Racine et al, Histone H2B ubiquitylation promotes activity of the intact Set1 histone methyltransferase complex in fission yeast
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1 Supplemental Materials Racine et al, Histone H2B ubiquitylation promotes activity of the intact Set histone methyltransferase complex in fission yeast Supplemental Experimental Procedures Table S Figures S-S5
2 Supplemental Experimental Procedures Denaturing whole-cell extract preparation- Extracts were prepared using a trichloroacetic acid lysis method as previously described (). Chromatin immunoprecipitation (ChIP) analysis of H3K4me3-ChIP was carried out using a H3K4me3-specific antibody purchased from Abcam (ab8580). The total H3 antibody was a generous gift from A. Verreault. Quantitative RT-PCR analysis-total S. pombe RNA was isolated using a hot phenol method as described previously (). RNA was converted to cdna using a RevertAid kit (Fermentas) and analyzed by qpcr using SYBR Green PCR mix (Biorad). Primers for act + were as described (2). Table S. S. pombe strains used in this study. Name Genotype Source JTB204 h- ade6-m26 Tanny et al., 2007 JTB24 spf-tap::kanmx6 leu-32 ura4-d8 his3-d ade6-m20 h- Roguev et al., 2003 JTB242 set-tap::kanmx6 leu-32 ura4-d8 his3-d ade6-m20 h- Roguev et al., 2003 JTB405 set-tap::kanmx6 brl2δ::hphmx4 leu- This study 32 ura4-d8 ade6 h? JTB404 set-tap::kanmx6 htb-k9r::kanmx6 ade6 h? JTB450 swd2.-tap::kanmx6 ade6-m26 h- This study JTB454 swd2.-tap::kanmx6 brl2δ::hphmx4 This study ade6-m26 h- JTB253-3 spf-tap::kanmx6 htb-k9r- FLAG::kanMX6 ade6 h? This study JTB46 spf-tap::kanmx6 brl2δ::hphmx4 ade6 This study h? JTB478 swd2.-tap::kanmx6 htb-k9r- This study FLAG::kanMX6 ade6 h? JTB202 rtf-tap::kanmx6 ade6-m26 h- This study JTB367 rtf-tap::kanmx6 htb-k9r::kanmx6 ade6 h- This study SUPPLEMENTAL REFERENCES. Tanny, J. C., Erdjument-Bromage, H., Tempst, P., and Allis, C. D. (2007) Genes Dev 2, Guiguen, A., Soutourina, J., Dewez, M., Tafforeau, L., Dieu, M., Raes, M., Vandenhaute, J., Werner, M., and Hermand, D. (2007) EMBO J 26, Figure S. The spf-tap strain has wild-type levels of H3K4me in vivo. Denaturing whole-cell extracts from untagged (JTB204) and spf-tap (JTB24) strains were analyzed by western blotting with the indicated antibodies. Figure S2. Extended time course of in vitro SetC methyltransferase reactions. The indicated mononucleosome substrates were incubated in the presence of SetC at 30 o C and analyzed by western blotting. Incubation times and antibodies are indicated on the right.
3 Figure S3. Expression of SetC subunits in wild-type and H2Bub-deficient strains. (A) Denaturing whole-cell extracts from the indicated TAP strains ( WT -wild-type; K -htb- K9R; B -brl2δ) were analyzed by western blotting with an anti-tap antibody. Corresponding strain numbers are JTB24, JTB253-3, JTB46, JTB450, JTB478, JTB454, JTB242, JTB405. (B) Quantification of band intensities for independent replicates of the blot shown in (A). For each sample [numbered -8 as shown in (A)] the TAP signal was normalized to the tubulin signal, and then normalized to the corresponding wild-type tagged strain. Figure S4. Chromatin immunoprecipitation (ChIP) analysis of H3K4me3 levels at act +. ChIP was carried out in a wild-type strain using antibodies against H3K4me3 and total histone H3 and analyzed by qpcr as described in Experimental Procedures. Relative enrichment of H3K4me3 at each position is expressed as a fraction of the total H3 enrichment. Figure S5. Transcription of act + is not defective in H2Bub-deficient strains. (A) Equal amounts of total RNA from each strain (JTB204, JTB86-3, JTB33, respectively) was converted into cdna and analyzed by qpcr for act + transcript. Transcript abundance in the indicated strains relative to wild-type was measured in two independent experiments; error bars denote standard deviations. (B) ChIP was carried out in the indicated TAP-tagged strains (JTB202, JTB367) and analyzed by qpcr as described in Materials and Methods. ChIP signals are expressed as a percentage of the input signal for each primer pair. Normalization was carried out by subtracting the background %IP obtained from an untagged strain. Error bars denote standard deviations from 3 independent experiments.
