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Supplementary Figure Legends
Supplementary Figure 1
Homology alignment of NIR from different species. N.c.: Neurospora crassa (Accession number
XP_329256), S.c.: Saccharomyces cerevisiae (Accession number NP_014849), D.r.: Danio rerio
(Accession number NP_001003830), G.g.: Gallus gallus (Accession number XP_417592), M.m.:
Mus musculus (Accession number AF155546), R.n.: Rattus norvegicus (Accession number
XP_233726), H.s.: Homo sapiens (Accession number BC003555), P.t.: Pan troglodytes (Accession number XP_520841), C.f.: Canis familiaris (Accession number XP_546728), D.m.: Drosophila melanogaster (Accession number NP_610095), A.t.: Arabidopsis thaliana (Accession number
NP_179414). The alignment was performed using ClustalX.
Supplementary Figure 2
Murine Nir cDNA. The full length mouse Nir cDNA sequence was determined by screening mouse
EST-databases (upper panel). The cDNA codes for a 750aa protein (lower panel). The
INHAT homology regions are shaded grey, the putative NLS is boxed and in bold. The
sequence is in accordance with a partial cDNA obtained by the mouse genome sequencing
project (deposited accession number AF155546).
Supplementary Figure 3
Homology alignment of NIR INHAT regions. Protein sequences of human INHATs. NIR
(Accession number BC003555), SET/TAF1(Accession number Q01105), pp32 (Accession number NP_006296), ATAXIN-3 (Accession number BAB55646.1), and PELP1 (Accession number AAC17708) were aligned using ClustalX.
Hublitz et al. 1 Supplementary Figure 4
NIR expression in human tissues. Northern analyses demonstrate a single NIR transcript of 3.3 kb.
A human multiple tissue Northern blot (MTN, BD-Clontech) was hybridized with N-terminal NIR or a -ACTIN probes. sk.m.: skeletal muscle.
Supplementary Figure 5
NIR is a functional INHAT. Bacterially expressed NIR INHAT domains prevent acetylation of
recombinantly expressed Drosophila melanogaster histones dH3 and dH4 by the histone
acetyltransferase p/CAF. To demonstrate specificity, the unrelated protein Nix1 is used as a
control. The INHAT assay is visualized by autoradiography.
Supplementary Figure 6
Functionality of HDAC-inhibitors. BHK cells were transiently transfected with 50ng of the
indicated expression plasmids. Cells were treated with TSA (330nM), Na-butyrate (5mM), or
Nicotinamide (10mM) for 24 hours. The extent of derepression mediated by the inhibitors is
exactly as reported (Minucci et al. 1997, Yang et al. 2005), demonstrating functionality of the
HDAC inhibitors. Error bars represent the standard error of the mean (SEM).
Supplementary Figure 7
NIR does not interact with classical co-repressor complexes. (A) NIR does not interact with members of the HDAC family. [35S]methionine-labeled in vitro translated LSD1, NIR, and HDAC
(HDAC1, 4, 5, and 6) proteins were immunoprecipitated with -NIR (2719) or -Flag (Sigma) antibody. The co-precipitation of Flag-LSD1 with myc-HDAC1 serves as an internal control for the hot IP assay system (Lee et al. 2005). Proteins were visualized by autoradiography. Co- immunoprecipitation fails to detect any interaction between NIR and HDAC1, 4, 5, or 6. (B) NIR
Hublitz et al. 2 does not interact with members of co-repressor complexes. Co-immunoprecipitations were performed with -NIR (2719) antibody. Proteins were visualized by autoradiography.
Supplementary Figure 8
HDAC1 and NIR are not present in the same complex on the p21 promoter. ChIP in
HCT116/p53+/+ cells using -NIR (2910) or -HDAC1 (C19, Santa Cruz) antibody demonstrates that NIR and HDAC1 are both present on the p21 promoter. Re-ChIP demonstrates that NIR and
HDAC1 are not present in the same complex. First ChIP was performed using either -NIR or -
HDAC1 antibody. Re-ChIP was performed using either -HDAC1 antibody and gIgG or -NIR
(2910) antibody and rIgG. The efficient p53/NIR Re-ChIP serves as a control.
