1. Volume 60, Issue 3, Pages 408-421 (November 2015)
Serine and SAM Responsive Complex SESAME Regulates Histone Modification Crosstalk by Sensing Cellular Metabolism 
Shanshan Li, Selene K. Swanson, Madelaine Gogol, Laurence Florens, Michael P. Washburn, Jerry L. Workman, Tamaki Suganuma 
Molecular Cell 
Volume 60, Issue 3, Pages (November 2015)
DOI: /j.molcel
Copyright © 2015 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2015 60, 408-421DOI: (10.1016/j.molcel.2015.09.024)
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. Figure 1 Identification of SESAME
(A) Western blot showing that global levels of H3pT11 were reduced in PYK1 ts mutant (cdc19-1) compared with WT.
(B) Purified recombinant Pyk1 (rPyk1) phosphorylates purified recombinant H3T11 (rH3T11).
(C) Table of peptides co-purifying with Pyk1, Sam1, Ser33, and controls from MudPIT analysis. PEP, unique peptides; (S), spectrum count; and SC, sequence coverage.
(D) Diagram displaying glycolysis and serine metabolism and the location of the co-purified proteins.
(E and G) The interactions of endogenous Pyk1 with Sam1, Shm2, and Acs2. Cytoplasm and nuclear extracts from cells expressing Shm2-FLAG and Sam1-HA from their endogenous promoters (E) were immunoprecipitated with anti-Pyk1 (E), anti-Sam1 (G), anti-Shm2 (G) antibodies, or IgG. The precipitates were analyzed by western blots for indicated antibodies, including anti-HA and anti-FLAG antibodies. Pgk1 was probed for a cytoplasmic marker. TBP was used for a nuclear marker.
(F) Fraction profiles of FLAG purified Sam1 or Ser33 by Superose 6 HR size-exclusion chromatography. Fraction numbers are indicated.
(H) Endogenously expressed Ser33-FLAG co-immunoprecipitated with endogenously expressed Sam1-HA. See also Figures S1 and S2.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions

4. Figure 2 SESAME Regulates H3pT11 and H3K4me3
(A) Western blot showing that the purified native SESAME complex phosphorylated rH3T11. The relative intensities of H3pT11/H3 quantified with standard error (SE) and p values (P), and biological repeat numbers (n) are indicated (the same with panels B–D and Figures 4A and 4I).
(B) Global levels of the indicated histone modifications in WT, sam1Δ, and sam2Δ strains, cultured in YPD medium were analyzed by western blots (left panels). The relative intensities were indicated (right of each bar graph). The asterisk indicates non-specific bands.
(C) Global levels of the indicated histone modifications in WT and PYK1 temperature sensitive mutant (cdc19-1) were analyzed by western blots. Cells were grown in YPD medium at 37°C for 3 hr.
(D) Ser33 and Shm2, enzymes functioning in the methionine synthesis, were found to be required for maximum H3pT11 and H3K4me3 levels by western blots. See also Figures S1 and S2.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions

5. Figure 3 Serine Metabolism Pathway Regulates H3pT11 and H3K4me3
(A) Western blot showing that addition of serine to media stimulated global levels of H3T11 phosphorylation in vivo.
(B) Western blot showing that addition of serine promoted phosphorylation of rH3T11 by recombinant Pyk1 in vitro.
(C) Western blot showing that addition of serine specifically enhanced rH3T11 phosphorylation by SESAME.
(D) In vitro pyruvate kinase assay showing that addition of serine but not glycine enhanced conversion of PEP to pyruvate by purified rPyk1.
(E) Western blot showing that addition of 2-Deoxy-D-glucose to media inhibited levels of H3pT11 and H3K4me3.
(F) Western blot showing that enzymes functioning in the methionine cycle, Met13 and Met6, are required for H3pT11 and H3K4me3 levels. See also Figure S3.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions

