1. Biochemistry and Chemical Biology
  2. Chromosomes and Gene Expression

Sumoylation of the human histone H4 tail inhibits p300-mediated transcription by RNA polymerase II in cellular extracts

  1. Calvin Jon A Leonen
  2. Miho Shimada
  3. Caroline E Weller
  4. Tomoyoshi Nakadai
  5. Peter L Hsu
  6. Elizabeth L Tyson
  7. Arpit Mishra
  8. Patrick MM Shelton
  9. Martin Sadilek
  10. R David Hawkins
  11. Ning Zheng
  12. Robert G Roeder  Is a corresponding author
  13. Champak Chatterjee  Is a corresponding author
  1. Department of Chemistry, University of Washington, United States
  2. Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, United States
  3. Project for Cancer Epigenomics, Cancer Institute of JFCR, Japan
  4. Department of Pharmacology, University of Washington, United States
  5. Howard Hughes Medical Institute, University of Washington, United States
  6. Department of Genome Sciences, Department of Medicine, University of Washington, United States
https://doi.org/10.7554/eLife.67952
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Cite this article
  1. Calvin Jon A Leonen
  2. Miho Shimada
  3. Caroline E Weller
  4. Tomoyoshi Nakadai
  5. Peter L Hsu
  6. Elizabeth L Tyson
  7. Arpit Mishra
  8. Patrick MM Shelton
  9. Martin Sadilek
  10. R David Hawkins
  11. Ning Zheng
  12. Robert G Roeder
  13. Champak Chatterjee
(2021)
Sumoylation of the human histone H4 tail inhibits p300-mediated transcription by RNA polymerase II in cellular extracts
eLife 10:e67952.
https://doi.org/10.7554/eLife.67952
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7 figures, 5 tables and 1 additional file

Figures

Figure 1 with 1 supplement
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Sumoylation inhibits p300-mediated H4 acetylation in octamer and mononucleosome substrates.

(A) Synthetic scheme for H4K12su. (i) An H4(1–14)K12aux peptide was ligated with a SUMO-3 (2–91) C47S α-thioester. (ii) The sumoylated H4(1–14) peptidyl hydrazide containing the auxiliary was …

Figure 1—source data 1

Unedited intact SDS-PAGE gels for all gel images shown in Figure 1.

https://cdn.elifesciences.org/articles/67952/elife-67952-fig1-data1-v2.pdf
Figure 1—figure supplement 1
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Histone octamer and mononucleosome acetylation by p300.

(A) Scheme outlining the p300 histone acetyltransferase assay with octamer and mononucleosome substrates. (B) Cartoon representation of SUMO-3 showing all surface-exposed lysine residues in stick …

Figure 1—figure supplement 1—source data 1

Unedited intact SDS-PAGE gels and western blot membranes for all gel and western blot images shown in Figure 1—figure supplement 1.

https://cdn.elifesciences.org/articles/67952/elife-67952-fig1-figsupp1-data1-v2.pdf
Figure 2 with 1 supplement
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Histone H4 sumoylation inhibits in vitro transcription from chromatinized plasmid templates.

(A) Scheme outlining steps during the in vitro transcription assay with chromatinized plasmids, nuclear extracts, activator Gal4-VP16 and p300. (B) Micrococcal nuclease digestion analysis of …

Figure 2—source data 1

Unedited intact TBE gel for chromatin digestion gel shown in Figure 2.

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Figure 2—figure supplement 1
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Coomassie-stained SDS-PAGE of chromatin assembly proteins and in vitro transcription components.

Asterisk indicates BSA used as a stabilizer.

Figure 2—figure supplement 1—source data 1

Unedited intact SDS-PAGE gel showing protein components of the in vitro transcription assay.

https://cdn.elifesciences.org/articles/67952/elife-67952-fig2-figsupp1-data1-v2.pdf
Figure 3 with 7 supplements
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Comparison of H4 tail acetylation by p300 in chromatinized plasmid templates with activator Gal4-VP16.

