1
Mersman, Du, Fingerman, South and Briggs
Supplemental Information
Supplemental Tables
Table S1. Yeast strains
Yeast Strain / Genotype / ReferenceBY4741 /
MATα his3∆ leu2∆0 LYS2 met15∆0 ura3∆0
/ Open BiosystemsMBY1282 / MATα his3∆200 ade2::hisG leu2∆0 ura3∆0 met15∆0 trp163 Ty1his3AI-236, Ty1ade2AI-515, SET1-N-3XMYC / This Study
MBY1217 / MATα his3∆200 ade2::hisG leu2∆0 ura2∆0 met15∆0 trp163 Ty1his3AI-236r , Ty1adeAI515 set1::TRP1 / (Briggs et al. 2001)
not4 /
BY4741: MATα his3∆ leu2∆0 LYS2 met15∆0 ura3∆0 not4∆::KanMX
/ Open Biosystemsrad6 /
BY4741: MATα his3∆ leu2∆0 LYS2 met15∆0 ura3∆0 rad6∆::KanMX
/ Open Biosystemsset2 /
BY4741: MATα his3∆ leu2∆0 LYS2 met15∆0 ura3∆0 set2∆::KanMX
/ Open BiosystemsSDBY1065 /
BY4741: MATα his3∆ leu2∆0 LYS2 met15∆0 ura3∆0 jhd2∆::HygMX
/ This StudySDBY1066 / BY4741: MATα his3∆ leu2∆0 LYS2 met15∆0 ura3∆0 not4::KanMX jhd2∆::HygMX / This Study
SDBY1067 / BY4741: MATα his3∆ leu2∆0 LYS2 met15∆0 ura3∆0 rad6::KanMX jhd2∆::HygMX / This Study
SDBY1068 / BY4741: MATα his3∆ leu2∆0 LYS2 met15∆0 ura3∆0 pdr5::KanMX jhd2∆::HygMX / This Study
SDBY1069 / as MBY1217 with pRS415/ADH1p / This Study
SDBY1070 / as MBY1282 with pRS415/ADH1p / This Study
SDBY1071 / as MBY1282 with pDPM2 / This Study
SDBY1072 / as MBY1282 with pDPM3 / This Study
SDBY1073 / as MBY1282 with pDPM4 / This Study
SDBY1074 / as MBY1282 with pDPM5 / This Study
SDBY1075 / as MBY1282 with pDPM6 / This Study
SDBY1076 / as BY4741 with pRS415/ADH1p / This Study
SDBY1077 / as SDBY1065 with pRS415/ADH1p / This Study
SDBY1078 / as SDBY1065 with pDPM3 / This Study
SDBY1079 / as SDBY1068 with pRS415/ADH1p / This Study
SDBY1080 / as SDBY1068 with pDPM3 / This Study
SDBY1081 / as MBY1217 with pRS415 / This Study
SDBY1082 / as BY4741 with pRS415 / This Study
SDBY1083 / as SDBY1065 with pRS415 / This Study
SDBY1084 / as not4 with pRS415 / This Study
SDBY1085 / as SDBY1066 with pRS415 / This Study
SDBY1086 / as SDBY1066 with pDPM2 / This Study
SDBY1087 / as SDBY1066 with pDPM7 / This Study
SDBY1088 / as SDBY1066 with pDPM8 / This Study
SDBY1089 / as MBY1217 with pRS416 / This Study
SDBY1090 / as BY4741 with pRS416 / This Study
Table S1 (Continued). Yeast strains
Yeast Strain / Genotype / ReferenceSDBY1092 / as not4 with pDPM9 / This Study
SDBY1093 / as not4 with pDPM10 / This Study
SDBY1094 / as not4 with pDPM11 / This Study
SDBY1095 / as MBY1217 with pRS415 and pRS416 / This Study
SDBY1096 / as SDBY1066 with pRS415 and pRS416 / This Study
SDBY1097 / as SDBY1066 with pDPM2 and pRS416 / This Study
SDBY1098 / as SDBY1066 with pRS415 and pDPM9 / This Study
SDBY1099 / as SDBY1066 with pRS415 and pDPM10 / This Study
SDBY1100 / as SDBY1066 with pRS415 and pDPM11 / This Study
SDBY1101 / as SDBY1066 with pDPM2 and pDPM9 / This Study
SDBY1102 / as SDBY1066 with pDPM2 and pDPM10 / This Study
