Diverse nucleosome Site-Selectivity among histone deacetylase complexes
- Division of Genetics, Department of Medicine, Brigham and Women’s Hospital, United States
- Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, United States
- Leicester Institute of Structural and Chemical Biology, Department of Molecular and Cell Biology, University of Leicester, United Kingdom
Figures
Figure 1
Histone H3 tail acetylation and HDAC complexes.
(A) Different acetylation sites on H3 N-terminal tail studied in this manuscript; (B) Components of four well- established HDAC1 complexes CoREST (LSD1, HDAC1, CoREST1), NuRD (MTA1, HDAC1, RBBP4), …
Figure 2 with 3 supplements
The semi-synthesis of full-length histone H3 with site-specific acetylations.
(A) Synthesis of H3 proteins with site-specific acetylations; gH3: globular region of histone H3; (B) MALDI-MS for a semi-synthetic histone H3 product, using H3K27ac as an example, *: an unknown …
Figure 2—figure supplement 1
MALDI-TOF spectra for H3K9/14/18/23/27ac 1–34 TOG peptides.
Figure 2—figure supplement 2
MALDI-TOF spectra for H3K9/14/18/23/27ac full length histone proteins.
Final product with single charged peak (1H+) and double charged peak (2H+) are labeled; minor gH3 impurity is also labeled; For H3K9ac(C110A) and H3K27ac, *: an unknown minor impurity.
Figure 2—figure supplement 3
The assembly of H3 proteins with acetylations into corresponding nucleosomes.
(A) Assembly of nucleosome with site-specific acetylated H3 in vitro with 146 bp Widom 601 DNA; (B) SDS-PAGE for octamer, using H3K9ac and H3K14ac as examples.
Figure 3 with 2 supplements
Typical results for Western-blot based deacetylation activity assay using different complexes on nucleosome with H3K9ac (n = 2 for each assay).
Figure 3—figure supplement 1
Primary antibody specificity test for anti-H3K23ac and anti-H3K27ac.
SDS-PAGE with histone H3K9ac, H3K23ac and H3K27ac was stained by (A) Coomassie, or transferred to membrane followed by blotting with (B) anti-H3K9ac, (C) anti-H3K23ac, or (D) anti-H3K27ac, separately.
Figure 3—figure supplement 2
The expression of HDAC complexes.
CoREST including LSD1 (1–852), HDAC1 (1–482), Flag tagged CoREST1(86–485); NuRD including Flag tagged MTA1 (1–715), HDAC1 (1–482), RBBP4 (1–425); Sin3B including Flag tagged Sin3B (1–1162), HDAC1 …
Figure 4 with 6 supplements
Typical results for Western-blot based deacetylation activity assay with 5 nM MiDAC on different nucleosomes with site-specific acetylation.
(A) nucleosome with H3K9ac, n = 2, V/E = 1.2 ± 0.19 min−1; (B) nucleosome with H3K14ac, n = 3, V/E = 0.52 ± 0.034 min−1; (C) nucleosome with H3K18ac, n = 2, V/E = 0.17 ± 0.0051 min−1; (D) nucleosome …
Figure 4—figure supplement 1
CoREST (CoREST-LSD1-HDAC1) activity assay over nucleosomes or histones with acetylation at different sites.
(A) H3K9ac (n = 2 for nucleosome assay; n = 3 for histone assay), (B) H3K14ac (n = 3 for nucleosome assay; n = 2 for histone assay); n = 3 for histone assay), (C) H3K18ac (n = 2 for nucleosome assay;…
Figure 4—figure supplement 2
MiDAC (MiDEAS-DNTTIP1-HDAC1) activity assay over nucleosomes or histones with acetylation at different sites.
(A) H3K9ac (n = 2 for nucleosome assay; n = 2 for histone assay), (B) H3K14ac (n = 3 for nucleosome assay; n = 3 for histone assay), (C) H3K18ac (n = 2 for nucleosome assay; n = 2 for histone …
Figure 4—figure supplement 3
NuRD (MTA1-RBBP4-HDAC1) activity assay over nucleosomes or histones with acetylation at different sites.
(A) H3K9ac (n = 2 for nucleosome assay; n = 2 for histone assay), (B) H3K14ac (n = 2 for nucleosome assay; n = 2 for histone assay), (C) H3K18ac (n = 2 for nucleosome assay; n = 2 for histone …
Figure 4—figure supplement 4
Sin3B (Sin3-HDAC1-RBBP4) activity assay over nucleosomes or histones with acetylation at different sites.
(A) H3K9ac (n = 2 for nucleosome assay; n = 2 for histone assay), (B) H3K14ac (n = 3 for nucleosome assay; n = 3 for histone assay), (C) H3K18ac (n = 2 for nucleosome assay; n = 2 for histone …
Figure 4—figure supplement 5
SMRT (GPS2-NCoR2 chimera-HDAC3-TBL1) activity assay over nucleosomes or histones with acetylation at different sites.
