Journal: Nature Neuroscience

Transcription

1 Journal: Nature Neuroscience Article Title: Corresponding Author: Yy1 as molecular link between neuregulin and transcriptional modulation of peripheral myelination. Patrizia Casaccia Supplementary Item & Number (add rows as necessary) Supplementary Figure 1 Supplementary Figure 2 Supplementary Figure 3 Supplementary Figure 4 Supplementary Figure 5 Supplementary Figure 6 Supplementary Table 1 Supplementary Table 2 Supplementary Table 3 Supplementary Table 4 Title or Caption Mortality rate and growth curve of Yy1 mutants DRG explant cultures from E13.5 embryos of Yy1 mutants and control mice. Proliferation of Schwann cells in Yy1 mutant DRG co-cultures is only moderately increased compared to controls after 21 days in vitro Conserved potential YY1 binding sites in the promoter and MSE of Egr2. Uncropped figures of the western blots shown in the indicated panels Schematic Model of Schwann cell differentiation and the role of YY1 as an important downstream effector of NRG-1 signaling List of antibodies List of plasmids List of primers used in qrt- PCR experiments Primers used in qpcr for Egr2 promoter and MSE after ChIP with anti-yy1 antibody 1

2 Yy1 as molecular link between neuregulin and transcriptional modulation of peripheral myelination. Ye He 1, Jin Young Kim 1, Jeffrey Dupree 2, Ambika Tewari 3, Carmen Melendez-Vasquez 3, John Svaren 4, Patrizia Casaccia 1* Supplementary Figure 1. Mortality rate and growth curve of Yy1 mutant mice. (a) The number of surviving Yy1 mutant mice at each time point and is shown in a bar graph on top of each bar. (b) The body weight chart during postnatal development shows a slower growth curve of Yy1 mutants compared to controls. At least 5 animals were analyzed for each genotype at each time point with the exception of p64 when only two Yy1 mutant mice were available 2

3 Supplementary Figure 2. DRG explant cultures from E13.5 embryos of Yy1 mutants and control mice. (a) Normal axonal outgrowth and Schwann cell migration in the cultures from Yy1 mutants, examined by bright field at day 1, 3 and 5 and by immunofluorescence and confocal microscopy at day 14 (staining for neurofilament medium chain NFM in green and nuclear DAPI in blue). Scale bar, 500 µm for bright field pictures and 50 µm for immunofluorescence micrographs. (b) In vitro myelination assay in cultures from DRGs of Yy1 and control embryos (E13.5) kept in myelinating conditions in vitro for 14 and 21 days and then immunostained for MBP (green). Note the fewer myelin segments in cultures from yy1 mutants compared to controls. Scale bar, 200 m. (c) DRG neuron-schwann cell coculture from E13.5 transgenic embryos (Yy1 flox/flox ; Plp-creER T ) treated with 4OH-tamoxifen (4OH-TM) for 48 h and stained for YY1 (red) and Cre (green). Arrowheads indicate Cre + Schwann cells with excised yy1, while arrows indicate Cre fibroblasts, retaining YY1 expression. Scale bar, 20 m. 3

4 Supplementary Figure 3. Proliferation of Schwann cells in Yy1 mutant DRG co-cultures is only moderately increased compared to controls after 21 days in vitro. (a) DRG neuron-schwann cell co-cultures from E13.5 Yy1 and from control embryos were maintained in myelinating conditions for 7 or 9 days and then pulsed with 10 µm BrdU for 6 h, immediately fixed and stained with antibodies specific for BrdU (green) and DAPI counterstaining (blue). Scale bar, 50µm. (b) The proliferative rate of Schwann cells was calculated as the percentage of BrdU+ cells relative to DAPI + nuclei. Note that the number of proliferating cells in Yy1 mutant cultures was greater than controls only if cells were maintained for at least 21 days in culture. Error bar, s.d., *P<0.05.(n=3) 4

5 Supplementary Figure 4. Conserved potential YY1 binding sites in the promoter and MSE of Egr2. Rat (R), mouse (M) and human (H) DNA sequences for Egr2 promoter and MSE were aligned and the potential YY1 binding sites that are conserved between species are indicated. Green denotes forward orientation, pink indicates reverse orientation, and yellow marks the overlap between two adjacent binding sites in opposite directions. 5