SDBY1103 / as SDBY1066 with pDPM2 and pDPM11 / This Study
SDBY1104 / as SDBY1066 with pRS415/ADH1p / This Study
SDBY1105 / as SDBY1066 with pDPM3 / This Study
SDBY1106 / MATα his3∆ leu2∆0 LYS2 met15∆0 ura3∆0 not4::HygMX / This Study
SDBY1107 / MATα his3∆ leu2∆0 LYS2 met15∆0 ura3∆0, JHD2-C-3XFLAG / This Study
SDBY1108 / MATα his3∆ leu2∆0 LYS2 met15∆0 ura3∆0 not4::HygMX, JHD2-C-3XFLAG / This Study
SDBY1109 / as SDBY1065 with pDPM16 / This Study
SDBY1110 / as SDBY1065 with pDPM17 / This Study
SDBY1111 / as SDBY1065 with pDPM18 / This Study
SDBY1112 / as SDBY1065 with pDPM19 / This Study
SDBY1113 / as SDBY1065 with pDPM20 / This Study
SDBY1114 / as SDBY1065 with pDPM21 / This Study
SDBY1115 / MATα his3∆200 ade2::hisG leu2∆0 ura3∆0 met15∆0 trp163 Ty1his3AI-236, Ty1ade2AI-515, SET1-N-MYC3#2a jhd2::HygMX / This Study
Table S2. Plasmids
Plasmid / InsertedGene / Promoter / Vector / SourcepRS415 / None / None / pRS415 / (Sikorski and Hieter 1989)
pRS416 / None / None / pRS416 / (Sikorski and Hieter 1989)
pRS415/ADH1p / None / ADH1p / pRS415 / (Mumberg et al. 1995)
pDPM1/PYK1p / None / PYK1p / pRS415 / This Study
pDPM2 / JHD2-FLAG / JHD2p / pRS415 / This Study
pDPM3 /
FLAG-JHD2
/ ADH1p / pRS415/ADH1p / This StudypDPM4 /
FLAG-JHD2
/ PYK1p / pDPM1/PYK1p / This StudypDPM5 /
FLAG-JHD2 H427A
/ PYK1p / pDPM1/PYK1p / This StudypDPM6 / FLAG-JHD2 PHD / PYK1p / pDPM1/PYK1p / This Study
pDPM7 /
JHD2 H427A-FLAG
/ JHD2p / pRS415 / This StudypDPM8 / JHD2 PHD-FLAG / JHD2p / pRS415 / This Study
pDPM9 /
NOT4-HA
/ NOT4p / pRS416 / This StudypDPM10 / NOT4 RING-HA / NOT4p / pRS416 / This Study
pDPM11 / NOT4 RRM-HA / NOT4p / pRS416 / This Study
pDPM12 /
CBP-JHD2
/ T7 / pCal-n / This StudyTable S2 (Continued). Plasmids
Plasmid / InsertedGene / Promoter / Vector / SourcepDPM13 / 6XHIS-NOT4 (yeast) / T7 / pET28b-9 / This Study
pDPM14 / CBP-JARID1C (1-700 a.a.) / T7 / pCAL-n / This Study
pDPM15 / 6XHIS-NOT4 (human) / T7 / pET28b-9 / This Study
pDPM16 / FLAG-JHD2 1 - 368 / ADH1p / pRS415/ADH1p / This Study
pDPM17 / FLAG-JHD2 1 - 548 / ADH1p / pRS415/ADH1p / This Study
pDPM18 / FLAG-JHD2 1 - 560 / ADH1p / pRS415/ADH1p / This Study
pDPM19 / FLAG-JHD2 233 - 728 / ADH1p / pRS415/ADH1p / This Study
pDPM20 / FLAG-JHD2 376 - 728 / ADH1p / pRS415/ADH1p / This Study
pDPM21 /
FLAG-JHD2PHD
/ ADH1p / pRS415/ADH1p / This StudypPFS42 /
CBP-BRE2
/ T7 / pCAL-n / This StudyTable S3. Fold Change Analysis of GUA1 Expression (See Fig. 7A)
Samples Compared / Gene / Fold Changenot4 + vector / WT / GUA1 / -2.5 +/- 0.02
jhd2 + vector / WT / GUA1 / 1.08 +/- 0.05
not4jhd2 + vector /WT / GUA1 / -1.07 +/- 0.07
not4 jhd2 + JHD2/ WT / GUA1 / -2.13 +/- 0.04
not4 jhd2 + JHD2 H427A/ WT / GUA1 / 1.16 +/- 0.12
not4 jhd2 + NOT4/ WT / GUA1 / -1.15 +/- 0.07
*Data represents six biological repeats with four technical repeats each.
