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Journal: Nature Neuroscience

Article Title: Yy1 as molecular link between neuregulin and transcriptional modulation of peripheral myelination.

Corresponding Author:

Patrizia Casaccia

Supplementary Item & Number (add rows as necessary)

Title or Caption

Supplementary Figure 1 Mortality rate and growth curve of Yy1 mutants

Supplementary Figure 2 DRG explant cultures from E13.5 embryos of Yy1 mutants and control mice.

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

Supplementary Figure 4 Conserved potential YY1 binding sites in the promoter and MSE of Egr2.

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

Supplementary Figure 6 Schematic Model of Schwann cell differentiation and the role of YY1 as an important downstream effector of NRG-1 signaling

Supplementary Table 1 List of antibodies

Supplementary Table 2 List of plasmids

Supplementary Table 3 List of primers used in qRT- PCR experiments

Supplementary Table 4 Primers used in qPCR for Egr2 promoter and MSE after ChIP with anti-YY1 antibody

Nature Neuroscience: doi:10.1038/nn.2686

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Yy1 as molecular link between neuregulin and transcriptional modulation of peripheral

myelination.

Ye He1, Jin Young Kim1, Jeffrey Dupree2, Ambika Tewari3, Carmen Melendez-Vasquez3, John Svaren4,

Patrizia Casaccia1*

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

Nature Neuroscience: doi:10.1038/nn.2686

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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 co-

culture from E13.5 transgenic embryos (Yy1flox/flox; Plp-creERT) 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.

Nature Neuroscience: doi:10.1038/nn.2686

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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)

Nature Neuroscience: doi:10.1038/nn.2686

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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.

Nature Neuroscience: doi:10.1038/nn.2686

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

Nature Neuroscience: doi:10.1038/nn.2686

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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.

Nature Neuroscience: doi:10.1038/nn.2686

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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 # 34-6200)

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-13036

NFM: mouse monoclonal; 1:400 for ICC; Upstate#05-744

NICD: mouse monoclonal; 1:500 for WB; Millipore#MAB5352

Phospho-serine: mouse monoclonal; 1:200 for WB, Upstate#05-1000

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 # Ab12132

2 g for ChIP

Nature Neuroscience: doi:10.1038/nn.2686

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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

University19).

Egr2-MSE-luci luciferase reporter driven by 1.3 kb myelin specific enhancer of mouse Egr2 (Dr.

Crabtree, Stanford University19).

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).

Nature Neuroscience: doi:10.1038/nn.2686

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Supplementary Table 3. List of primers used in qRT- PCR experiments

Gene Forward ReverseMouse

CnB1 5’-TATTCGACACAGACGGCAAC-3’ 5’-CGCCTTTGACACTGAACTGA -3’

Cdk4 5’-AACTGATCGGGACATCAAGG -3’ 5’-CAGGCCGCTTAGAAACTGAC -3’

Cyclin D1 5’-CACAACGCACTTTCTTTCCA -3’ 5’-TCCAGAAGGGCTTCAATCTG -3’

Egr1 5’-CCACAACAACAGGGAGACCT-3’ 5’-ACTGAGTGGCGAAGGCTTTA -3

Egr2 5’-CAGTGACTGCCACCCCTTAT-3 5’- TACTTTCGAGGTCGCAGGAT-3’

Gapdh 5’-ACCCAGAAGACTGTGGATGG-3’ 5’-CACATTGGGGGTAGGAACAC-3’

Jagged1 5’-GGAAGTGGAGGAGGATGACA -3’ 5’-GGGCCTTCTCCTCTCTGTCT -3’

Mpz 5’-CTGGTCCAGTGAATGGGTCT -3’ 5’-GTCCCTTGGCATAGTGGAAA -3’

Nab1 5’-TCTATGGGCGATTTGACTCC -3’ 5’-CAGGGCAAAAAGCTCATCTC -3’

