Will_4-13-15_GroupMtng_Final

1. A proteomic comparison of Mediator in HeLa and SJSA cells Will Daggett Group Meeting April 13th, 2015

2. CDK8 / CDK19 associate with mediator through a four-protein module CDK8 module MED13/LMED12/L cyclin C CDK8/19 wt CDK8 module CDK8 Mediator Mediator

3. SJSA-1 cells have CDK19, but not CDK8 by western blot CDK8 and 19 are very similar – hard to study in isolation SJSA-1 (osteosarcoma) cells are easy to work with Galbraith et al. Cell. 2013.

4. 1. Do SJSA-1 cells truly have CDK19, but not CDK8? How about other differences in Mediator subunits? 2. Are there unique factors associated with CDK19-Mediator in SJSAs? Questions

5. Hypotheses 1. Unique factors are associated with Mediator in SJSAs 2. SJSA-1 cells have less CDK8, but do have CDK19

6. Approach 1. IP MED1 from SJSA (CDK19) and HeLa (CDK8/19) NE’s a. Submit samples for mass spectrometry b. Compare Mediator subunits in both cell lines c. Other unique factors? 2. IP CDK19 from SJSAs and HeLa a. Evaluate CDK19 IP efficiency – CDK8/19 antibodies b. Compare CDK19-Mediator associated proteins

7. Silver Stain HeLa NE and CDK8 antibody 250 kD 150 100 75 50 Silver Stain HeLa NE and CDK8 antibody 250 kD 150 100 75 50 37 37 Antibody amount optimization and IP practice – CDK8

8. Antibody amount optimization and IP practice – MED1 250 kD 150 100 75 50 25 37 Silver Stain HeLa NE and MED1 antibody

9. MED1 IPs submitted for MS analysis – total protein Sypro Ruby 10 BSA (ng) 20 50 100 150 250 150 100 75 50 37 25 For IP: -20ug MED1 antibody on 20ul dry protein G beads -Equivalent amounts of total protein in NE (10mg/ml HeLa; 2mg/ml SJSA) -2.5 bead volume elutions in low pH glycine ***Found about a 4x difference between the samples***

10. MED1 IPs submitted for MS analysis - Mediator 250 150 100 75 50 37 25 MED12 (1:1k) MED23 (1:1k) CDK8 (1:1k) Cyc C (1:1k) HeLa E1+2 1, 2, 5% HeLa E3 1, 2% SJSA E1+2 1, 2, 5% SJSA E3 1, 2% SJSA IP has less total protein Submit all of SJSA IP for MS – equivalent amount of HeLa/beads SJSA IP has less Mediator SJSA IP has no CDK8 and MED12! Western ***8x difference according to MED23 – used 5x less HeLa eluate***

11. Mass spectrometry sample prep. workflow

12. MS standardization with spectral counts - NSAF Allows us to directly compare proteins across samples! Must standardize for total spectral counts per sample Zhang et al. Anal. Chem. 2010. Where, SAF (spectral abundance factor) = prot. spec. cts / length i = protein identity N = total number of proteins Must standardize for protein length