Table S4. Fold Change Analysis of GUA1 Expression (See Fig. 7B)
Samples Compared / Gene / Fold Changeset1/ WT / GUA1 / -2.04 +/- 0.1
*Data represents three biological repeats with three technical repeats each.
Table S5. Fold Change Analysis of GUA1 H3 K4me3 ChIP Analysis (See Fig. 7C, S5A, B)
Samples Compared / Location / Fold Changenot4/ WT / 5’ / -2.5 +/- 0.08
jhd2/ WT / 5’ / -1.1 +/- 0.12
not4 jhd2/ WT / 5’ / -1.36 +/- 0.09
not4/not4 jhd2 / 5’ / -1.8 +/- 0.09
jhd2/not4 jhd2 / 5’ / 1.2 +/- 0.11
not4/jhd2 / 5’ / -2.3 +/- 0.08
not4/ WT / 3’ / -4.5 +/- 0.06
jhd2/ WT / 3’ / 1.45 +/- 0.33
not4 jhd2/WT / 3’ / -1.6 +/- 0.06
not4/not4 jhd2 / 3’ / -2.9 +/- 0.06
jhd2/not4 jhd2 / 3’ / 2.3 +/- 0.33
not4/jhd2 / 3’ / -6.5 +/- 0.06
*Data represents three biological repeats with three technical repeats each.
Table S6. Fold Change Analysis of GUA1 H3 K4me2 ChIP Analysis (See Fig. 7D, S5C, D)
Samples Compared / Location / Fold Changenot4/ WT / 5’ / 3.2 +/- 0.5
jhd2/ WT / 5’ / -1.1 +/- 0.13
not4 jhd2/ WT / 5’ / 1.18 +/- 0.02
not4/not4 jhd2 / 5’ / 2.7 +/- 0.5
jhd2/not4 jhd2 / 5’ / -1.3 +/- 0.13
not4/jhd2 / 5’ / 3.4 +/- 0.5
not4/ WT / 3’ / -3.4 +/- 0.05
jhd2/ WT / 3’ / -1.1 +/- 0.5
not4 jhd2/ WT / 3’ / -2.5 +/- 0.05
not4/not4 jhd2 / 3’ / -1.4 +/- 0.05
jhd2/not4 jhd2 / 3’ / 2.25 +/- 0.05
not4/jhd2 / 3’ / -3.1 +/- 0.05
*Data represents three biological repeats with three technical repeats each.
Supplemental Materials and Methods
Histone antibodies
Primary antibodies -H3K4me1 (07-436,lot number 30218),-H3K4me2 (07-030,lot number 26335), and -H3K4me3 (07-473, lot number 24503) were obtained from Millipore and were used at 1:2500, 1:5000, and 1:5000 dilutions, respectively. -H3K36me3 (Ab9050, lot number 134025) and -histone H3 (Abcam, Ab1791,lot number 172452) were obtained from Abcam and were used at 1:5000 and 1:10,000 dilutions, respectively.
