Supplementary Notes - Word file (197 KB )

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Supplementary Information
Methods
Materials and plasmids
Biotinylated histone peptides were synthesized at Stanford PAN facility or purchased
from Upstate Biotechnology. Antibodies used in study: H2A, H2B, H4, H3-diMeK9, H3triMeK9, H3-AcK14, H4-meK20, tubulin, HDAC1, SAP30, and Sin3a (Upstate
Biotechnology); H3, H3-diMeK4, H3-triMeK4, and H4-AcK8 (Abcam); Cyclin D1 and
GST (Santa Cruz); Flag M5 (Sigma); ING2 1. Taqman Gene Expression Assay
primer/probe sets for Cyclin D1 (FAM/MGB probe) and human GAPDH (VIC/MGB
probe) are from Applied Biosystems. WDR5 shRNAi constructs were purchased from
Open Biosystems and ING2 shRNAi constructs were cloned into pSuper and pSRP as
previously described 1. Targeting sequences: WDR5: RNAi1: 5’GAGAGTGGCTGGCAAGTTC-3’, RNAi2: 5’-GTGGAAGAGTGACTGCTAA-3’;
ING2: RNAi1: 5’-TCGGGCAAGACAAATGGAG-3’; RNAi2: 5’TGGAGTTACACTCACAGTG-3’. ING2 and derivative constructs were either
previously described 1 or cloned into p3xFLAG (Sigma) or pBABE vectors with 3xFLAG-HA epitopes. Mutants were generated by PCR mediated site-directed
mutagenesis. GST-ING1(PHD), GST-Mi2(PHD) and bromodomain of BDF1 were
previously described 1, 2. The PHD domain fusions of ING3 (aa 347-418), ING4 (aa 184249), ING5 (aa 174-240), YNG1 (aa 149-220), YNG2 (aa 202-282), PHO23 (aa 274-331)
and chromodomain of Drosophila HP1 (aa 15-76), EAF3 (66-137), CHD1(251-467) were
cloned into pGEX-6P (Amersham).
In vitro binding assays
2 µg protein were incubated with 1-10 µg of calf thymus total histones (Worthington) in
binding buffer (50 mM Tris-HCl 7.5, 1M NaCl, 1% NP-40, 0.5 mM EDTA, 1 mM
phenylmethyl sulphonyl fluoride (PMSF) plus protease inhibitors (Roche)) at 4 °C for 4
h, followed by an additional 1 h w/glutathione beads (Amersham), similar to as described
in 3. Bound protein were analyzed by SDS-PAGE and detected by Coomassie stain or
Western analysis. For histone peptide binding, 1.0 µg of biotinylated histone peptides
were incubated with 1 g of protein in binding buffer (50 mM Tris-HCl 7.5, 300 mM
NaCl, 0.1% NP-40, 1 mM PMSF plus protease inhibitors) overnight at 4 °C with rotation.
After 1 h incubation with Streptavidin beads (Amersham) and extensive washing, bound
proteins were analyzed by SDS-PAGE and Western blotting. Mononucleosome and
chromatin assembly, purification and binding assays were performed as described 4-6.
Generation of ING2 stable cell lines and ING2 complex purification
HeLa S3 cells stably expressing FLAG-HA-ING2 or ING2 mutants were generated by
retroviral transduction, and nuclear extracts (NE) prepared from ~5X109 cells. ING2
complexes were affinity-purified from NE with anti-FLAG m2 mAb-conjugated agarose
beads (Sigma) in buffer P containing 20 mM Tris-HCl, pH 7.9, 150 mM NaCl, 2 mM
MgCl2, 10% glycerol, 1 mM PMSF, 0.1% NP-40, and eluted with 0.4 mg/ml FLAG
peptides in buffer P. The purified complex was used for enzymatic assays or further
purified with anti-HA agarose.