13. Normalized Mediator comparison with IP-MS – NSAF x 10^3 Gene Description beads HeLa SJSA (HeLa/SJSA) (SJSA/HeLa) MED1_HUMAN Mediator of RNA polymerase II transcription subunit 1 0 5.0 4.7 1.07 0.93 MED4_HUMAN Mediator of RNA polymerase II transcription subunit 4 0 5.9 8.2 0.71 1.40 MED6_HUMAN Mediator of RNA polymerase II transcription subunit 6 0 5.7 7.6 0.76 1.32 MED7_HUMAN Mediator of RNA polymerase II transcription subunit 7 0 3.1 7.6 0.40 2.49 MED8_HUMAN Mediator of RNA polymerase II transcription subunit 8 0 3.5 13.3 0.26 3.80 MED9_HUMAN Mediator of RNA polymerase II transcription subunit 9 0 2.2 2.7 0.82 1.22 MED10_HUMAN Mediator of RNA polymerase II transcription subunit 10 0 6.4 11.1 0.58 1.73 MED11_HUMAN Mediator of RNA polymerase II transcription subunit 11 0 1.0 1.0 1.00 1.00 MED14_HUMAN Mediator of RNA polymerase II transcription subunit 14 0 3.5 7.2 0.48 2.07 MED15_HUMAN Isoform 3 of Mediator of RNA polymerase II transcription subunit 15 0 4.1 4.9 0.84 1.19 MED16_HUMAN Isoform 2 of Mediator of RNA polymerase II transcription subunit 16 0 2.7 7.2 0.38 2.64 MED17_HUMAN Mediator of RNA polymerase II transcription subunit 17 0 4.8 8.8 0.54 1.84 MED18_HUMAN Mediator of RNA polymerase II transcription subunit 18 0 2.6 1.7 1.53 0.65 MED19_HUMAN Mediator of RNA polymerase II transcription subunit 19 0 1.9 1.3 1.45 0.69 MED20_HUMAN Mediator of RNA polymerase II transcription subunit 20 0 6.8 7.7 0.89 1.12 MED21_HUMAN Mediator of RNA polymerase II transcription subunit 21 0 2.5 8.2 0.30 3.32 MED22_HUMAN Isoform Surf5A of Mediator of RNA polymerase II transcription subunit 22 0 4.0 9.0 0.44 2.25 MED23_HUMAN Isoform 4 of Mediator of RNA polymerase II transcription subunit 23 0 1.6 5.8 0.27 3.69 MED24_HUMAN Isoform 2 of Mediator of RNA polymerase II transcription subunit 24 0 2.8 9.8 0.28 3.52 MED25_HUMAN Isoform 5 of Mediator of RNA polymerase II transcription subunit 25 0 0.8 2.1 0.41 2.45 MED26_HUMAN Mediator of RNA polymerase II transcription subunit 26 0 1.8 0 X X MED27_HUMAN Mediator of RNA polymerase II transcription subunit 27 0 2.2 15.3 0.15 6.81 MED28_HUMAN Mediator of RNA polymerase II transcription subunit 28 0 1.2 1.8 0.70 1.43 MED29_HUMAN Mediator of RNA polymerase II transcription subunit 29 0 3.9 5.7 0.68 1.47 MED30_HUMAN Mediator of RNA polymerase II transcription subunit 30 0 3.9 17.3 0.23 4.42 MED31_HUMAN Mediator of RNA polymerase II transcription subunit 31 0 4.4 4.5 0.97 1.03 CDK8_HUMAN Isoform 2 of Cyclin-dependent kinase 8 0 1.3 0 X X CDK19_HUMAN Cyclin-dependent kinase 19 0 1.1 0 X X CCNC_HUMAN Isoform 2 of Cyclin-C 0 2.1 0 X X MED12_HUMAN Isoform 3 of Mediator of RNA polymerase II transcription subunit 12 0 2.3 0 X X MED13_HUMAN Mediator of RNA polymerase II transcription subunit 13 0 1.2 1.2 0.99 1.01 MD13L_HUMAN Mediator of RNA polymerase II transcription subunit 13-like 0 0.2 0.3 0.65 1.55 Sum of SAFs per sample: Beads (HeLa) = 45.7 HeLa MED1 = 29.4 SJSA MED1 = 12.6

