inactivation impacts on the level of expression of other E2F/DP-regulated genes. Exponentially growing human U2Os cells were transfected with either scrambled control siRNA or siRNA directed against human hCARM1, analyzed and 48 h later for mRNA contents of indicated genes by quantitative RT-PCR (Q-RTPCR).

Supporting Figure 7 Fig. 7.
G₀-G₁/S activation of the mDHFR gene coincides with the CARM1/PRMT4-stimulated arginine-methylation of nucleosomal histones located at the CCNE1 promoter. ChIP analyses of the mouse DHFR promoter with antibodies against CARM1/PRMT4 and modified histone residues. ChIP analyses were performed as described in Fig. 1. PCR amplifications on gradually diluted (1, 0.1, 0.01) input chromatin confirmed that equal amounts of materials were used.

Supporting Figure 8 Fig. 8.
ACTR protein depletion in shRNA-expressing cells. Total protein extracts of U2Os cells expressing either shRNA directed against hACTR or scrambled control shRNA were probed for the presence of actin or ACTR by immunoblotting.

Supporting Figure 9 Fig. 9.
CARM1 inactivation in MEFs impacts cell cycle reentry after quiescence but have little effect on their capacity to grow exponentially in high serum. (A) Growth curve of exponentially growing CARM1^–/– and control (WT, CARM1^–/+) cells maintained in high FCS. (B) G₀-S phase progression is delayed in CARM1^–/– MEFs. CARM1^–/– or control (WT, CARM1^–/+). MEFs, synchronized in G₀ by serum depletion, were serum restimulated and, at indicated times, were scored for BrdU incorporation (S-phase entry) by indirect immunofluorescence. The diagram is the average of three independent experiments

Supporting Matrials and Methods SiRNA-Mediated Depletions of Human ACTR and CARM1.
All synthetic oligonucleotides were ordered from Eurogentec (Brussels, Belgium). hACTRs, 5'-AACUUGGAUCCACUGGCCAdTdT; hACTRas, 5'-UGGCCAGUGGAUCCAAGUUdTdT (1); hCARM1s, 5'-CAGCUCUACAUGGAGCAGUdTdT; hCARM1as, 5'-ACUGCUCCAUGUAGAGCUGdTdT (2). siRNAs of corresponding scrambled sequences were used as controls. shRNA-mediated depletions of human ACTR/AIB1 were obtained as described previously (1) Primers Used for CHIP.
mCE12s, 5'-TGAGGGGCTCGCAGCCCTCG; mCE12as, 5'-CCCGGCTTCGAGCGGGACAT; hCEs, 5'-TGATTCCCCGTCCCTGCGCCT; hCEas, 5'-CGCTGGCGGCCGAGCCGGCTG; hCD1s, 5'-TCTGCCCCTCGCTGCTCCC; hCD1as, 5'-GACAGCCCTCTGGAGGCTCC; mDHFR s, 5'-GCC TAA GCT GCG CAA GTG GT; mDHFR s, 5'-as GTC TCC GTT CTT GCC AAT CC. PCR parameters are available upon request. Primers Used for Quantitative RT-PCR.
Reverse transcription was performed with M-MLV reverse transcriptase (Invitrogen, Carlsbad, CA) and quantitative PCR with the following primers: S26: s5'-GAA CAT TGT AGA AGC CGT TGC TGT C, as5'-AAC CTT GCT ATG GAT GGC ACA GCT C; RPLPO, s5'-cga cct gga agt cca act ac, as5'-ATC TGC TGC ATC TGC TTG. 28S: S28s, 5'-GAACATTGTAGAAGCCGTTGCTGCC; S28as, 5'-AACCTTGCTATGGATGGCACAGCTC. Mouse CCNE1: mCEs, 5'-CCTTAAGTGGCGTCTAAGCCCT; mCEas, 5'-CCCGTGTCGTTGACATAGGC. human CCNE1: hCEs, 5'-GAGCCAGCCTTGGGACAATA; hCEas, 5'-CGGTC ATCATCTTCTTTGTCAGG. Human ACTR/AIB1: hACTRs, 5'-TCCAATATGCAT GGGTCACTGT; hACTRas, 5'-TGGCTACCTCAGCTGGTGAATT. Mouse ACTR: mACTRs, 5'-CCCCGTGTATCGTTTCTCCTT; mACTRas, 5'-GTCTGCGCACTCACAATAGTTC, human CARM1 : hCARM1s/5'-5GCCATCCTGCAAAACCACA; hCARM1as, 5'-CGGCAAAAAACGACAGGATC. Human b-actin : hactins, 5'-GCG GGA AAT CGT GGC TGA CAT T; hactinas, 5'-GAT GGA GGT GAA GGT AGT TTC GT. Proliferation Assays
: MEFs were seeded in triplicate, in 96-well plates (2 ´ 10³ cells per well) and analyzed at various time points with CellTiter96 AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI), which was used according to manufacturer’s instructions. MEFs entry into S phase following quiescence (Fig. 9) was monitored on cells grown on coverslips and incubated with BrdU (Sigma, St. Quentin Fallavier, France) during 90 min before fixation/permeabilization and probing with a-BrdU Ab (Becton Dickinson, Erembodegem, Belgium) as described previously (3).

1. Louie, M. C., Zou, J. X., Rabinovich, A. & Chen, H. W. (2004) Mol. Cell Biol. 24, 5157–5171.

2. Chevillard-Briet. M., Trouche, D. & Vandel, L. (2002) EMBO J. 21, 5457–5466.

3. Le Cam, L., Polanowska, J., Fabbrizio, E., Olivier, M., Philips, A., Ng Eaton, E., Classon, M., Geng, Y. & Sardet, C. (1999) EMBO J. 18, 1878–1890.