Supplemental Information. Anti-Inflammatory Chromatinscape. Suggests Alternative Mechanisms. of Glucocorticoid Receptor Action

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1 Immunity, Voume 7 Suppementa Information nti-infammatory Chromatinscape Suggests ternative Mechanisms of Gucocorticoid Receptor ction Kyu-Seon Oh, Heta Pate, Rache. Gottschak, Wai Shing Lee, Songjoon aek, Iain D.C. Fraser, Gordon L. Hager, and Myong-Hee Sung

C h LPS vs no LPS Figure S. RN-seq reproducibiity, reated to Figure. Two bioogica repicates were prepared for RN-seq of MDMs in a the conditions shown in Figure. Log (FPKM +.) vaues are used.

2 C h LPS vs no LPS Figure S. RN-seq reproducibiity, reated to Figure. Two bioogica repicates were prepared for RN-seq of MDMs in a the conditions shown in Figure. Log (FPKM +.) vaues are used. () Repicate reproducibiity for a given LPS time point. () LPS response at h for the two repicates. (C) edger anaysis showing og FC (fod change) (LPS h - h) versus og CPM (counts per miion reads). Differentiay expressed genes are marked in red (FDR <.5). The gray ines indicate the fod changes vaues of + and -. The pot shows that many differentiay expressed genes with FDR <.5 have fod change vaues ess than -fod (red points between the two gray ines).

3 . Cxcr. 5 Sep 5 Nos 5 mount reative to basa eve Cd55 Cxc 8 Mmp... -treated Cc h h h h LPS -treated /LPS h /LPS h LPS h 59 Dex-reguated genes C LPS h LPS h /LPS h LPS h /LPS h h h h h LPS -treated Cec5a Cec7a -treated mount reative to basa eve Ib LPS h Figure S. Genes differentiay reguated by - versus ate-treatment. (Reated to Figure ) (-) RT-qPCR conﬁrmation of differentiay expressed genes identiﬁed from RN-seq data. MDMs were treated with LPS and Dex as indicated on the horizonta axes. Transcript eves were normaized to the LPS h eve. Genes in the ower in ate Dex () and ower in eary Dex () categories. Two bioogica repicates were prepared and each repicate was measured in two or three technica repicates. Error bars, s.d. (C) Heatmap of RN-seq data for additiona genes (not incuded in Figure ) which were reguated by Dex. The coor scae dispays og FPKM ratios with respect to the LPS h vaues. The repicate coumns are arranged the same way as in Figure. Four gene custers were obtained by a robust variant of K-means custering agorithm.

4 -h h No Dex No GR activation LPS h 8h Re ChIP-seq samping GR ChIP-seq samping DNase-seq samping Dex Eary GR activation LPS Late GR activation LPS Dex Re binding 8h vs h after LPS in MDMs without Dex No Re binding hrs after LPS in -treated vs Dex-untreated MDMs No Re binding 8hrs after LPS in -treated vs Dex-untreated MDMs Reduced Re binding 8hrs after LPS in -treated vs Dex-untreated MDMs Figure S. Genomic occupancy of Re in Dex treated MDMs. (Reated to Figures -) () Experimenta design for MDM treatment and sampe coection for ChIP-seq and DNase-seq. The timeine is with respect to the onset of LPS stimuation (t = ). () Pairwise comparison of a the Re ChIP-seq data, density normaized for sequencing depth.

5 LPS ony C verage nucear Re occupancy Time to peak 5 Time (hrs) EGFP-Re (a.u.) Mean nucear intensity LPS ony 8 LPS ony D Dex ony LPS ony No LPS Mean nucear intensity Re p8 p-p8 5 ERK p-erk - 8 STT- RhoGDI Dex ony E 5 LPS ony Whoe ce ysate No LPS Mean nucear intensity Iκ-α Re hnrnp 8 Time after LPS (hrs) Nucear extract Figure S. Monitoring the nucear eve of Re by ive ce imaging of RW.7 cone expressing EGFP-Re and mcherry reporter and by Western bot anaysis of MDMs. (Reated to Figure ) () The time course of nucear EGFP-Re intensity in individua singe ces. The green arrows mark the time of Dex treatment. Live ce imaging of ces for each condition was performed in bioogica dupicates in separate days, and the repicate data were pooed. The tota number of ces quantiﬁed:,, for LPS ony,, and treatment, respectivey. () The time-averaged nucear occupancy of EGFP-Re (deﬁned by the area under the nucear EGFP curve divided by the tota time span), from data shown in (). (C) The time to the ﬁrst peak nucear transocation of EGFP-Re, identiﬁed as previousy described (Sung MH et a. Sci Signa ), from data shown in (). In the box pots in -C, top and bottom ends of the box indicate 5th- and 75th-percentie vaues, the red bars indicate the median vaues, the crosses are outier data points. (D) Western bot of Re and MPK signaing factors using MDMs. RhoGDI, oading contro. (E) Western bot of Re in nucear extracts of MDMs. hnrnp, nucear fractionation contro. In D-E, three midde anes show MDM sampes treated with LPS for ten hours, either without Dex, pre-treated with Dex, or ate-treated with Dex. Each pane shows a representative resut from bioogica repicates.