17. Normalized Mediator comparison with IP-MS – NSAF x 10^3 Gene Description beads HeLa SJSA (HeLa/SJSA) (SJSA/HeLa) MED1_HUMAN Mediator of RNA polymerase II transcription subunit 1 0 5.0 4.7 1.07 0.93 MED4_HUMAN Mediator of RNA polymerase II transcription subunit 4 0 5.9 8.2 0.71 1.40 MED6_HUMAN Mediator of RNA polymerase II transcription subunit 6 0 5.7 7.6 0.76 1.32 MED7_HUMAN Mediator of RNA polymerase II transcription subunit 7 0 3.1 7.6 0.40 2.49 MED8_HUMAN Mediator of RNA polymerase II transcription subunit 8 0 3.5 13.3 0.26 3.80 MED9_HUMAN Mediator of RNA polymerase II transcription subunit 9 0 2.2 2.7 0.82 1.22 MED10_HUMAN Mediator of RNA polymerase II transcription subunit 10 0 6.4 11.1 0.58 1.73 MED11_HUMAN Mediator of RNA polymerase II transcription subunit 11 0 1.0 1.0 1.00 1.00 MED14_HUMAN Mediator of RNA polymerase II transcription subunit 14 0 3.5 7.2 0.48 2.07 MED15_HUMAN Isoform 3 of Mediator of RNA polymerase II transcription subunit 15 0 4.1 4.9 0.84 1.19 MED16_HUMAN Isoform 2 of Mediator of RNA polymerase II transcription subunit 16 0 2.7 7.2 0.38 2.64 MED17_HUMAN Mediator of RNA polymerase II transcription subunit 17 0 4.8 8.8 0.54 1.84 MED18_HUMAN Mediator of RNA polymerase II transcription subunit 18 0 2.6 1.7 1.53 0.65 MED19_HUMAN Mediator of RNA polymerase II transcription subunit 19 0 1.9 1.3 1.45 0.69 MED20_HUMAN Mediator of RNA polymerase II transcription subunit 20 0 6.8 7.7 0.89 1.12 MED21_HUMAN Mediator of RNA polymerase II transcription subunit 21 0 2.5 8.2 0.30 3.32 MED22_HUMAN Isoform Surf5A of Mediator of RNA polymerase II transcription subunit 22 0 4.0 9.0 0.44 2.25 MED23_HUMAN Isoform 4 of Mediator of RNA polymerase II transcription subunit 23 0 1.6 5.8 0.27 3.69 MED24_HUMAN Isoform 2 of Mediator of RNA polymerase II transcription subunit 24 0 2.8 9.8 0.28 3.52 MED25_HUMAN Isoform 5 of Mediator of RNA polymerase II transcription subunit 25 0 0.8 2.1 0.41 2.45 MED26_HUMAN Mediator of RNA polymerase II transcription subunit 26 0 1.8 0 X X MED27_HUMAN Mediator of RNA polymerase II transcription subunit 27 0 2.2 15.3 0.15 6.81 MED28_HUMAN Mediator of RNA polymerase II transcription subunit 28 0 1.2 1.8 0.70 1.43 MED29_HUMAN Mediator of RNA polymerase II transcription subunit 29 0 3.9 5.7 0.68 1.47 MED30_HUMAN Mediator of RNA polymerase II transcription subunit 30 0 3.9 17.3 0.23 4.42 MED31_HUMAN Mediator of RNA polymerase II transcription subunit 31 0 4.4 4.5 0.97 1.03 CDK8_HUMAN Isoform 2 of Cyclin-dependent kinase 8 0 1.3 0 X X CDK19_HUMAN Cyclin-dependent kinase 19 0 1.1 0 X X CCNC_HUMAN Isoform 2 of Cyclin-C 0 2.1 0 X X MED12_HUMAN Isoform 3 of Mediator of RNA polymerase II transcription subunit 12 0 2.3 0 X X MED13_HUMAN Mediator of RNA polymerase II transcription subunit 13 0 1.2 1.2 0.99 1.01 MD13L_HUMAN Mediator of RNA polymerase II transcription subunit 13-like 0 0.2 0.3 0.65 1.55 Sum of SAFs per sample: Beads (HeLa) = 45.7 HeLa MED1 = 29.4 SJSA MED1 = 12.6 ?