(A) Extracted ion chromatograms of all H4(4–17) tryptic peptides obtained after SDS-PAGE resolution and in-gel trypsination of acetylated chromatin containing wild-type (wt) H4. (B) Extracted ion …

Figure 3—source data 1

Excel file listing all mass spectral data plotted in Figure 3, Tables 1–4; and Figure 3—figure supplements 2–7.

https://cdn.elifesciences.org/articles/67952/elife-67952-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
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Coomassie-stained SDS-PAGE of histone acetylation assay on chromatinized plasmids containing wild-type (wt) H4 or H4K12su with p300 and activator Gal4-VP16.

Gel bands excised for tandem mass spectrometry (MS-MS) analysis are indicated.

Figure 3—figure supplement 2
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Tandem MS of tetra-acetylated tryptic peptide H4(4–17).

(A) Representative tandem mass spectrometry (MS-MS) spectrum of tetra-acetylated tryptic peptide H4(4–17) generated after in vitro acetylation of chromatinized plasmids containing wild-type (wt) H4 …

Figure 3—figure supplement 3
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Tandem MS of tri-acetylated tryptic peptide H4(4–17).

(A) Representative tandem mass spectrometry (MS-MS) spectrum of tri-acetylated tryptic peptide H4(4–17) generated after in vitro acetylation of chromatinized plasmids containing wild-type (wt) H4 …

Figure 3—figure supplement 4
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Tandem MS of di-acetylated tryptic peptide H4(4–17).

(A) Representative tandem mass spectrometry (MS-MS) spectrum of di-acetylated tryptic peptide H4(4–17) generated after in vitro acetylation of chromatinized plasmids containing wild-type (wt) H4 …

Figure 3—figure supplement 5
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Representative tandem mass spectrometry (MS-MS) spectrum of tri-acetylated tryptic peptide H4(4–17) generated after in vitro acetylation of chromatinized plasmids containing H4K12su with p300 and activator Gal4-VP16 followed by in-gel desumoylation.

(B) Peptide fragment-ion maps of the four possible tri-acetylated H4(4–17) peptide patterns, indicating all ions identified over three spectra. Acetylation on K12 in H4K12su is not possible due to …

Figure 3—figure supplement 6
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Representative tandem mass spectrometry (MS-MS) spectrum of di-acetylated tryptic peptide H4(4–17) generated after in vitro acetylation of chromatinized plasmids containing H4K12su with p300 and activator Gal4-VP16 followed by desumoylation.

(B) Peptide fragment-ion maps of the six possible di-acetylated H4(4–17) peptide species, indicating all ions identified over three spectra.

Figure 3—figure supplement 7
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The tandem mass spectrometry (MS-MS) spectrum of unacetylated tryptic peptide H4(4–17) after in vitro acetylation of chromatinized plasmids containing H4K12su with p300 and activator Gal4-VP16 followed by desumoylation.

(B) Peptide fragment-ion map of the unmodified H4(4–17) peptide, indicating identified ions.

Figure 4 with 1 supplement
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Biochemical crosstalk between H4 sumoylation and acetylation in HEK293T cells.

(A) Extended micrococcal nuclease digestion of chromatin to generate mononucleosomes that were detected by the presence of ~150 bp DNA in 1.5% agarose gels. (B) Immunoprecipitation (IP) from HEK293 …

Figure 4—source data 1

Unedited intact SDS-PAGE gels and western blot membranes for all gels and western blot images shown in Figure 4.

https://cdn.elifesciences.org/articles/67952/elife-67952-fig4-data1-v2.pdf
Figure 4—figure supplement 1
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Coomassie-stained SDS-PAGE gel of input (I) and elution (E) samples from immunoprecipitation with anti-HA magnetic beads of micrococcal nuclease digested nuclear extracts prepared from HEK293T cells transfected with HA-Su3(ΔGG)-H4.
Figure 5 with 1 supplement
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H3K4 methylation by the extended catalytic module (eCM) of the complex of proteins associated with Set1 (COMPASS) methyltransferase complex is inhibited by H4 sumoylation.