6. Figure 4
Interaction of SESAME with the Set1 Complex Facilitates H3T11 Phosphorylation
(A) Western blot showing global levels of H3pT11 were lost in H3T11A and H3K4A mutants. Three repeats are quantitated with SE (Figure 2A).
(B) Western blots showing the effects of Set1 complex mutants on H3pT11 and H3K4 methylation.
(C) Western blots showing addition of SAM to media stimulates H3pT11 in a Set1-dependent manner.
(D) Diagram shows dual roles of serine in SESAME-mediated H3pT11.
(E) MudPIT analysis showing that SESAME components co-purified with Set1and Bre2.
(F–H) Western blots showing the interactions of endogenous Ser33 with Set1 (F) and the requirements of Ser33 and Shm2 (H) for the interaction of SESAME with Set1 complex (G).
(I) Western blot showing that rSet1 enhanced rH3T11 phosphorylation by SESAME in vitro.
(J) Genetic interaction between SET1 and PYK1 (CDC19).
(K) SHM2 genetically interacted with SET1. See also Figures S4 and S5.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions

7. Figure 5 Genome-wide Co-localization among Subunits of SESAME
(A–D) Composite binding profiles of Sam1 (A), Ser33 (B), Pyk1 (C), and Set1 (D) at 5,065 genes, which were divided into five groups according to their transcriptional rates (transcription rate rages are indicated in each panel). The log2 ratios of normalized enrichment (immunoprecipitation/input) for each gene region, including 500 bp upstream and downstream from the gene, were used for average gene analysis. The transcription start site (TSS) and termination site (TES) are indicated. Anti-FLAG antibody was used for ChIP.
(E) Venn diagram showing the overlap between peaks enriched for Sam1 (orange), Ser33 (blue), and Pyk1 (green). The numbers of binding sites are represented. See also Figure S6.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions

8. Figure 6
Set1 Facilitates Recruitment of SESAME to the Target Genes in Respond to Serine Metabolism
(A) Diagram of the qPCR amplicons for ChIP assay.
(B–H) ChIP assays were performed with anti-FLAG (B, C, D, and E), -Pyk1 (B), -H3pT11 (G and H), -H3K4me3 (F) antibodies using the amplicons at PYK1 indicated in (A). Immunoprecipitated chromatin were measured by qPCR and normalized to input or histone H3. The cells expressing FLAG only were used as negative control (B, left panel). Error bars represent SE. Data represent the mean ± SE of three independent experiments (B–L).
(I and J) ChIP assays for H3K4me3 (I), H3pT11 (I), and Sam1 (J) at PYK1 when 110 mM glucose was present or was depleted from cell culture medium.
(K and L) ChIP analysis of H3K4me3, H3pT11 (K), and Sam1 (L) at PYK1 when cells were treated with/without 10 mM serine. See also Figures S7A–S7E.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions

9. Figure 7 SESAME Suppresses PYK1 Transcription
(A) qRT-PCR analysis of PYK1 transcription in WT, sam1Δ, ser33Δ, and shm2Δ cells, grown in SD minimal medium. Data represent the mean ± SE of six independent experiments.∗p < 0.05.
(B) Pyk1 protein levels in WT, sam1Δ, ser33Δ, and shm2Δ cells were analyzed by western blots. FLAG-Pyk1 was expressed under its own promoter.
(C) PYK1 transcription was increased in H3T11A cells, compared with WT by qRT-PCR analysis. Data represent mean ± SE of three independent experiments.∗p < 0.05.
(D) H3T11A cells were susceptible to oxidative stress when grown in the presence of H2O2.
(E) Summary model proposes that the supplying sufficient glucose promotes serving a substrate from PEP to Pyk1 in SESAME. Set1 is recruited at the promoter of PYK1 and recruits SESAME, which synthesizes SAM for H3K4 methylation. SESAME phosphorylates H3T11 at 5 prime of PYK1 and suppresses the PYK1 transcription. See also Figures S7F–S7H.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2015 Elsevier Inc. Terms and Conditions