18. Normalized Mediator comparison with IP-MS – NSAF x 10^3 Gene Description beads HeLa SJSA (HeLa/SJSA) (SJSA/HeLa) MED1_HUMAN Mediator of RNA polymerase II transcription subunit 1 0 5.0 4.7 1.07 0.93 MED4_HUMAN Mediator of RNA polymerase II transcription subunit 4 0 5.9 8.2 0.71 1.40 MED6_HUMAN Mediator of RNA polymerase II transcription subunit 6 0 5.7 7.6 0.76 1.32 MED7_HUMAN Mediator of RNA polymerase II transcription subunit 7 0 3.1 7.6 0.40 2.49 MED8_HUMAN Mediator of RNA polymerase II transcription subunit 8 0 3.5 13.3 0.26 3.80 MED9_HUMAN Mediator of RNA polymerase II transcription subunit 9 0 2.2 2.7 0.82 1.22 MED10_HUMAN Mediator of RNA polymerase II transcription subunit 10 0 6.4 11.1 0.58 1.73 MED11_HUMAN Mediator of RNA polymerase II transcription subunit 11 0 1.0 1.0 1.00 1.00 MED14_HUMAN Mediator of RNA polymerase II transcription subunit 14 0 3.5 7.2 0.48 2.07 MED15_HUMAN Isoform 3 of Mediator of RNA polymerase II transcription subunit 15 0 4.1 4.9 0.84 1.19 MED16_HUMAN Isoform 2 of Mediator of RNA polymerase II transcription subunit 16 0 2.7 7.2 0.38 2.64 MED17_HUMAN Mediator of RNA polymerase II transcription subunit 17 0 4.8 8.8 0.54 1.84 MED18_HUMAN Mediator of RNA polymerase II transcription subunit 18 0 2.6 1.7 1.53 0.65 MED19_HUMAN Mediator of RNA polymerase II transcription subunit 19 0 1.9 1.3 1.45 0.69 MED20_HUMAN Mediator of RNA polymerase II transcription subunit 20 0 6.8 7.7 0.89 1.12 MED21_HUMAN Mediator of RNA polymerase II transcription subunit 21 0 2.5 8.2 0.30 3.32 MED22_HUMAN Isoform Surf5A of Mediator of RNA polymerase II transcription subunit 22 0 4.0 9.0 0.44 2.25 MED23_HUMAN Isoform 4 of Mediator of RNA polymerase II transcription subunit 23 0 1.6 5.8 0.27 3.69 MED24_HUMAN Isoform 2 of Mediator of RNA polymerase II transcription subunit 24 0 2.8 9.8 0.28 3.52 MED25_HUMAN Isoform 5 of Mediator of RNA polymerase II transcription subunit 25 0 0.8 2.1 0.41 2.45 MED26_HUMAN Mediator of RNA polymerase II transcription subunit 26 0 1.8 0 X X MED27_HUMAN Mediator of RNA polymerase II transcription subunit 27 0 2.2 15.3 0.15 6.81 MED28_HUMAN Mediator of RNA polymerase II transcription subunit 28 0 1.2 1.8 0.70 1.43 MED29_HUMAN Mediator of RNA polymerase II transcription subunit 29 0 3.9 5.7 0.68 1.47 MED30_HUMAN Mediator of RNA polymerase II transcription subunit 30 0 3.9 17.3 0.23 4.42 MED31_HUMAN Mediator of RNA polymerase II transcription subunit 31 0 4.4 4.5 0.97 1.03 CDK8_HUMAN Isoform 2 of Cyclin-dependent kinase 8 0 1.3 0 X X CDK19_HUMAN Cyclin-dependent kinase 19 0 1.1 0 X X CCNC_HUMAN Isoform 2 of Cyclin-C 0 2.1 0 X X MED12_HUMAN Isoform 3 of Mediator of RNA polymerase II transcription subunit 12 0 2.3 0 X X MED13_HUMAN Mediator of RNA polymerase II transcription subunit 13 0 1.2 1.2 0.99 1.01 MD13L_HUMAN Mediator of RNA polymerase II transcription subunit 13-like 0 0.2 0.3 0.65 1.55 Sum of SAFs per sample: Beads (HeLa) = 45.7 HeLa MED1 = 29.4 SJSA MED1 = 12.6 ?