6 5 kb chr5:,,,5,,, 5 kb chr: 9,5, LPS h LPS h LPS 8h Re LPS h/ LPS h/ LPS 8h/ LPS 8h/ GR _ Dex aone (before LPS) _ (after LPS) _ DNase LPS aone _ K95 Ifnar LPS 8h Dex aone (before LPS) (after LPS) Re. to input DN GR ChIP-qPCR LPS h LPS 8h LPS h/ LPS 8h/ LPS 8h/ _ K8 I Re ChIP-qPCR 5 Re. to input DN _ 8 Rea Tnf I Ib Dusp Neg Rea Tnf I Ib Dusp I upstream Neg Figure S5. Genomic occupancy of Re and GR in Dex treated MDMs. (Reated to Figures -5) () Genome browser shots of I (custer ) and Ifnar (custer ) oci. Data tracks show density normaized for sequencing depth. Genomic coordinates in mm9. () Re and GR ChIP-qPCR at binding sites identiﬁed from ChIP-seq. MDMs were treated with LPS and/or Dex as indicated. Neg, negative contro ocus. Error bars, s.d.

h _ PU.:.8E-5 (5%) Dex aone (before LPS).5 GR:.E- (%) _..5 (after LPS).

by Dex Expression enhanced by Dex by Dex Expression enhanced by Dex Figure S.

(Reated to Figure ) () GR ChIP-qPCR of MDMs treated with Dex for min, min, h, h.

neg, negative contro ocus. Error bars, s.d.

Tomtom was used to match motifs to known TF binding speciﬁcity.

are shown for each enriched TF. Top, GR ChIP sites were anayzed for both sets.

7 chr:,, ChIP-qPCR,5,.5. No Dex Dex min Dex hr Dex hr Constant GR ChIP sites (before and after LPS) LPS h _ PU.:.8E-5 (5%) Dex aone (before LPS).5 GR:.E- (%) _..5 (after LPS). Per Lcn Neg _ GR ChIP sites gained after LPS LPS aone PU.:.5E-5 (%) _ Re:.E-8 (%) _ GR:.E-7 (%) Lcn GR ChIP density at dista sites near LPS-induced genes (after LPS) C (after LPS) Re. to input DN (%). Dex aone (before LPS) RN-seq FPKM ogratio ( versus no Dex) Expression suppressed by Dex Expression enhanced by Dex Dex aone (before LPS) RN-seq FPKM ogratio ( versus no Dex) Expression suppressed by Dex Expression enhanced by Dex Figure S. Gain of GR binding and ack of correation with Dex-dependent gene reguation. (Reated to Figure ) () GR ChIP-qPCR of MDMs treated with Dex for min, min, h, h. No speciﬁc binding is observed at the Lcn promoter site where GR ChIP-seq peaks are detected ony after LPS. Per serves as a positive contro site, showing GR binding after Dex aone. neg, negative contro ocus. Error bars, s.d. () Motifs enriched in the indicated GR binding sites. Tomtom was used to match motifs to known TF binding speciﬁcity. MEME E-vaue and the proportion (in parenthesis) of ChIP sites containing the motif are shown for each enriched TF. Top, GR ChIP sites were anayzed for both sets. (C) GR ChIP-seq density at GR binding sites after LPS versus before LPS stimuation of MDMs. Coored points mark dista (> kb from TSS) sites within kb of LPS-induced genes. Logratios of RN-seq FPKM vaues are coor-coded as indicated in the scae bar. The ratios FPKM(LPS h) / FPKM(LPS h, -treated) and FPKM(LPS h) / FPKM(LPS h, -treated) are shown in eft and right, respectivey.

8 DNase/ * DNase density og ratio +/ Dex DNase/ Repicate Repicate DNase/LPS aone Repicate De x at e D HS s, ar D HS y on d un bo y LP LP af te r e fo r be d un bo G R G R De x S Repicate s, e Repicate S Repicate Figure S7. Chromatin accessibiity changes after Dex treatments. (Reated to Figures 5-) () Reproducibiity of DNase-seq intensity. ioogica repicates of each DNase-seq sampe were sequenced and their intensity proﬁes were compared over a the DHSs detected in at east one sampe. () Vioin pots of Dex-speciﬁc changes in DNase-seq intensity at GR bound sites and at a DHSs. White dots mark median vaues. Thick bars end at 5 percentie (top) and 75 percentie (bottom) vaues. sterisk marks statisticay signiﬁcant difference of the two groups (Wech two-sampe t-test p = 9.5-5).

Supplementary Material: Quantifying evolutionary constraints on B cell affinity maturation

Supplementary Material: Quantifying evolutionary constraints on B cell affinity maturation QUANTIFYING EVOLUTIONARY CONSTRAINTS ON B CELL AFFINITY MATURATION 1 Suppementary Materia: Quantifying evoutionary constraints on B ce affinity maturation CONNOR O. MCCOY, TREVOR BEDFORD, VLADIMIR N. MININ,