(A) Structure of the COMPASS eCM bound to a mononucleosome (PDB code 6UGM). The disordered H3 and H4 tails are shown in gold and blue, respectively, with the last observable amino acid indicated. …

Figure 5—source data 1

Unedited western blot membranes for all western blot images shown in Figure 5.

https://cdn.elifesciences.org/articles/67952/elife-67952-fig5-data1-v2.pdf
Figure 5—figure supplement 1
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Coomassie-stained SDS-PAGE gel of recombinant Kluyveromyces lactis complex of proteins associated with Set1 (COMPASS) catalytic module (CM) and COMPASS extended catalytic module (eCM) sub-complexes used in H3K4 methylation assays.

The Sdc1 subunit (10 kDa) is not observed on this gel due to its size. Set1-SET is the catalytic domain. Set1-N674 includes the nSET domain, beginning at residue 674.

Figure 6
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Biochemical crosstalk between H4 sumoylation and H3 methylation in HEK293 cells.

(A) Immunoprecipitation (IP) from HEK293 cells transfected with HA-Su3(ΔGG)-H4 (HSH4). Input (I) and eluate (E) lanes correspond to undigested bulk chromatin and eluted HA-tagged mononucleosomes …

Figure 6—source data 1

Unedited intact gels and western blot membranes for all gels and western blot images shown in Figure 6.

https://cdn.elifesciences.org/articles/67952/elife-67952-fig6-data1-v2.pdf
Figure 7
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Mechanisms of chromatin regulation by H4K12su.

(A) Transcription from chromatinized templates containing wild-type (wt) H4 is accompanied by acetylation of all four histones by p300, and with the methylation of the H3 tail by the complex of …

Tables

Table 1
H4(4–17) tail peptides acetylated by p300 in chromatinized plasmid templates with activator Gal4-VP16*,†.
H4(4–17) peptide[M][M + 2 H]2+PSMH4PSMH4K12su
prGKprGGKprGLGKprGGAKprR1549.89775.95n.d.1
prGKprGGKacGLGKprGGAKprR1535.88768.95n.d.n.d.
prGKacGGKacGLGKprGGAKprR1521.86761.9423
prGKacGGKacGLGKacGGAKprR1507.85754.935n.d.‡
prGKacGGKacGLGKprGGAKacR1507.85754.93n.d.3§
prGKacGGKacGLGKacGGAKacR1493.83747.927n.d.‡
  1. *

    Peptides were chemically propionylated before and after trypsinization to cap unmodified lysine side-chains and newly generated N-termini.

  2. †

    Tandem mass spectrometry (MS-MS) spectra observed contained major fragments for the shown modification pattern over other potential patterns, however, no singly acetylated peptides were observed for wild-type (wt) H4.

  3. ‡

    Acetylation at K12 is not possible for H4K12su.

  4. §

    The triply acetylated peptide from H4K12su is blocked from acetylation at K12, but is propionylated after in-gel desumoylation. PSM = peptide spectral match. n.d. = not detected.

Table 2
Comparisons of relative ion intensities of characteristic fragment ions from an enzymatically di-acetylated and chemically propionylated H4(4–17) peptide, [M + 2 H]2+ = 762 Da, after activator and p300-mediated acetylation of chromatinized plasmids containing wild-type (wt) H4*.
SpeciesIon% of Total ion intensityAvg. ratio
K5ac, K8acb5+1.3161.65412.4
K5pr, K8pr0.0720.253
K12ac, K16acy9+0.0420.3060.2
K12pr, K16pr3.1760.971
  1. *

    Only two unique spectra were observed and analyzed for the doubly acetylated and propionylated H4(4–17) tail peptide from wild-type (wt) H4 chromatin.