19. Mediator abundance with MaxQuant CDK19/CCNC are likely limit of detection issues! Few peptides ID’d in HeLa MED12 and MED26 are likely NOT limit of detection issues – not there in SJSAs! Gene names Protein names Un. peps-beads Intensity-beads Un. peps-HeLa Intensity-HeLa Un. peps-SJSA Intensity-SJSA HeLa/SJSA SJSA/HeLa MED1 Mediator of RNA polymerase II transcription subunit 1 0 0 36 1137800000 19 761510000 1.49 0.67 MED4 Mediator of RNA polymerase II transcription subunit 4 0 0 8 281240000 7 376590000 0.75 1.34 MED6 Mediator of RNA polymerase II transcription subunit 6 0 0 4 135590000 4 247150000 0.55 1.82 MED7 Mediator of RNA polymerase II transcription subunit 7 0 0 4 35440000 3 61751000 0.57 1.74 MED8 Mediator of RNA polymerase II transcription subunit 8 0 0 6 131310000 7 473170000 0.28 3.60 MED9 Mediator of RNA polymerase II transcription subunit 9 0 0 3 58757000 2 33613000 1.75 0.57 MED10 Mediator of RNA polymerase II transcription subunit 10 0 0 1 14584000 3 89406000 0.16 6.13 MED11 Mediator of RNA polymerase II transcription subunit 11 0 0 1 11409000 1 5959700 1.91 0.52 MED14 Mediator of RNA polymerase II transcription subunit 14 0 0 18 325090000 30 1106300000 0.29 3.40 MED15 Mediator of RNA polymerase II transcription subunit 15 0 0 10 210060000 6 308430000 0.68 1.47 MED16 Mediator of RNA polymerase II transcription subunit 16 0 0 12 466610000 14 743740000 0.63 1.59 MED17 Mediator of RNA polymerase II transcription subunit 17 0 0 13 280930000 15 528550000 0.53 1.88 MED18 Mediator of RNA polymerase II transcription subunit 18 0 0 5 111340000 2 66021000 1.69 0.59 MED19 Mediator of RNA polymerase II transcription subunit 19 0 0 3 62846000 2 27582000 2.28 0.44 MED20 Mediator of RNA polymerase II transcription subunit 20 0 0 6 216990000 6 307540000 0.71 1.42 MED21 Mediator of RNA polymerase II transcription subunit 21 0 0 1 4579300 2 296360000 0.02 64.72 MED22 Mediator of RNA polymerase II transcription subunit 22 0 0 4 90392000 3 102540000 0.88 1.13 MED23 Mediator of RNA polymerase II transcription subunit 23 0 0 13 142590000 19 543390000 0.26 3.81 MED24 Mediator of RNA polymerase II transcription subunit 24 0 0 13 234470000 19 672050000 0.35 2.87 MED25 Mediator of RNA polymerase II transcription subunit 25 0 0 4 19285000 2 54583000 0.35 2.83 MED26 Mediator of RNA polymerase II transcription subunit 26 0 0 9 88457000 0 0 X 0 MED27 Mediator of RNA polymerase II transcription subunit 27 0 0 5 139100000 9 400350000 0.35 2.88 MED28 Mediator of RNA polymerase II transcription subunit 28 0 0 1 8762400 1 14993000 0.58 1.71 MED29 Mediator of RNA polymerase II transcription subunit 29 0 0 5 104590000 4 258630000 0.40 2.47 MED30S;MED30 Mediator of RNA polymerase II transcription subunit 30 0 0 4 44101000 5 128540000 0.34 2.91 MED31 Mediator of RNA polymerase II transcription subunit 31 0 0 2 42416000 1 36983000 1.15 0.87 CDK8 Cyclin-dependent kinase 8 0 0 1 4874300 0 0 X 0 CDK19 Cyclin-dependent kinase 19 0 0 2 19045000 0 0 X 0 MED12 Mediator of RNA polymerase II transcription subunit 12 0 0 27 351070000 0 0 X 0 MED13 Mediator of RNA polymerase II transcription subunit 13 0 0 19 176110000 6 78870000 2.23 0.45 MED13L Mediator of RNA polymerase II transcription subunit 13-like 0 0 4 21724000 3 24112000 0.90 1.11 Cyclin-C CCNC 0 0 2 50450000 0 0 X 0

20. Conclusions 1. SJSAs have lower amounts of CDK8, MED12 and MED26 than HeLa 2. CDK19 and Cyc C are likely limit of detection issues 4. CDK19 IPs will help with limit of detection issues a. So will bead-clearing IPs in the future a. Why? 3. HeLa and SJSA IPs were well-matched for total protein and Mediator

21. Future Directions • Validate Med26 is actually lower in SJSA by Western Blot • Search specific proteins between HeLa and SJSA • Validate any specific proteins with Western Blot • Replicates and growth of SJSA cells and experiments • Eventually complete CDK19-IP experiments with comparisons

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