Construction of plasmids and yeast strains
SDBY1065 strain was generated as previously described (Goldstein and McCusker 1999). SDBY1066, SDBY1067, and SDBY1068 deletion strains were generated by standard protocols using PCR amplification of the KanMX cassette from not4, rad6, or pdr5 strains obtained from Open Biosystems. SDBY1106 strain was generated as previously described (Goldstein and McCusker 1999). SDBY1107 and SDBY1108 strains were generated as previously described (Gelbart et al. 2001). HA-tagged Not4 and FLAG-tagged Jhd2 constructs containing their own promoters (NOT4p and JHD2p) were generated by PCR amplification of their open reading frames plus 496 bp sequences upstream and engineered with single HA or FLAG epitope-tags at their C-terminus. The Not4-HAPCR product was cloned with the restriction sites XbaI and XhoI into pRS416 to make pDPM9. The Jhd2-FLAGPCR product was cloned with the restriction sites HindIII and XhoI into pRS415 to make pDPM2. Not4RING (pDPM10) and Not4 RRM (pDPM11) mutants were generated by standard site-directed mutagenesis (Stratagene) using the pDPM9 as a template. Jhd2 under the control of the ADH1 promoter (ADH1p) was constructed by PCR amplification of the open reading frame and engineered with single N-terminal FLAG epitope-tag. The PCR product was designed with the restriction sites BglII and XhoI and was cloned into the vector pRS415/ADH1p to make pDPM3. Jhd2 under the control of the PYK1 promoter (PYK1p) was constructed in a similar manner using the vector pDPM1/PYK1p to make pDPM4. Jhd2 H427A (catalytically inactive; pDPM5 and pDPM7) and Jhd2 PHD (pDPM6 and pDPM8) mutants were generated by standard site-directed mutagenesis (Stratagene) using pDPM2 or pDPM4 as templates. pDPM18, pDPM19, and pDPM20 were generated by PCR amplification of the open reading frame and were engineered with a single N-terminal FLAG epitope-tag. pDPM16, pDPM17, and pDPM21 were generated by standard site-directed mutagenesis (Stratagene) using pDPM3 as a template. Full-length yeast and human Not4 were PCR amplified using genomic DNA and human Not4 cDNA (accession number BC035590), respectively, as templates and both were cloned into pET28b-9, a modified pET28 (Novagen) bacterial expression vector. Yeast Not4 was cloned using the restriction sites EcoRI and XhoI to make pDPM13. Human Not4 was digested with the restriction enzymes BglII and XhoI and was cloned into pET28b-9 that was digested with the enzymes BamHI and XhoI to make pDPM15. Full-length Jhd2 and JARID1C (1-700 a.a.; encoding amino acid residues 1 – 700)PCR products were cloned into the pCAL-n (Stratagene) bacterial expression vector using genomic DNA and JARID1C cDNA (accession number BC054499), respectively, as templates. Jhd2 was digested with the restriction enzymes BglII andXhoI and was cloned into pCAL-n that was digested with the enzymes BamHI and XhoI to make pDPM12. JARID1C(1-700 a.a.) was cloned into pCAL-n using the restriction sites EcoRI and HindIII to make pDPM14. Full-length Bre2 was PCR amplified using genomic DNA as a template. Bre2 was digested with the restriction enzymes BamHI and XhoI and was cloned into the vector pCAL-n (Stratagene) that was digested with BamHI and XhoI to make pPFS42.
Bacterial expression and purification.
HIS-Ubc4 (yeast), HIS-Not4 (yeast), HIS-Not4 (human), CBP-Jhd2, CBP-JARID1C (1-700 a.a.), and CBP-Bre2 were expressed in bacteria as described(Fingerman et al. 2007).Soluble protein was purified using Ni-NTA resin (Qiagen) or Calmodulin Affinity resin (Stratagene) according to the manufacturers’ protocols. HIS-Ubc4 (yeast), HIS-Not4 (yeast), and HIS-Not4 (human) cultures were induced with 0.4 mM IPTG for four hours at room temperature and were purified according to manufacturer’s protocol (Qiagen). CPB-Jhd2 cultures were induced with 0.01 mM IPTG at 18ºC overnight and CBP-Jhd2 was purified according to manufacturer’s protocol (Stratagene). CBP-JARID1C (1-700 a.a.) and CBP-Bre2 cultures were induced with 0.05 mM IPTG at 18ºC overnight and were purified according to manufacturer’s protocol (Stratagene). Purified HIS-Ubc4 (yeast), HIS-Not4 (yeast), HIS-Not4 (human), CBP-Jhd2, CBP-JARID1C1-700, and CBP-Bre2were dialyzed in dialysis buffer (50 mM Tris, pH 7.5; 150 mM KCl, 2.5 mM MgCl2, 0.5 mM EDTA, 0.25 mM DTT, 10% Glycerol). Ube1 (E1, human; E-305) and UbcH5a (E2, human; E2-616) proteins were purchased from Boston Biochem for in vitro ubiquitination assays.