Nab2 5’- AGCTTTAGCCCCAAGAGTCC-3’ 5’- CCCTGCTCCAACATCAGATT-3’

Notch1 5’-GTAGCTGCCGAACCAGTAGC -3’ 5’-CAAAAGGCCAGAAAGAGCTG -3’

Cdkn1a (p21)

5’-CGGTGGAACTTTGACTTCGT -3’ 5’-CAGGGCAGAGGAAGTACTGG -3’

Plp 5’-CCCACCCCTATCCGCTAGTT -3’ 5’-AGGAAAAAAAGCACCATTGTG-3’

Pmp22 5’- TCCTCATCAGTGAGCGAATG-3’ 5’-GCTGGCAGAAGAACAGGAAC -3’

Pou3f 5’-CAAGCAGTTCAAGCAACGAC-3’ 5’-TGGTCTGCGAGAACACGTTA -3’

Sox2 5’-CACAACTCGGAGATCAGCAA -3 5’-CTCCGGGAAGCGTGTACTTA -3’

Sox11 5’-GCTGGAAGATGCTGAAGGAC-3’ 5’-GTCGGGATAATCAGCCATGT -3’

Rat

Egr1 5’-CAGGAGTGATGAACGCAAGA -3’ 5’-AGCCCGGAGAGGAGTAAGAG -3’

Egr2 5’-GCTTCAACCACAGCCTCTTC -3’ 5’-TGGGACATGGTACACACACC -3’

Nab1 5’-CACCACTGAAAGCGAGCATA -3’ 5’-GGCTTCACTGCTTTCCTTTG-3’

Nab2 5’-CGACAGCCAGAAAGAAGAGG -3’ 5’-GCTAAGCTGTTTGCCCTCAC -3’

Yy1 5’-GAGCAGATCATTGGGGAGAA-3’ 5’-AGGGAGTTTCTTGCCTGTCA -3’

Nature Neuroscience: doi:10.1038/nn.2686

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Supplementary Table 4. Primers used in qPCR for Egr2 promoter and MSE after ChIP with anti-

YY1 antibody

Region position Forward Reversepromoter I -965 / -882 5’-GTCCAAGCTTCCATCTGGTC -3’ 5’-CTCCAGCTGCTGCACAAAG -3’

II -741 / -584 5’-ACAAACAAACAGCCCAGACC -3’ 5’-AAAAATTACCCGCACTCACG -3’

III -373 / -317 5’-AAGTCTCGGAGAACCGGAAT -3’ 5’-GCAGCGATGGTAGCTCTGTC -3’

IV -148 / -97 5’- CCAAGCCCGTATGCAAAT-3’ 5’- CTGGGCCTGGTAGCTTTTG-3’

MSE

V 108 / 286 5’- CCAGCCTGTTCCTGTTCATT-3’ 5’- CTGGGAGGGACAGAATTCAC-3’

VI 547 / 738 5’- CGGACTTGCATTGCATAGAA-3’ 5’-CCCGTACATCCACTCACACA -3’

VII 797 / 847 5’-TCCTGACCAGAAAGATTGTTATTGAG -3’ 5’-TGCAGGATTTCAGCTTTGTGA-3’

VIII 821 / 1071 5’-AGCCCTTCACAAAGCTGAAA -3’ 5’-GGATTTCATCCTTGGCTTCA -3’

The sequence of the Egr2 1.3 kb promoter (chr20: 21889477-21888178) and of the 1.3 kb MSE

(chr20: 21849014-21847714), used for the design of the ChIP primers, were obtained from ENSEMBL

according to the homology of human or mouse sequences1,2. 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, 155-66 (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, 2749-57 (1990).

3. Shrivastava, A. & Calame, K. An analysis of genes regulated by the multi-functional transcriptional regulator Yin Yang-1. Nucleic Acids Res 22, 5151-5 (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, 4353-62 (1995).

Nature Neuroscience: doi:10.1038/nn.2686