Table 3
Comparisons of relative ion intensities of characteristic fragment ions from an enzymatically tri-acetylated and chemically propionylated H4(4–17) peptide, [M + 2 H]2+ = 755 Da, after activator and p300-mediated acetylation of chromatinized plasmids containing wild-type (wt) H4*.
SpeciesIon% of Total ion intensityAvg. ratio
K5ac, K8ac, K12acb9+3.0324.9383.98124.1 ± 13.3
K5ac, K8ac, K12pr0.3390.0960.147
K5ac, K8ac, K12acb10+0.8750.8621.10910.3 ± 5.1
K5ac, K8ac, K12pr0.1120.1520.064
K12pr, K16acy8+0.271n.d.0.0120.2
K12ac, K16pr0.8851.0051.263
K12pr, K16acy9+n.d.0.1230.0540.02
K12ac, K16pr2.5874.4426.296
  1. *

    Three unique spectra corresponding to the tri-acetylated and propionylated H4(4–17) tail peptide from wild-type (wt) H4 chromatin were analyzed. Error reported is standard deviation of the mean. n.d. = not detected.

Table 4
Comparisons of relative ion intensities of characteristic fragment ions from an enzymatically di-acetylated, desumoylated, and chemically propionylated H4(4–17) peptide, [M + 2 H]2+ = 762 Da, after activator and p300-mediated acetylation of chromatinized plasmids containing H4K12su*.
SpeciesIon% of Total ion intensityAvg. ratio
K5ac, K8acb5+2.0962.3081.77071.6
K5pr, K8prn.d.0.0710.016
K5ac, K8acb6+2.6032.8352.90033.1
K5pr, K8pr0.053n.d.0.170
K12pr, K16acy6+0.0380.080n.d.0.04
K12pr, K16pr1.2271.3781.375
K12pr, K16acy8+0.0270.0130.1380.1 ± 0.09
K12pr, K16pr0.8150.7320.677
  1. *

    Three unique spectra corresponding to the di-acetylated and propionylated H4(4–17) tail peptide from H4K12su chromatin were analyzed. Error reported is standard deviation of the mean. n.d. = not detected.