Immunoprecipitation of Jhd2 and Set1 from yeast cell extracts.
For immunoprecipitation of episomally expressedFLAG-tagged Jhd2 (JHD2p, ADH1p,and PYK1p) or 3XFLAG-tagged Jhd2 (endogenous locus), 50 mL cultures were grown to mid-log phase. Whole cell lysates were prepared by bead beating in Buffer A (40 mM HEPES-NaOH pH 7.5, 350 mM NaCl, 0.1% Tween-20, 10% glycerol, 1 g leupeptin, aprotinin, and pepstatin A, 1 mM PMSF) and protein levels were normalized by Bradford assay (Bio-Rad). 2X SDS sample buffer was added to an aliquot of lysate to analyze for protein loading by immunoblotting for H3(Abcam, Ab1791). M2 -FLAG resin (Sigma, A2220) was added tonormalized lysate and incubated at 4ºC for 2 hours with rotation. The immunoprecipitated resin was washed twice with lysis buffer and then resuspended in 2X SDS sample buffer. Samples were boiled, centrifuged and loaded on an 8% SDS-PAGE gel. After transfer to PVDF membrane,-FLAG antibody (Sigma, F7425) was used at a 1:5000 dilution to detect FLAG-tagged Jhd2.
To detect polyubiquitinatedJhd2 expressed from the ADH1 promoter, FLAG-Jhd2 was immunoprecipitated and detected as described above. The immunoprecipitated samples were run on an 8% SDS-PAGE gel for detection of unmodified Jhd2 and 6% SDS-PAGE gel for detection of polyubiquitinated Jhd2. After transfer, Western Blots to be used for detecting ubiquitin were boiled in ddH2O for 5 min. Blots were probed with an -FLAG antibody (Sigma, F7425)used at a 1:5000 dilution or -Ubiquitin antibody used at a 1:500 dilution (Santa Cruz Biotech., SC-8017)(Laney and Hochstrasser 2002).
To detect polyubiquitinated Jhd2-FLAG that was expressed episomally under the control of the Jhd2 promoter (JHD2p), jhd2 pdr5 strains expressing either empty vector or JHD2p-JHD2-FLAG and a wild-type strain expressing empty vector were grown mid-log phase and treated with either 0.1 mM MG132 (Peptides International, IZL-3175-v) or DMSO as indicated in Fig. 3B for 30 min. Whole cell lysates were prepared in Buffer A containing 20 mM NEM. Immunoprecipitation of Jhd2-FLAG and detection of ubiquitin, FLAG, and H3 were performed as described above.
To detect polyubiquitinated Jhd2-3XFLAG that was expressed from its endogenous locus, wild-type or not4 strains expressing endogenous levels of Pdr5 were grown overnight in 35 mL cultures in SC media without ammonium sulfate supplemented with 0.1% proline. Cultures were backdiluted in 50 mL to OD600 = 0.5 and sodium dodecyl sulfate (SDS) was added to 0.003%(Liu et al. 2007). After 3 hrs of incubation at 30ºC, cultures were treated with either 0.1 mM MG132 (Peptides International, IZL-3175-v) or DMSO as indicated in Fig. 3C and Fig. 6B for 30 min. Whole cell lysates were prepared in Buffer A containing 20 mM NEM. Immunoprecipitation of Jhd2-3XFLAG and detection of ubiquitin, FLAG, and H3 were performed as described above.
