SUPPLEMENTAL MATERIAL

Title:

SETDB1 HISTONE METHYLTRANSFERASE REGULATES MOOD-RELATED

BEHAVIORS AND EXPRESSION OF THE NMDA RECEPTOR SUBUNIT NR2B

Yan Jiang1,2, Mira Jakovcevski1, Rahul Bharadwaj1,2, Caroline Connor1,2, Frederick A. Schroeder1, Cong L. Lin1 , Juerg Straubhaar3, Gilles Martin1 and Schahram Akbarian1

1Brudnick Neuropsychiatric Research Institute, Department of Psychiatry, 2Graduate School of Biomedical Sciences, 3Program in Molecular Medicine, University of Massachusetts Medical School, 303 Belmont Street, 01604, Worcester, MA

This file includes:

Supplemental Figures and Tables

Figure S1. Global H3K9 methylation in CK-Setdb1 brain.

Figure S2. Agarose gel images of PCR product from

conformation capture (3C).

Figure S3. General neurological functions and novelty- and anxiety-related

behaviors in CK-Setdb1 and littermate control mice.

Table S1. Primer list.

Table S2. mycSetdb1 occupancies across 6, 8 and 16

There isnodifferencebetweengenotypes(Mann-Whitneytest). of CK-Setdb1(Set)andwiltype(w)mice.anti-GAPDHwasusedasloadingcontrol.N=6/group. showing anti-H3K9me3signalfrom(A)prefontalcortex,(B)striatum,and(C)hippocampus Supplemental FigureS1:GlobalH3K9methylationin A H3K9me3 GAPDH

H3K9me3/GAPDH 0.0 1.0 1.5 2.0 0.5 e e w Set w Set e w Set B H3K9me3 GAPDH

H3K9me3/GAPDH 0.0 1.0 1.5 0.5 e e w Set w Set e w Set CK-Setdb1 brain.Immunoblotsandbargraphs C H3K9me3 GAPDH

H3K9me3/GAPDH 0.0 1.0 1.5 2.0 0.5 e e w Set w Set e w Set BAC 3C BAC 3C + + + + + + T4 ligase + + -

Supplemental Figure S2: Chromosome conformation capture (3C). Representative gel images showing PCR products from primers 3 and 10 that represent the interaction between Grin2b 5’ end and 3rd intron close to Setdb1-target site (Fig. 4Ba). DNA templates from Bacterial Artifical Chromosome (BAC) and 3C libraries (3C) treated with (+) or without (-) T4 ligase. A B C Weight Rotarod Locomotion

3 40 300 x 10 4 * 200 20 2 100 Weight (g) Weight Latency (sec) Total activity Total 0 0 0

CK-Setdb1 wildtype

D E Light/dark exploration Elevated plus maze Supplemental Figure S 3: General neurological functions and Tdark Tlight Tclose Topen novelty- and anxiety-related behaviors are not altered in CK- 550 Setdb1 mice. (A) Body weight, (B) Rotarod performance and (C) novelty-induced locomotor activity, and performance in two 200 anxiety-related paradigms, the (D) light/dark box and (E) elevated 450 plus maze are shown for adult CK-Setdb1 and wildtype littermate mice. Data shown as mean ± SEM. N = 12-31 per genotype. *p < Time (sec) Time 100 (sec) Time 0.05 as compared to wildtype, Mann Whitney test. 20

0 0 CK-Setdb1 wildtype CK-Setdb1 wildtype Supplemental Table 1:

RT-PCR primers (Mus musculus): Primer sequence symbol Forward Reverse Setdb1 CTTCTGGCTCTGACGGTGAT GGAAGCCATGTTGGTTGATT Grin2b CCCAGATCCTCGATTTCATT GCCAAACTGGAAGAACATGG Grin2a TGCAAGTTACACAGCCAACC ATCGGAAAGGCGGAGAATAG Grin1 GCCACCCTCACTTTTGAGAA AGGCGATCTCGATGAAAATG 18S rRNA CATGGCCGTTCTTAGTTGGT GAACGCCACTTGTCCCTCTA