Supplementary Figure 9
NIR and p53 interact in vivo. p53 was isolated as a NIR-interacting protein using TAP purification in 293 cells. Tryptic digestion of the corresponding bands and subsequent MALDI-
TOF/TOF analyses identified p53 as one of several NIR interaction partners (18% matched mass values with a sequence coverage of 44%).
Supplementary Figure 10
NIR protein levels are not altered by genotoxic stress. While p53 protein level is induced by addition of doxorubicin in HCT116/p53+/+ cells, the amount of NIR remains unchanged in both p53- containing and p53-deficient HCT116 cells. The Western blot was decorated with -NIR (2719) or
-p53 (DO-1) antibody. -ACTIN serves as an internal control.
Hublitz et al. 3 Supplementary Figure 11
NIR and p53 interact in vivo. (A) Co-immunoprecipitation of TAP-tagged NIR and myc-tagged p53 in HCT116/p53+/+ cells. Membranes were decorated with -NIR (2719) or -p53 (DO-1) antibody. 5% of whole cell extract was used as input. (B) Co-immunoprecipitation of Flag-tagged
NIR and myc-tagged p53 in BHK cells. IPs were performed with either -p53 specific antibodies
DO-1 and DO-7, or the -myc specific 9E10 antibody. Membranes were decorated with the -NIR
2719 or the rabbit -p53 CM1 antibody (Biocare Medical, 1:5000). 5 % of whole cell extract was used as input.
Supplementary Figure 12
Influence of NIR and p53 on reporter constructs lacking p53 binding sites. p53-deficient
Saos-2 and HCT116 cells were co-transfected with luciferase reporters containing mutated
p53 binding sites together with NIR and p53 expression plasmids as indicated. Bars represent
mean +SD (n>6).
Supplementary Figure 13
NIR specifically modulates transcriptional activity of p53. (A) Co-transfection of NIR does not influence the transcriptional properties of Gal-CBF1 in BHK cells. (B) Co-transfection of NIR does not influence the 5-Dihydrotestosterone (DHT)-dependent transcriptional activity of androgen receptor (AR) in BHK cells. (C) Co-transfection of NIR does not influence the DHT-dependent transcriptional activity of AR on a complex promoter in LNCaP cells. (D) Co-transfection of NIR does not influence the thyroid hormone (T3)-dependent transcriptional activity of a Gal-thyroid hormone receptor (TR) fusion protein in BHK cells. BHK or LNCaP cells were co-transfected with
500ng reporter and the indicated amounts of expression plasmids. Error bars indicate the standard error of the mean (SEM).
Hublitz et al. 4 Supplementary Figure 14
NIR knockdown alters p21 expression. NIR-knockdown by a second, independent siRNA (NIR2) results in increased p21 protein level in HCT116/p53+/+ cells. Cells were either untreated (-) or treated (+) with doxorubicin for 6 hours. Western blots were performed at day three post transfection with siRNA. NIR knockdown does not alter p53 protein levels.
Supplementary Figure 15
Depletion of NIR does not increase expression of the p53 target genes BAX, NOXA, and PIG3.
HCT116 cells proficient or deficient in wildtype p53 were depleted for NIR by siRNA (NIR1)
and subsequently challenged with the genotoxic agent doxorubicin. RT-PCR analyses were
performed at day three post transfection with siRNA.
Supplementary Figure 16
Knockdown of NIR by a different siRNA (NIR2) in HCT116/p53+/+ cells also leads to histone hyper-acetylation at p53 target promoters, as shown by ChIP assays using a combination of - acetylated H3 and H4 antibodies. Unrelated promoters (U6 and GAPDH) are not enriched, thus demonstrating specificity.
Supplementary Figure 17
NIR does not influence DNA binding of p53. HCT116/p53+/+cells were treated with siRNA
targeting NIR (NIR1) or with an unrelated control siRNA targeting firefly luciferase.
Occupancy of the endogenous p21 and PIG3 promoters by p53 in presence or absence of NIR
is demonstrated by ChIP using-p53 (DO-7).
Hublitz et al. 5