(A) H3K9ac (n = 2 for nucleosome assay; n = 3 for histone assay), (B) H3K14ac (n = 3 for nucleosome assay; n = 4 for histone assay), (C) H3K18ac (n = 5 for nucleosome assay; n = 4 for histone …
Figure 4—figure supplement 6
HDAC1 activity assay over nucleosomes or histones with acetylation at different sites.
(A) H3K9ac (n = 3 for nucleosome assay; n = 3 for histone assay), (B) H3K14ac (n = 5 for nucleosome assay; n = 3 for histone assay), (C) H3K18ac (n = 2 for nucleosome assay; n = 4 for histone …
Figure 5 with 3 supplements
Scheme and kinetics for CoREST and MiDAC complex activity on nucleosome with mutations H3K9acR8G (n = 3 for CoREST assay, n = 5 for MiDAC assay) and 14acG13R (n = 6 for CoREST assay, n = 3 for MiDAC assay).
(A) The sequence of H3K9acR8G comparing with WT-H3K9ac; (B) The sequence of H3K14acG13R comparing with WT-H3K14ac; (C) Table for the rate constants of different complexes on nucleosomes with …
Figure 5—figure supplement 1
MALDI-TOF spectra for H3K9acR8G H3K14acG13R 1–34 peptides, and their corresponding full length histone proteins.
Figure 5—figure supplement 2
CoREST activity assay over nucleosomes with H3K9acR8G or H3K14acG13R.
(A) H3K9acR8G (n = 3 for nucleosome assay), (B) H3K14acG13R (n = 6 for nucleosome assay). (C) Curve fitting for H3K9acR8G nucleosome kinetics with 30 nM CoREST, and (D) Curve fitting for H3K14acG13R …
Figure 5—figure supplement 3
MiDAC activity assay over nucleosomes with H3K9acR8G or H3K14acG13R.
(A) H3K9acR8G (n = 5 for nucleosome assay), (B) H3K14acG13R (n = 3 for nucleosome assay). (C) Curve fitting for H3K9acR8G nucleosome kinetics with 5 nM MiDAC, and (D) Curve fitting for H3K14acG13R …
Figure 6 with 1 supplement
Western-blot and kinetics of NuRD complex activity on nucleosome with H4K16ac.
(A) Western-blot based deacetylation activity assay with 40 nM NuRD complex (HDAC1-MTA1-RBBP4) on H4K16ac nucleosomes blot with site-specific acetylation antibody and anti-H3 antibody to show the …
Figure 6—figure supplement 1
NuRD activity assay over H4K16ac nucleosome.
(A) H4K16ac nucleosome (n = 3 for nucleosome assay), and (B) Curve fitting for the nucleosome kinetics with 40 nM NuRD.
Tables
Table 1
Rate constants of different HDAC1/HDAC3 complexes on various histone proteins with site-specific acetylation.
Kinetic values shown are ± S.E.M. For each enzymatic reaction on histone proteins: the semi-synthetic H3K9/14/18/23/27ac histone H3 proteins (1.0 μM) were incubated with different HDAC1 and HDAC3 …
| Normalized V/[E] (min−1) | HDAC1 | CoREST(RCOR1) | MiDAC(MiDEAS) | NuRD(MTA1) | Sin3B(Sin3) | SMRT(NCoR2) |
|---|---|---|---|---|---|---|
| H3K9ac | 0.30 ± 0.046 | 2.1 ± 0.38 | 0.42 ± 0.086 | 0.84 ± 0.38 | 0.56 ± 0.15 | 0.44 ± 0.10 |
| H3K14ac | 0.65 ± 0.40 | 2.8 ± 0.18 | 0.79 ± 0.29 | 1.5 ± 0.58 | 1.1 ± 0.093 | 0.78 ± 0.34 |
| H3K18ac | 1.1 ± 0.27 | 1.8 ± 0.37 | 0.45 ± 0.29 | 0.86 ± 0.32 | 1.2 ± 0.24 | 1.5 ± 0.57 |
| H3K23ac | 0.64 ± 0.10 | 2.6 ± 0.55 | 0.22 ± 0.037 | 0.36 ± 0.12 | 0.93 ± 0.20 | 0.65 ± 0.32 |
| H3K27ac | 1.1 ± 0.25 | 2.0 ± 0.35 | 0.43 ± 0.038 | 1.5 ± 0.48 | 1.1 ± 0.31 | 1.8 ± 0.80 |
Table 2
Rate constants of different HDAC1 and HDAC3 complexes on various nucleosomes with site-specific acetylation on H3.