ChIP primers at human GRIN2B locus: Homo sapiens, chromosome 12, Ensembl, ENSG00000150086 Forward: CAACGGGGACTGTTATTGAGA; Reverse: CCCACACGGAATGTGTTAGA

ChIP primers at mouse Grin2b locus: Mus musculus chromosome 6, reference assembly (C57BL/6J), NC_000072.5, GI:149361431 Primer Product Primer sequence pair location Forward Reverse 1 - 1 KB TTTGTCTCGGGAAAGAATGG GGGTTCTCCACAGCAGTGTT 2 0 KB GAGACTGGGTTAGCGGAGAG GTGGAACGCATCCTTGAAGT 3 + 1 KB GCTTTGCAGGAAAGAAAACG GTTAGCAGTGCCAAGGCTTC 4 + 15 KB TTGGACCGCTGACATTCATA AAGTGAGGCTGTCTGGCAGT 5 + 31.5 KB CAGCACCCACATTGTACGTC GGCATATTCATGCCCAAAAG 6 + 60 KB GCCAGACACATGGTCCTTTT AAGAAACCAGGGAGCCATTT

3C primers at mouse Grin2b locus (all primers are 5’ to 3’ on Grin2b sense strand) Mus musculus chromosome 6, reference assembly (C57BL/6J), NC_000072.5, GI:149361431 Primer Prime Primer sequence (5’ to 3’) Name location 1 - 4.4 KB GGCTCGGGAGTTTGGAAATGAGCCC 2 - 231 bp TTAAGCCGCCCCATCTCGCTGCTATT 3 + 5.2 KB CAATGGCTACCTTTGAGAGAGACACG 4 + 8.1 KB GGGTGACATCTCGGTCACCTTT 5 + 9.0 KB CTCATTCTTCTTTTTGTAGACCCTCTC 6 + 14.0 KB TTTGAGTGGAGAATGACAGCCGGG 7 + 20.2 KB GGGAAAAACATTGCTCCTGATAGCAC 8 + 22.2 KB CCTCAAACCTGATGTTTCTGGTATCT 9 + 27.6 KB CTAAGTGGGAAAAGTGTGTTTTGCTT 10 + 28.6 KB CCCACCATGAAGTAATGTCCTGTCTC 11 + 33.3 KB ACTATAACGCTTGCATACTCCTGGAA 12 + 35.6 KB GGGCTGTGCTTTGTCTGAACAC

Supplemental Table S2: mycSetdb1 occupancies across chromosomes 6, 8 and 16.