To detect endogenously 3XMYC-tagged Set1, wild-type and jhd2 strains expressing endogenous levels of Pdr5 in which Set1 is triple-MYC tagged at its endogenous locus were grown in SC media without ammonium sulfate containing 0.1% proline overnight as described above. 120 mL cultures were backdiluted toOD600 = 0.5 and SDS was added to 0.003%. After 3 hrs of incubation at 30ºC, cultures were treated with 0.1 mM MG132 (Peptides International, IZL-3175-v) as indicated in Fig. 3D. Aliquots were taken immediately before addition of MG132 and 30, 60, and 90 min after addition of MG132 as follows: 5 mL aliquots were taken for analysis of modified histones and 25 mL aliquots were taken for immunoprecipitation of Set1. Immunoprecipitiation and detection of MYC-tagged Set1 was performed similarly to the procedure described for FLAG-tagged Jhd2 with the exception that lysates were incubated with 2 g -Myc antibody (9E10, Roche, 11 667 203 001) for 2 hrs rotating at4ºC. 12 L of protein G sepharose (GE Healthcare, 17-0618-01) were added and the lysates were rotated for 1 hr at 4ºC. The immunoprecipitated resin was washed twice with 1 mL lysis buffer and then resuspended in 12 L 2X SDS sample buffer. Samples were boiled, centrifuged, and loaded on an 8% SDS-PAGE gel. After transfer to PVDF membrane, an -MYC antibody (9E10, Roche, 11 667 203 001) was used at a 1:5000 dilution to detect MYC-tagged Set1.
Gene expression analysis.
All quantitative real-time PCR(Q-PCR)reactions were performed using a StepOne Real-Time PCR System (Applied Biosystems). To determine GUA1 expression in the indicated strains, 6 mL cultures of each strain were grown to mid-log phase in either YPD or selective media. Total RNA extraction was done with the Qiagen RNEasy kit as per manufacturer’s protocol. Total RNA (2g) was digested with RNase-free DNase (Promega) according to manufacturer’s instructions. Digested RNA (1g) was subjected to a reverse transcription reaction using the iScript cDNA synthesis kit according to manufacturer’s protocol (BioRad). Q-PCR was performed as follows: A 12.5 L reaction was run using 6.25 L SYBR Green master mix (Applied Biosystems), 0.125 L each forward primer and reverse primer (either ACT1-001F and ACT1-002R or GUA1-001F and GUA1-002R) from a 5 M stock, 1 L cDNA of the appropriate concentration, and 5 L water. DNA was amplified with the following program: an initial hold of 95 C for 10 min was followed by 40 cycles at 95 C for 15 s and 60 C for 1 min. Data was analyzed using the Ct method in which actin was used as the endogenous control and expression of GUA1 in each deletion strain was compared to GUA1 expression in a wild-type strain. To detect Jhd2 transcript, cDNA was prepared as described above. Q-PCRwas performed as follows: A 20 L reaction was run using 10 L TaqMan Universal PCR Master Mix (Applied Biosystems), 1 L 20X ACT1 or JHD2 TaqMan gene expression assay mix (Applied Biosystems), 1 L cDNA, and 8 L water. DNA was amplified with the following program: an initial hold of 50 C for 2 min and 95 C for 10 min was followed by 40 cycles at 95 C for 15 s and 60 C for 1 min. Data was analyzed using the Ct method in which actin was used as the endogenous control and expression of JHD2 in experimental strains was compared to expression of JHD2 in a wild-type strain. Primer and probe sequences are indicated below.
Determination of primer PCR efficiency for gene expression analysis using SYBR Green
To determine PCR efficiency of the ACT1 (endogenous control) and GUA1 primer sets, quantitative real-time PCR was performed on a DNA dilution series (1 g, 100 ng, 10 ng, 1 ng, and 0.1 ng genomic DNA from a wild type strain). The PCR efficiency of the ACT1 primer set was 85.9% and the PCR efficiency of the GUA1 primer set was 87.1%. A 12.5 L reaction was run using 6.25 uL SYBR Green master mix (Applied Biosystems), 0.125 L each forward primer and reverse primer from a 5 M stock, 1 L cDNA of the appropriate concentration, and 5 L water. PCR was performed in a StepOne Real-Time PCR System (Applied Biosystems) with the following program: an initial hold of 95 C for 10 min was followed by 40 cycles at 95 C for 15 s and 60 C for 1 min. Three technical repeats were performed for each reaction and a standard curve was generated for both the ACT1 and GUA1 primer sets.