6 Supplementary Figure 5. Uncropped figures of the western blots shown in the indicated panels 6

7 Supplementary Figure 6. Schematic Model of Schwann cell differentiation and the role of YY1 as an important downstream effector of NRG-1 signaling. Schematic model of the intracellular events regulated by YY1 in Schwann cells during postnatal development. Solid lines represent previously established pathways and dashed lines indicate YY1-dependent pathways. 7

8 Supplementary Table 1. List of antibodies BrdU: mouse monoclonal; 1:100 for IHC and ICC; Dako Cytomation Caspr: rabbit polyclonal; 1:1,000 for IHC; from Dr. Martin Grumet, Rutgers University Cre: rabbit polyclonal; 1:3,000 for IHC and ICC; Covance # PRB-106C CNPase: mouse monoclonal; 1:500 for WB; Sternberger Monoclonals, Inc # SMI22 EGR2: rabbit polyclonal; 1:500 for IHC and WB; Covance # PRB-236B MAG: rabbit polyclonal; 1:100 for ICC; ZYMED # ) MBP: mouse monoclonal; 1:1,000 for IHC and WB; Sternberger Monoclonals # SMI99 MBP: Chicken antibody 1:400 for ICC; Aves Labs MPZ : Chicken antibody 1:400 for ICC; Aves Labs NFATc4: rabbit polyclonal; 1:150 for WB; Santa Cruz#sc NFM: mouse monoclonal; 1:400 for ICC; Upstate# NICD: mouse monoclonal; 1:500 for WB; Millipore#MAB5352 Phospho-serine: mouse monoclonal; 1:200 for WB, Upstate# POU3F1: rabbit polyclonal; 1:300 for IHC; from Dr. Dies Meijer, Erasmus University S100: mouse monoclonal; 1:200 for ICC; Sigma# S2532 Tubulin, alpha: mouse monoclonal; 1:5,000 for WB; Sigma#T9026 YY1 : mouse monoclonal, 1:100 for IHC and 1:200 for WB, Santa Cruz#sc-7341; rabbit polyclonal, 1:100 for IHC and 1:1,000 for WB, Santa Cruz# sc-1703; rabbit polyclonal, 1:500 for ICC and 1:1,000 for WB, Abcam # Ab g for ChIP 8

9 Supplementary Table 2. List of plasmids pcx-egfp vector containing EGFP gene under IRES pcx-fl-yy1 obtained by subcloning Human full length YY1-flag tagged (Dr. Seto, University of South Florida) into pcx-egfp pcx-yy1-phosphomutant (S118, S184 and S247 were changed to alanine using Stratagene's QuikChange II Site Directed Mutagenesis Kit and confirmed by DNA sequencing. pcx-egr2-ha-tagged by subcloning Egr2 30 into pcx-egfp vector. Egr2-luci luciferase reporter driven by1.3 kb promoter of mouse Egr2 (Dr. Crabtree, Stanford University 19 ). Egr2-MSE-luci luciferase reporter driven by 1.3 kb myelin specific enhancer of mouse Egr2 (Dr. Crabtree, Stanford University 19 ). YY1 TransLucent Reporter, a luciferase reporter under the control of an enhancer element containing multiple YY1 binding sequences upstream of the minimal Herpes Simplex thymidine kinase promoter (Panomics, CA) SureSilencing shrna plasmids for rat yy1 with puromycin resistance (SuperArray). 9