Chr Start End Gene *P MAT Fold_ FDR Length symbol value Score Change (%) 6 4321318 4321942 52.73 3.77 1.9 92.59 624 6 16076332 16076956 50.34 3.67 1.77 104.23 624 6 20350041 20350696 51.05 3.7 1.78 104.76 655 6 21309552 21310211 Kcnd2 63.99 4.23 1.98 160 659 6 22979916 22980540 Ptprz1 56.48 3.93 2.12 92.86 624 6 40726822 40727446 55.38 3.89 1.87 90.91 624 6 40866394 40867018 1700074P13 55.54 3.89 2.08 93.55 624 Rik 6 42523741 42524401 53.33 3.8 1.81 102.17 660 6 63247772 63248396 Grid2 52.63 3.77 1.97 87.93 624 6 76052255 76052879 50.33 3.67 2 104.23 624 6 79698627 79699251 52.87 3.78 1.9 92.45 624 6 101988140 101988764 56.29 3.92 1.89 92.86 624 6 102446084 102446708 54.22 3.84 1.93 102.63 624 6 106191076 106191700 Cntn4 51.87 3.74 1.88 98.33 624 6 106331139 106332144 Cntn4 85.53 5 2.32 0 1005 6 107095329 107095953 52.58 3.77 1.8 87.93 624 6 109803105 109803770 58.5 4.02 1.96 100 665 6 125756608 125757264 Ano2 59.02 4.04 2.27 105.56 656 6 129389966 129390729 Clec1a 54.04 3.83 1.81 102.56 763 6 129570792 129571450 Klrk1 56.69 3.94 1.84 96.15 658 6 129730659 129731424 Gm156 56.71 3.94 2.12 96.15 765 6 130056780 130057404 Klra1 50.7 3.69 1.81 109.23 624 6 130124004 130124773 Klra14 66.7 4.34 1.96 300 769 6 130337684 130338308 Klra1 50.54 3.68 1.81 107.46 624 6 131259803 131260427 Styk1 51.06 3.7 1.78 104.76 624 6 136091380 136092142 Grin2b 63.7 4.22 2.03 160 762 6 137570123 137570855 Eps8 54.36 3.84 1.82 102.7 732 6 138190758 138191493 59.93 4.07 1.88 107.14 735 6 138325063 138325687 Lmo3 50.24 3.67 1.81 104.17 624 6 139802803 139803427 Pik3c2g 50.33 3.67 1.77 104.23 624 8 15923894 15924554 Csmd1 55.43 3.89 1.83 93.75 660 8 18730052 18730676 Angpt2 60.26 4.09 1.99 92.31 624 8 18730052 18730676 Mcph1 60.26 4.09 1.99 92.31 624 8 29047421 29048245 61.6 4.14 2.09 92.31 824 8 35356408 35357032 55.95 3.91 2.09 93.1 624 8 41717604 41718228 Mtmr7 57.73 3.98 1.86 100 624 8 42788176 42788995 60.19 4.08 2.01 92.31 819 8 51043898 51044522 56.54 3.93 2.23 96.15 624 8 56137894 56138593 Gpm6a 53.49 3.81 1.81 102.22 699 8 61648334 61649146 61.89 4.15 1.9 137.5 812 8 78245462 78246086 1700007B14 50.52 3.68 1.77 107.46 624 Rik 8 84913056 84913680 Il15 52.71 3.77 1.84 87.93 624 8 84957957 84958581 50.95 3.7 2.12 106.25 624 8 88672076 88672885 53.89 3.82 1.81 102.38 809 8 88679789 88680413 50.04 3.66 1.92 104.11 624 8 93234147 93234771 54.5 3.85 2.1 102.78 624 8 98644504 98645209 58.52 4.02 1.91 100 705 16 9705088 9705712 Grin2a 54.89 3.87 1.92 100 624 16 27985466 27986090 51.5 3.72 1.87 98.36 624 16 38561451 38562075 Tmem39a 52.76 3.78 1.84 92.45 624 16 39727493 39728230 58.86 4.03 1.92 105.56 737 16 39803155 39803779 50.41 3.67 1.93 104.23 624 16 41079080 41079738 60.51 4.1 2.2 92.31 658 16 42460897 42461521 53.23 3.8 1.85 97.96 624 16 47389742 47390537 64.98 4.27 1.94 233.33 795 16 49348854 49349478 53.68 3.82 1.81 102.33 624 16 51276509 51277133 58.65 4.02 1.87 105.56 624 16 52194304 52195041 Cblb 59.56 4.06 1.88 113.33 737 16 53526982 53527606 52.95 3.78 2.04 92.45 624 16 57802247 57802902 57.23 3.96 1.89 100 655 16 59301397 59302021 Olfr203 53.31 3.8 1.97 97.96 624 16 63109560 63110184 55.62 3.9 1.83 93.55 624 16 63223509 63224133 53.88 3.82 2.17 102.38 624 16 63416792 63417527 63.58 4.22 2.36 128.57 735 16 66132908 66133571 60.9 4.11 1.9 92.31 663 16 67158056 67158717 Cadm2 52.03 3.74 2.14 98.31 661 16 70603393 70604117 53.8 3.82 1.81 102.38 724 16 74171251 74172165 Robo2 63.13 4.2 2.05 128.57 914 16 79915483 79916107 52.53 3.77 1.8 87.93 624 16 80456926 80457588 54.48 3.85 1.91 102.78 662 16 87325851 87326475 53.03 3.79 1.89 92.45 624 16 88517863 88518552 53.47 3.81 2.05 102.22 689 16 88717203 88717867 2310061N02 53.31 3.8 1.85 97.96 664 Rik 16 92927371 92928289 57.33 3.97 1.9 100 918

*-10log10_P value. The chromosomal location of hits is from NCBI Mus musculus Build 36.1.