Primer pairs for gene expression analysis using SYBR Green approach
GUA1 and Actin (ACT1) primer sequences used to determine GUA1 expression:
PExpress_GUA1-001F (+305 b.p.): 5’ AGTTGGTCGTGGTGACAAGAGA 3’
PExpress_GUA1-002R (+370 b.p.): 5’ TTGGAATCGTCAATCACCTTCA 3’
ACT1-001F (+766 b.p.): 5’ TGGATTCCGGTGATGGTGTT 3’
ACT1-002R (+837 b.p.): 5’ TCAAAATGGCGTGAGGTAGAGA 3’
Primer pairs for gene expression analysis using TaqMan approach
JHD2 and Actin (ACT1) primer and probe sequences used to determine Jhd2 expression:
JHD2-ANYF (+1023 b.p.): 5’ TCAGCACCGTATTTAACTGTCGTT 3’
JHD2-ANYR (+975 b.p.): 5’ GGAAGAGATGTTTTGGAGCTTAGTGA 3’
JHD2-ANYM1 (+1003 b.p. - +1017 b.p.): 5’ ACTGCGACGATTCTT 3’
Yeast-Acti-ANYF (+815 b.p.): 5’ CTCTCTACCTCACGCCATTTTGA 3’
Yeast-Acti-ANYR (+898 bp): 5’ CCACGTTCACTCAAGATCTTCATCA 3’
Yeast-Acti-ANYM2(+842 b.p. - +856 b.p.): 5’ CTACCGGCCAAATCG 3’
Primer pairs used for chromatin immunoprecipitation analysis using TaqMan approach
The primers and probe used to analyze the 5’ end of GUA1 were designed to amplify GUA1 between 257 bp and 342 bp downstream of the start codon and are the sequences are as follows:
GUA15’ end-ANYF (+257 b.p.): 5’ GGGTATCTGTTACGGTATGCAAGAAT 3’
GUA1_5’ end-ANYR (+342 b.p.): 5’ GGCAGGACCGTACTCTCTCTT 3’
GUA1_5’end-ANYM2(+304 b.p. - +318 b.p.): 5’ CACCACGACCAACTTG 3’
The primers and probe used to analyze the 3’ end of GUA1 were designed to amplify GUA1 between 1335 bp and 1400 bp downstream of the start codon and the sequences are as follows:
GUA13’ end-ANYF (+1335 b.p.): 5’ CCCAAGCCTTTGCCTGTTTG 3’
GUA13’ end-ANYR (+1400 b.p.): 5’ CATAGGTTCTTTGGTCACCCATAACA 3’
GUA1 3’ end-ANYM1(+1354 b.p. - +1368 b.p.): 5’ TTGCCCGTTAAATCC 3’
Primer pairs used for chromatin immunoprecipitation analysis using gel analysis
The sequences of the primers used to analyze the 5’ end of GUA1 are as follows:
GUA1_001F (+229 b.p.): 5’-GCTATCTTTGATTTGAACGTTCC-3’
GUA1_002R (+474 b.p.): 5’-GGAGTTATCGGAGGTGGCAAT-3’
The sequences of the primers used to analyze the 3’ end of GUA1 are as follows:
GUA1_003F (+1183 b.p.): 5’-CATGATCTAGTCTGGAGACAC-3’
GUA1_004R (+1507 b.p.): 5’-CATTGACAATTCTTGAGGCGAC-3’.
Supplemental figure legends
Figure S1. A decrease in Histone H3 K4 trimethylation is dependent on both the presence of Jhd2 and inhibition of the proteasome with the drug MG132. The double deletion strain jhd2 pdr5expressing either empty vector or Jhd2-FLAG was treated with the proteasome inhibitor MG132 for up to 90 minutes as indicated. H3 K4 trimethylation was analyzed in each strain. An antibody directed against H3 was used as a loading control.
