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

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
Research Article
Cite this article as: eLife 2021;10:e67952 doi: 10.7554/eLife.67952
7 figures, 5 tables and 1 additional file

Figures

Figure 1 with 1 supplement
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 converted to a C-terminal α-thioester and ligated with H4(15–102) A15C. The auxiliary was then reductively cleaved from the ligation product. Cys15 in the final ligation product was desulfurized to the native Ala15 to yield site-specifically sumoylated H4K12su. (B) C4 analytical RP-HPLC trace of purified H4K12su (top). ESI-MS of purified H4K12su (bottom). Calculated mass 21,596.7 Da. Observed, 21,594.2 ± 3.4 Da. (C) Coomassie-stained 15% SDS-PAGE of reconstituted octamers containing wild-type (wt) H4 or H4K12su. (D) Ethidium bromide stained 5% TBE gel of mononucleosomes containing wt H4 or H4K12su. (E) Western blots of p300 assay products with octamer substrates containing wt H4 or H4K12su, probed with a site-independent pan-acetyllysine antibody (top) and an H4K16ac-specific antibody (bottom). An asterisk indicates assays with heat-inactivated p300 to exclude non-enzymatic acetylation. (F) Fluorogram of p300 assay products with [3H]-acetyl-CoA as the co-factor and mononucleosome substrates containing wt H4 or H4K12su.

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
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 representation. The dashed line represents N-terminal residues not observed in the structure. PDB code 1U4A. (C) Coomassie-stained SDS-PAGE of purified catalytic domain of SENP2, cat.SENP2, consisting of residues 365–590. (D) Coomassie-stained SDS-PAGE of purified p300-FLAG from HEK293T cells. (E) Coomassie-stained SDS-PAGE corresponding to the HAT assay shown in Figure 1E. Asterisk indicates heat-inactivated p300 was used. (F) Histone acetylation assay with octamers containing wild-type (wt) H4 or H4K12su. Autoacetylation of p300 was observed with a pan-acetyllysine antibody. (G) Histone acetylation assay with p300 and wt H4 containing octamers and mononucleosomes. No cat.SENP2 was used in this assay. The asterisk indicates pre-incubation of p300-FLAG with acetyl-CoA for 1 hr to allow for the build-up of activating p300-autoacetylation prior to the addition of mononucleosome substrate (Thompson et al., 2004) (H) Histone acetylation assay with wt H4 octamer with or without equimolar amounts of 147 bp Widom 601 double-stranded DNA (dsDNA).

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
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 plasmids chromatinized with wild-type (wt) H4 or H4K12su indicating the similar occupancy and spacing of mononucleosomes. (C) Autoradiogram of 32P-labeled 365 base RNA transcript generated from p300-mediated transcription from chromatinized templates containing wt H4 or H4K12su in the presence or absence of the histone deacetylase (HDAC) inhibitor, trichostatin A.

Figure 2—source data 1

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

https://cdn.elifesciences.org/articles/67952/elife-67952-fig2-data1-v2.pdf
Figure 2—figure supplement 1
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
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 chromatograms of all H4(4–17) tryptic peptides obtained after SDS-PAGE resolution, in-gel desumoylation and trypsination of acetylated chromatin containing H4K12su. The extracted m/z of each spectrum is centered on the [M + 2 H]2+ precursor ion.

Figure 3—figure supplement 1
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
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 with p300 and activator Gal4-VP16. (B) Peptide fragment-ion map of the tetra-acetylated H4(4–17) peptide indicating all ions identified over three spectra.

Figure 3—figure supplement 3
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 with p300 and activator Gal4-VP16. (B) Peptide fragment-ion maps of the four possible tri-acetylated H4(4–17) peptide patterns, indicating all ions identified over three spectra.

Figure 3—figure supplement 4
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 with p300 and activator Gal4-VP16. (B) Peptide fragment-ion maps of the six possible di-acetylated H4(4–17) peptide patterns, indicating all ions identified over two spectra.

Figure 3—figure supplement 5
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 presence of SUMO-3 at K12.

Figure 3—figure supplement 6
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
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
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 cells transfected with HA-Su3(ΔGG)-H4 (HSH4). Input (I) and eluate (E) lanes correspond to undigested bulk chromatin and eluted HA-tagged mononucleosomes containing HSH4. Antibodies targeting H4K12ac and H4K16ac were employed to detect the degree of wild-type (wt) H4 and HSH4 acetylation in HA-tagged mononucleosomes. Total histone H3 in each sample was employed as an equal loading control.

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
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
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. Dotted lines indicate missing N-terminal amino acids. Spp1 (orange) suppressor of PRP protein 1. Swd3 (light blue), Set1 complex WD40 repeat protein 3. Swd1 (red), Set1 complex WD40 repeat protein 1. Set1 (green), SET domain protein 1. Bre2 (pink), brefeldin-A sensitivity protein 2. Sdc1-A/B (yellow), suppressor of CDC25 protein 1. (B) Western blots of the products from methylation assays with mononucleosome substrates containing wild-type (wt) H4 or H4K12su and the COMPASS eCM complex. Mono-, di-, and trimethylated states of H3K4 were detected by the indicated modification-specific antibodies. (C) Western blots of the products from methylation assays with mononucleosome substrates containing wt H4 or H4K12su and the COMPASS catalytic module. Mono-, di-, and trimethylated states of H3K4 were detected by the indicated modification-specific antibodies.

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
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.

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 containing HSH4. Antibodies targeting H3K4me1/2/3 were employed to detect the degree of H3K4 methylation in HA-tagged mononucleosomes. Total histone H3 in each sample was employed as an equal loading control. (B) FLAG-HA-Su3(ΔGG)-H4(Δ1–11) (suH4) protein expression in HEK293 cells and its localization to chromatin within 4 hr. Lanes: 1, Cytoplasmic fraction. 2, Nucleoplasmic fraction. 3, Chromatin fraction. (C) RPMK normalized chromatin immunoprecipitation (ChIP)-seq signals for SUMO-H4 (red, right y-axis) and H3K4me3 (blue, left y-axis) are plotted across length normalized gene bodies for 19,531 UniProt annotated protein coding genes plus 3 kb upstream of the transcription start site (TSS) and 3 kb downstream of the transcription end site (TES).

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
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 proteins associated with Set1 (COMPASS)/SET1 complexes. (B) The inhibition of PolII-mediated transcription from chromatinized templates containing H4K12su is accompanied by reduced p300-mediated H4 tail acetylation. H4K12su also inhibits H3 tail methylation by the extended catalytic module of the COMPASS complex. For clarity, only one of two histone tails, each, is shown for H3 and H4.

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 McGintyPlasmid 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)-H4GenScriptThis study; generated plasmid containing indicated CDS
Recombinant DNA reagentpcDNA5-FLAG-HA-SUMO-3(ΔGG)H4(Δ1–11)This studyThis study; plasmid generated containing indicated CDS
Sequence-based reagentp300_Ctrm_FLAG_RIDT5’-ATC CTT GTA ATC GTG TAT GTC TAG TGT ACT C-3’
Sequence-based reagentp300_Ctrm_FLAG_FIDT5’-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

Additional files

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)