Enzymatic assays
Purified ING2 complex (5 µl) was incubated with calf thymus histones (5 µg) in
deacetylation buffer (50 mM Tris pH 8.0, 150 mM KCl, 1 mM MgCl2 and 10% glycerol)
supplemented with protease inhibitors for 1 h at 37 °C, and the reaction mixtures
analyzed by SDS-PAGE and Western blot. In vitro methylation of histones for HDAC
assays with SET7 protein was carried out for 30 min in the presence of S-adenosyl
methionine (SAM) as described7. Demethylation assays with LSD1 were performed as
described8.
Protein-protein ChIP, ChIP, RT-PCR, RNAi and cell viability assays
Modified protein-protein ChIP assays for detection of in situ ING2-histone interactions
was performed as described 9. ING2-specific and control shRNA transfection vectors
were purchased from Openbiosystems or previously characterized 1. RNA was prepared
using RNeasy plus kit (Qiagen) and reverse-transcribed using First Strand Synthesis kit
(Invitrogen). Quantitative Real-time RT-PCR was performed in triplicate on the ABI
PRISM 7700 Sequence Detection System. Cyclin D1 expression was calculated
following normalization to GAPDH levels by the comparative Ct (Cycle threshold)
method. ChIP assays were performed according to the protocol from Upstate
Biotechnology. Briefly, HT1080 cells stably expressing RNAi-resistant reconstituted
ING2 or derivatives and shRNA vectors for ING2 or control were transfected with ING2
or control shRNA for two days, then treated with 2 µM doxorubicin for 60 min, followed
by ChIP assays with the indicated antibodies. Semi-quantitative PCR reactions were
performed with ChIP-bound and input DNA, and amplifications within the linear range
for each primer pair were quantified using NIH Image. Primers used in the study are
available upon request. DNA damage sensitivity assays were performed as described 10.
Supplementary Figure Legends
Supplementary Figure S1. ING2 PHD domain specifically binds in vitro to
trimethylated Lysine 4 of histone H3. a. GST-ING2(PHD) pull-downs histone H3 from
HeLa nuclear extract. Shown is Coomassie stain of eluates from GST pull-down assays
resolved by SDS-PAGE. Histone H3 was identified by Western and MALDI mass
spectrometric analysis. WT, wild-type; CKA and 3KA, two lipid-binding deficient
ING2(PHD) domains 1. Asterisk indicates degradation products of GST- ING2(PHD), as
determined by western and mass spectrometry. b, ING2(PHD) does not bind to
nucleosomes reconstituted from E. coli-expressed recombinant histones. Shown are
radiolabeled nucleosomal DNA in intact mono-nucleosomes fractionated on nondenaturing gels, following incubation with GST-ING2(PHD) or GST control proteins 4. c,
Preferential binding of ING2(PHD) domain to trimethylated H3K4 peptides (indicated by
*) in in vitro peptide binding assay. Shown are Western blots of peptide-bound GSTING2(PHD) following histone peptide binding assays with the indicated histone H3
biotinylated peptides. Me, methyl; Ac, acetyl; ph, phospho. d and e, The
chromodomains of CHD1, HP1, and EAF3 bind preferentially to their known targets, H3diMeK4, H3-diMeK9, and H3-di/tri-MeK36 peptides 11-17, respectively, in histone
peptide binding assays.
Supplementary Figure S2. The ING2 PHD domain D230A mutation specifically
abrogates methylated H3K4 binding but not PtdIns(5)P-binding. The indicated GSTING2(PHD) fusion proteins were tested for binding to lipid blots containing serial dilutions
of PtdIns(5)P (PI5P) and PtdIns (PI) (in picomoles) as previously described 1. ING2(PHD)
and ING2(PHD-D230A), are described in the text. ING2(PHD-Y215A), ING2(PHD-V221A),
ING2(PHD-M226K), ING2(PHDY223A/E225A), ING2(PHD-E237A/W238A) and ING2(PHD-W254A) are
described in 18, and ING2(PHD-CKA) and ING2(PHD-264trunc) are PtdIns(5)P-binding mutants
previously described 1.