10 Supplementary Table 3. List of primers used in qrt- PCR experiments Gene Forward Reverse Mouse CnB1 5 -TATTCGACACAGACGGCAAC-3 5 -CGCCTTTGACACTGAACTGA -3 Cdk4 5 -AACTGATCGGGACATCAAGG CAGGCCGCTTAGAAACTGAC -3 Cyclin D1 5 -CACAACGCACTTTCTTTCCA TCCAGAAGGGCTTCAATCTG -3 Egr1 5 -CCACAACAACAGGGAGACCT-3 5 -ACTGAGTGGCGAAGGCTTTA -3 Egr2 5 -CAGTGACTGCCACCCCTTAT TACTTTCGAGGTCGCAGGAT-3 Gapdh 5 -ACCCAGAAGACTGTGGATGG-3 5 -CACATTGGGGGTAGGAACAC-3 Jagged1 5 -GGAAGTGGAGGAGGATGACA GGGCCTTCTCCTCTCTGTCT -3 Mpz 5 -CTGGTCCAGTGAATGGGTCT GTCCCTTGGCATAGTGGAAA -3 Nab1 5 -TCTATGGGCGATTTGACTCC CAGGGCAAAAAGCTCATCTC -3 Nab2 5 - AGCTTTAGCCCCAAGAGTCC CCCTGCTCCAACATCAGATT-3 Notch1 5 -GTAGCTGCCGAACCAGTAGC CAAAAGGCCAGAAAGAGCTG -3 Cdkn1a 5 -CGGTGGAACTTTGACTTCGT CAGGGCAGAGGAAGTACTGG -3 (p21) Plp 5 -CCCACCCCTATCCGCTAGTT AGGAAAAAAAGCACCATTGTG-3 Pmp TCCTCATCAGTGAGCGAATG-3 5 -GCTGGCAGAAGAACAGGAAC -3 Pou3f 5 -CAAGCAGTTCAAGCAACGAC-3 5 -TGGTCTGCGAGAACACGTTA -3 Sox2 5 -CACAACTCGGAGATCAGCAA CTCCGGGAAGCGTGTACTTA -3 Sox11 5 -GCTGGAAGATGCTGAAGGAC-3 5 -GTCGGGATAATCAGCCATGT -3 Rat Egr1 5 -CAGGAGTGATGAACGCAAGA AGCCCGGAGAGGAGTAAGAG -3 Egr2 5 -GCTTCAACCACAGCCTCTTC TGGGACATGGTACACACACC -3 Nab1 5 -CACCACTGAAAGCGAGCATA GGCTTCACTGCTTTCCTTTG-3 Nab2 5 -CGACAGCCAGAAAGAAGAGG GCTAAGCTGTTTGCCCTCAC -3 Yy1 5 -GAGCAGATCATTGGGGAGAA-3 5 -AGGGAGTTTCTTGCCTGTCA -3 10

11 Supplementary Table 4. Primers used in qpcr for Egr2 promoter and MSE after ChIP with anti- YY1 antibody Region position Forward Reverse promoter I -965 / GTCCAAGCTTCCATCTGGTC CTCCAGCTGCTGCACAAAG -3 II -741 / ACAAACAAACAGCCCAGACC AAAAATTACCCGCACTCACG -3 III -373 / AAGTCTCGGAGAACCGGAAT GCAGCGATGGTAGCTCTGTC -3 IV -148 / CCAAGCCCGTATGCAAAT CTGGGCCTGGTAGCTTTTG-3 MSE V 108 / CCAGCCTGTTCCTGTTCATT CTGGGAGGGACAGAATTCAC-3 VI 547 / CGGACTTGCATTGCATAGAA-3 5 -CCCGTACATCCACTCACACA -3 VII 797 / TCCTGACCAGAAAGATTGTTATTGAG TGCAGGATTTCAGCTTTGTGA-3 VIII 821 / AGCCCTTCACAAAGCTGAAA GGATTTCATCCTTGGCTTCA -3 The sequence of the Egr2 1.3 kb promoter (chr20: ) and of the 1.3 kb MSE (chr20: ), used for the design of the ChIP primers, were obtained from ENSEMBL according to the homology of human or mouse sequences 1,2. The presence of YY1 consensus binding sites (C/g/a)(G/t)(C/t/a)CATN(T/a)(T/g/c) 3,4 in these sequences was determined by using TESS (Transcription Element Search System) and Genomatix software. 1. Ghislain, J. et al. Characterisation of cis-acting sequences reveals a biphasic, axon-dependent regulation of Krox20 during Schwann cell development. Development 129, (2002). 2. Rangnekar, V.M., Aplin, A.C. & Sukhatme, V.P. The serum and TPA responsive promoter and intron-exon structure of EGR2, a human early growth response gene encoding a zinc finger protein. Nucleic Acids Res 18, (1990). 3. Shrivastava, A. & Calame, K. An analysis of genes regulated by the multi-functional transcriptional regulator Yin Yang-1. Nucleic Acids Res 22, (1994). 4. Yant, S.R. et al. High affinity YY1 binding motifs: identification of two core types (ACAT and CCAT) and distribution of potential binding sites within the human beta globin cluster. Nucleic Acids Res 23, (1995). 11