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Chemical compound, drugAcetyl-CoARoche10101893001Co-factor for p300 enzyme
Commercial assay or kitCalcium Phosphate Transfection KitThermoK278001Mammalian cell transfection
Commercial assay or kitLipofectamine 3000InvitrogenL3000001Mammalian cell transfection
OtherAnti-DYKDDDDK G1 (mouse monoclonal) Affinity ResinGenScriptL00432 Antibody-conjugated resin for IP
OtherHisPur Ni-NTA resinThermo88221Affinity purification resin
OtherAnti-HA (mouse monoclonal) magnetic beadsPierce88836; RRID:AB_2861399Antibody-conjugated resin for IP
Peptide, recombinant proteinMicrococcal nuclease solutionThermo88216Digestion of dsDNA
OthercOmplete, Mini, EDTA-free protease inhibitor cocktailRoche11836170001Protease inhibitor cocktail
Chemical compound, drug[3H]-acetyl-CoAAmerican Radiolabeled ChemicalsART0213BRadioactive co-factor for p300
Peptide, recombinant proteinPierce Trypsin Protease, MS GradeThermo90057Protein cleavage C-terminal to Arg/Lys
Chemical compound, drugPropionic anhydrideSigma-AldrichP51478Propionylation of peptide lysines and N-terminus
OtherDMEMGibco11956118Cell culture medium
OtherDPBSGibco14190250Cell culture PBS buffer
OtherFetal bovine serumGibco16000044Cell culture medium additive
OtherAmplify fluorographic reagentGE AmershamNAMP100Tritium decay signal amplifier
OtherKodak GBX developer and fixerCarestream Health1900943Immunoblot imaging reagents
Chemical compound, drugTrifluoroacetic acidAlfa AesarAA31771-36Peptide synthesis reagent
Chemical compound, drugFormic acidAcros OrganicsAC147932500Ion-pairing agent for HPLC
Chemical compound, drugAcetonitrile (ACN)FisherA996Solvent for HPLC
OtherC18 Zip tipMilliporeZTC18S096Peptide purification
Chemical compound, drugGlacial acetic acidFisherA38C-212Additive for HPLC solvent
Strain, strain background (Escherichia coli)E. coli BL21(DE3) competent cellsThermoFEREC0114Chemically competent cells
Strain, strain background (Escherichia coli)E. coli DH5α competent cellsNEBC2987HVIALChemically competent cells
Cell line (Homo sapiens)HEK 293TATCCCRL-3216; RRID:CVCL_0063For transient transfection
Cell line (Homo sapiens)Flp-In T-Rex 293 cell lineInvitrogenR78007Stable cell line generation
Recombinant DNA reagentpST100-20xNCP601aGift from Dr Robert K McGinty–Plasmid containing 20 repeats of Widom 601 sequence
Recombinant DNA reagentpcDNA3.1-p300-His6Addgene23252; RRID:Addgene_23252Plasmid for full-length p300
Recombinant DNA reagentpET28a-His6-SENP2(365–590)Addgene16357; RRID:Addgene_16357Plasmid for SUMO protease catalytic domain
Recombinant DNA reagentpcDNA3.1-HA-SUMO-3(ΔGG)-H4GenScript–This study; generated plasmid containing indicated CDS
Recombinant DNA reagentpcDNA5-FLAG-HA-SUMO-3(ΔGG)H4(Δ1–11)This study–This study; plasmid generated containing indicated CDS
Sequence-based reagentp300_Ctrm_FLAG_RIDT–5’-ATC CTT GTA ATC GTG TAT GTC TAG TGT ACT C-3’
Sequence-based reagentp300_Ctrm_FLAG_FIDT–5’-GAT GAC GAT AAA TAG TGA TAC TAA GCT TAA GTT TAA AC-3’
AntibodyRabbit polyclonal anti-acetyllysine antibodyMilliporeAB3879; RRID:AB_11214410WB (1:2000) dilution
AntibodyRabbit polyclonal anti-H4K16ac antibodyActive Motif39167; RRID:AB_2636968WB (1:2000) dilution
AntibodyRabbit polyclonal anti-H4K12ac antibodyActive Motif39066WB (1:2000) dilution
AntibodyRabbit monoclonal anti-H3K4me1Cell Signaling Technology5326; RRID:AB_10695148WB (1:2000) dilution
AntibodyRabbit polyclonal anti-H3K4me2Abcamab7766; RRID:AB_2560996WB (1:2000) dilution
AntibodyRabbit polyclonal anti-H3K4me3Abcamab8580; RRID:AB_306649WB 1:2000 dilution
AntibodyRabbit polyclonal anti-Histone H3Abcamab1791; RRID:AB_302613WB (1:2000) dilution
AntibodyMouse monoclonal anti-Histone H3Abcamab24834; RRID:AB_470335WB (1:2000) dilution
AntibodyRabbit monoclonal anti-HACell Signaling Technology3724; RRID:AB_1549585WB (1:2000) dilution
AntibodyMouse monoclonal anti-FLAGSigma-AldrichF1804; RRID:AB_262044WB (1:2000) dilution
AntibodyAnti-rabbit monoclonal, HRP conjugatedGE HealthcareNA934; RRID:AB_2722659WB (1:40000) dilution
AntibodyIRDye 680RD Goat polyclonal anti-Rabbit IgGLi-COR Biosciences926–68071; RRID:AB_10956166WB (1:15000) dilution
AntibodyIRDye 800CW Goat polyclonal anti-Rabbit IgGLi-COR Biosciences926–32211; RRID:AB_621843WB (1:15000) dilution
AntibodyIRDye 800CW Goat polyclonal anti-Mouse IgGLi-COR Biosciences926–32210; RRID:AB_621842WB (1:15000) dilution

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