4 Figure S H3K4me H3K4me2 H3K4me3 H3
5 Figure S2 Unmodified H2Bub Time (hours) H3K4me2 H3
6 A Figure S3 spf-tap swd2.-tap set-tap WT K B WT K B WT B TAP tubulin B.2 Relative normalized intensity
7 Figure S4 2 3 act + Relative enrichment (H3K4me3/H
8 Figure S5 A.4.2 Relative mrna level WT htb-k9r brl2!" 2 3 B act Normalized % IP rtf-tap rtf-tap htb-k9r
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P-Box Dsf -----------------MG-TAG--DRLLD-IPCKVCGDRSSGKHYGIYSCDGCSGFFKR 39 NR2E1 -----------------MSKPAGSTSRILD-IPCKVCGDRSSGKHYGVYACDGCSGFFKR 42 Tll MQSSEGSPDMMDQKYNSVRLSPAASSRILYHVPCKVCRDHSSGKHYGIYACDGCAGFFKR
Supporting Information
Supporting Information Rougemaille et al. 1.173/pnas.12494719 SI Methods Strain Construction. Homologous replacement of DNA was accomplished by lithium acetate transformation of PCR products containing
Figure S1: NUN preparation yields nascent, unadenylated RNA with a different profile from Total RNA.
Summary of Supplemental Information Figure S1: NUN preparation yields nascent, unadenylated RNA with a different profile from Total RNA. Figure S2: rrna removal procedure is effective for clearing out
To determine the effect of N1IC in the susceptibility of T cells to the tolerogenic effect of tumorassociated
Supplementary Methods Tolerogenic effect of MDSC To determine the effect of N1IC in the susceptibility of T cells to the tolerogenic effect of tumorassociated MDSC in vivo, we used a model described previously
Supplementary Information
Supplementary Information Supplementary Figure 1. ZBTB20 expression in the developing DRG. ZBTB20 expression in the developing DRG was detected by immunohistochemistry using anti-zbtb20 antibody 9A10 on
Schematic representation of the endogenous PALB2 locus and gene-disruption constructs
Supplementary Figures Supplementary Figure 1. Generation of PALB2 -/- and BRCA2 -/- /PALB2 -/- DT40 cells. (A) Schematic representation of the endogenous PALB2 locus and gene-disruption constructs carrying
Figure S1. Replication initiation sites at efficient ORIs do not coincide with nucleosomedepleted
Figure S1. Replication initiation sites at efficient ORIs do not coincide with nucleosomedepleted regions in the mouse genome. Typical examples of SNS profiles (Cayrou et al., 11) and nucleosome occupancies
Nature Structural and Molecular Biology: doi: /nsmb Supplementary Figure 1. Validation of CDK9-inhibitor treatment.
Supplementary Figure 1 Validation of CDK9-inhibitor treatment. (a) Schematic of GAPDH with the middle of the amplicons indicated in base pairs. The transcription start site (TSS) and the terminal polyadenylation
Nature Immunology: doi: /ni Supplementary Figure 1. Zranb1 gene targeting.
Supplementary Figure 1 Zranb1 gene targeting. (a) Schematic picture of Zranb1 gene targeting using an FRT-LoxP vector, showing the first 6 exons of Zranb1 gene (exons 7-9 are not shown). Targeted mice
Supplementary Materials for
www.sciencesignaling.org/cgi/content/full/5/244/ra72/dc1 Supplementary Materials for An Interaction Between BZR1 and DELLAs Mediates Direct Signaling Crosstalk Between Brassinosteroids and Gibberellins
Engineering splicing factors with designed specificities
nature methods Engineering splicing factors with designed specificities Yang Wang, Cheom-Gil Cheong, Traci M Tanaka Hall & Zefeng Wang Supplementary figures and text: Supplementary Figure 1 Supplementary