Supplementary Figure S3. ING2(PHD) association with H3 is correlated with K4
methylation level in vitro.
a, Methylation of H3K4 by SET7 increases ING2(PHD)
binding to H3. Shown are Western blots of calf thymus histones bound to GST-ING2(
PHD)
or GST control protein. SAM, S-adenosyl-methionine, co-factor for SET7. b,
Demethylation of H3K4 by LSD1 decreases ING2(PHD) binding to H3, assayed as in (a).
Supplementary Figure S4. Methyl-lysine recognition is a property of at least a
subset of PHD domains. a, PHD domains of yeast and human ING family preferentially
bind H3-diMeK4 and H3-triMeK4 in vitro. Shown is Western detection of the indicated
peptide-bound GST-PHD domains in histone peptide binding assays using the indicated
biotinylated peptides (Upstate). Me, methyl; Ac, acetyl; Ph, phospho; aa, amino acids.
Assays were performed in 150 mM NaCl; note 300 mM NaCl was used for all other
experiments. b, PHD domain of Mi2 preferentially binds to trimethylated H3K36
peptides (indicated by *) in in vitro histone peptide binding assays.
Supplementary Figure S5. Silver-stained gels of affinity-purified wild-type and
mutant ING2 macromolecular complexes. Bands corresponding to known subunits of
TAP-ING2 complex are indicated 19. We note that the ING2 protein does not silver stain
well.
Supplementary Figure S6. ING2 occupancy across the cyclin D1 gene correlates
with the presence of methylated-H3K4. a, ChIP analyses with indicated antibodies at
the cyclin D1 promoter and 3’ coding region in the indicated cell lines, in the presence or
absence of doxorubicin (2 µM, 1 hr). Flag-ING2, diMe-H3K4 and triMeH3K4 are
detected at the promoter region, but not the 3´ coding region of the cyclin D1 gene.
%input = ChIP/input x 100. b, ChIP analyses with antibodies to H3-diMeK4, H3triMeK4 and total H3 at the cyclin D1 promoter are shown as controls to Figure 4d, in the
presence or absence of dox. %input = ChIP/input x 100 was determined by semiquantitative PCR and represents the average of three to four independent experiments.
Error bars indicate the S.E.M. All p values <0.05.
Supplementary Figure 7. DNA damage-dependent increased ING2 occupancy at the
c-Myc promoter requires H3- triMeK4-binding activity. ChIP assays at the c-Myc
promoters as in Fig 4d, from ING2 shRNA knock-down HT1080 cells reconstituted with
RNAi-resistant flag-tagged wild-type and mutant ING2 proteins. The data are normalized
to untreated samples and represent the average of three independent experiments; error
bars indicate the S.E.M.
Supplementary Figure 8. Model of acute transcriptional repression mediated by
ING2 recognition of trimethylated H3K4. Active genes are marked by trimethylated
H3K4 via the activity of H3K4 histone methyltransferases. In response to a cellular
stress, such as DNA damage, pro-proliferative and pro-survival genes are repressed to
allow for repair of the DNA, or if the damage is too severe, apoptosis. ING2 recognition
of the H3-triMeK4 mark on these actively transcribed genes can stabilize a repressive
HDAC1 complex acutely at these genes, leading to deacetylation and transcriptional
inactivation. By focusing HDAC1 repressor complexes on actively transcribed genes,
recognition of H3-triMeK4 by ING2 may be important for the efficiency of acute gene
repression. Such a mechanism may be particularly important in the context of cellular
responses to acute stress, such as DNA damage insults, in which rapid shut-off of
proliferation genes is critical to prevent propagation of cells harboring damaged DNA.
Thus, the H3-triMeK4 mark can function both in transcriptional activation and
repression, depending on the protein effector that binds to it. Signaling mechanisms, such
as phosphoinositide and inositol polyphosphate signaling, may regulate dynamic
subnuclear trafficking of the ING2/HDAC1 complex to target promoters 20. In addition,
locus-specific transcription factors may target recruitment.
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