Kinetic values shown are ± S.E.M. For each enzymatic reaction on nucleosomes: the semi-synthetic H3K9/14/18/23/27ac nucleosomes (100 nM) were incubated with different HDAC1 and HDAC3 complexes at …
| V/[E] (min−1) | HDAC1 | CoREST(RCOR1) | MiDAC(MiDEAS) | NuRD(MTA1) | Sin3B(Sin3) | SMRT(NCoR2) |
|---|---|---|---|---|---|---|
| H3K9ac | N.D. | 0.10 ± 0.012 | 1.2 ± 0.19 | 0.012 ± 0.0032 | 0.0050 ± 0.0043 | 0.046 ± 0.0045 |
| H3K14ac | N.D. | <0.005 | 0.52 ± 0.034 | 0.0091 ± 0.00080 | <0.005 | 0.019 ± 0.0022 |
| H3K18ac | N.D. | 0.062 ± 0.0019 | 0.17 ± 0.0051 | 0.0046 ± 0.00088 | 0.0082 ± 0.0030 | 0.021 ± 0.0039 |
| H3K23ac | N.D. | 0.067 ± 0.010 | 0.048 ± 0.0071 | 0.0071 ± 0.00083 | 0.0084 ± 0.0033 | 0.0081 ± 0.0028 |
| H3K27ac | N.D. | 0.093 ± 0.0088 | 0.18 ± 0.036 | 0.0077 ± 0.0028 | 0.0089 ± 0.00053 | 0.039 ± 0.0027 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (X. laevis) | Histone H2A, H2B, H4 (X. laevis) | DOI: 10.7554/eLife.37231 | Construct shared by Cynthia Wolberger (Johns Hopkins) | |
| Gene (X. laevis) | Histone gH3 (X. laevis) | DOI: 10.7554/eLife.37231 DOI: 10.1021/ja205630g | Construct shared by Dirk Schwarzer (Tübingen) | |
| Gene (S. aureus) | F40-sortase (Originally mutated fromS. aureus sortase A) | DOI: 10.7554/eLife.37231 DOI: 10.1021/ja205630g | Construct shared by Dirk Schwarzer (Tübingen) | |
| Gene (E. coli) | 146 bp 601 DNA (Widom sequence forE. coliexpression) | DOI: 10.7554/eLife.37231 | Construct shared by Song Tan (Penn State) | |
| Strain, strain background (E. coli) | BL21 Rosetta (DE3) pLysS (E. coli) | Sigma-Aldrich | Cat# 70956–4 | Competent cells |
| Cell line (Homo-sapiens) | HEK293F (Homo-sapiens) | Thermo Fisher Scientific | RRID:CVCL_6642 | Transfected with plasmids to express different HDAC1 complexes. Mycoplasma tested negative. Authenticated by the venfor. Not on ICLAC misidentified cell line registry. |
| Antibody | Anti-H3K9ac (Rabbit) | Cell Signaling | Cat# 9671, RRID:AB_331532 | WB (1:2000) |
| Antibody | Anti-H3K14ac (Rabbit) | EMP Millipore | Cat# 07–353, RRID:AB_310545 | WB (1:2000) |
| Antibody | Anti-H3K18ac (Rabbit) | EMP Millipore | Cat# 07–354, RRID:AB_ 441945 | WB (1:2000) |
| Antibody | Anti-H3K23ac (Rabbit) | EMP Millipore | Cat# 07–355, RRID:AB_310546 | WB (1:2000) |
| Antibody | Anti-H3K27ac (Rabbit) | Cell Signaling | Cat# 8173, RRID:AB_10949503 | WB (1:2000) |
| Antibody | Anti-H3 (Rabbit) | Abcam | Cat# ab1791, RRID:AB_302613 | WB (1:5000) |
| Antibody | Anti-H4K16ac (Rabbit) | Active Motif | Cat# 39167, RRID:AB_2636968 | WB (1:5000) |
| Antibody | HRP conjugated anti-Rabbit secondary (Goat) | Cell Signaling | Technology Cat# 7074, RRID:AB_2099233 | WB (1:2000) |
| Peptide, recombinant protein | HDAC1/HSP70 | BPS Bioscience | Cat# 50051 | |
| Peptide, recombinant protein | Nucleosome with H4K16ac | Epicypher | Cat# 16–0354 | |
| Chemical compound | 20 Fmoc-amino acids | EMP Millipore DOI: 10.7554/eLife.37231 | Fmoc-Gly-OH as example: Cat# 852001 | |
| Software, algorithm | ImageJ | DOI: 10.7554/eLife.37231 Download fromimagej.nih.gov/ij/ | RRID:SCR_003070 | |
| Software, algorithm | GraphPad Prism Eight | DOI: 10.7554/eLife.37231 Download fromwww.graphpad.com | RRID:SCR_002798 |