Figure S2. CBP-tagged Bre2 is a non-specific substrate for the E3 ubiquitin ligase Not4 in an in vitro ubiquitination assay (A and B) An in vitro ubiquitination assay was performed by incubating CBP-tagged Bre2 and Not4 with Ube1, Ubc4, ATP and free ubiquitin for 0, 30 or 60 min. A and B represent in vitro ubiquitination assays using two different concentrations of CBP-tagged Bre2. CBP-tagged Bre2 was detected with a -CBP antibody. In vitro ubiquitination assays were performed using similar conditions used to generated specific polyubiquitination of Jhd2 and JARID1C (see figure 6A and 6C). In vitroubiquitination assays suggest that Bre2 is not a substrate for polyubiquitination by Not4 when assayed under the same conditions as Jhd2. Results indicate that either no specific bands are detected (A) or that only non-specific bands are detected (B).
Figure S3. Jhd2 is polyubiquitinated in vivo by the E3 ubiquitin ligase Not4. Jhd2 was immunoprecipitated from the indicated strains and analyzed by SDS-PAGE. Polyubiquitination and expression of Jhd2 were confirmed with a -Ubiquitin and -FLAG immunoblots. Bradford assays and immunoblots of histone H3 from whole cell lysates indicated equivalent amounts of lysate was used for each immunoprecipitation assay.
Figure S4. H3 K4 trimethylation is important for wild-type expression of the GMP Synthetase, GUA1. (A) Schematic of GUA1 indicating positions of primers used for gene expression analysis by SYBR Green approach, chromatin immunoprecipitation analysis by TaqMan approach, and chromatin immunoprecipitation analysis by PCR and agarose gel electrophoresis approach. (B) Expression of GUA1 was determined in wild-type, jhd2, not4, and not4 jhd2 strains grown to mid-log phase in YPD media by quantitative real-time PCR analysis. ACT1 (Actin) expression was used as an internal control to normalize expression levels. (C) Chromatin immunoprecipitations of whole cell lysates from wild-type, jhd2, not4, and not4 jhd2 strains grown to mid-log phase in YPD media were performed using antibodies specific to H3 K4 di- or trimethylation and H3. Chromatin immunoprecipitation eluates were PCR amplified using 5’ and 3’ GUA1 gene-specific primer sets and analyzed by agarose gel electrophoresis.
Figure S5. Not4 and Jhd2 control trimethylation at the GUA1 locus. (A – D) Data represented in Figure 7C and D are presented here as separate graphs for GUA1 ChIP analysis. All data are shown as realtive to wildtype. (A) Chromatin immunoprecipitations of whole cell lysates from wild-type, jhd2, not4, and not4 jhd2 strains grown to mid-log phase in YPD media were performed using antibodies specific to H3 K4 trimethylation and H3. Chromatin immunoprecipitation eluates were analyzed by quantitative real-time PCR using 5’ GUA1 gene-specific TaqManprimer sets. (B) Chromatin immunoprecipitations of whole cell lysates from wild-type, jhd2, not4, and not4 jhd2 strains grown to mid-log phase in YPD media were performed using antibodies specific to H3 K4 trimethylation and H3. Chromatin immunoprecipitation eluates were analyzed by quantitative real-time PCR using 3’ GUA1 gene-specific TaqManprimer sets. (C) Chromatin immunoprecipitations of whole cell lysates from wild-type, jhd2, not4, and not4 jhd2strains grown to mid-log phase in YPD media were performed using antibodies specific to H3 K4 dimethylation and H3. Chromatin immunoprecipitation eluates were analyzed by quantitative real-time PCR using 5’ GUA1 gene-specific TaqManprimer sets. (D) Chromatin immunoprecipitations of whole cell lysates from wild-type, jhd2, not4, and not4 jhd2 strains grown to mid-log phase in YPD media were performed using antibodies specific to H3 K4 dimethylation and H3. Chromatin immunoprecipitation eluates were analyzed by quantitative real-time PCR using 3’ GUA1TaqMan gene-specific primer sets.