<p>Supplementary Figure Legends</p><p>Supplementary Figure 1</p><p>Homology alignment of NIR from different species. N.c.: Neurospora crassa (Accession number</p><p>XP_329256), S.c.: Saccharomyces cerevisiae (Accession number NP_014849), D.r.: Danio rerio</p><p>(Accession number NP_001003830), G.g.: Gallus gallus (Accession number XP_417592), M.m.:</p><p>Mus musculus (Accession number AF155546), R.n.: Rattus norvegicus (Accession number</p><p>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</p><p>NP_179414). The alignment was performed using ClustalX. </p><p>Supplementary Figure 2</p><p>Murine Nir cDNA. The full length mouse Nir cDNA sequence was determined by screening mouse</p><p>EST-databases (upper panel). The cDNA codes for a 750aa protein (lower panel). The</p><p>INHAT homology regions are shaded grey, the putative NLS is boxed and in bold. The</p><p> sequence is in accordance with a partial cDNA obtained by the mouse genome sequencing</p><p> project (deposited accession number AF155546).</p><p>Supplementary Figure 3</p><p>Homology alignment of NIR INHAT regions. Protein sequences of human INHATs. NIR</p><p>(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. </p><p>Hublitz et al. 1 Supplementary Figure 4</p><p>NIR expression in human tissues. Northern analyses demonstrate a single NIR transcript of 3.3 kb.</p><p>A human multiple tissue Northern blot (MTN, BD-Clontech) was hybridized with N-terminal NIR or a -ACTIN probes. sk.m.: skeletal muscle.</p><p>Supplementary Figure 5</p><p>NIR is a functional INHAT. Bacterially expressed NIR INHAT domains prevent acetylation of</p><p> recombinantly expressed Drosophila melanogaster histones dH3 and dH4 by the histone</p><p> acetyltransferase p/CAF. To demonstrate specificity, the unrelated protein Nix1 is used as a</p><p> control. The INHAT assay is visualized by autoradiography.</p><p>Supplementary Figure 6</p><p>Functionality of HDAC-inhibitors. BHK cells were transiently transfected with 50ng of the</p><p> indicated expression plasmids. Cells were treated with TSA (330nM), Na-butyrate (5mM), or</p><p>Nicotinamide (10mM) for 24 hours. The extent of derepression mediated by the inhibitors is</p><p> exactly as reported (Minucci et al. 1997, Yang et al. 2005), demonstrating functionality of the</p><p>HDAC inhibitors. Error bars represent the standard error of the mean (SEM).</p><p>Supplementary Figure 7</p><p>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</p><p>(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</p><p>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.</p><p>Supplementary Figure 8</p><p>HDAC1 and NIR are not present in the same complex on the p21 promoter. ChIP in</p><p>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</p><p>HDAC1 are not present in the same complex. First ChIP was performed using either -NIR or -</p><p>HDAC1 antibody. Re-ChIP was performed using either -HDAC1 antibody and gIgG or -NIR</p><p>(2910) antibody and rIgG. The efficient p53/NIR Re-ChIP serves as a control.</p><p>Supplementary Figure 9</p><p>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-</p><p>TOF/TOF analyses identified p53 as one of several NIR interaction partners (18% matched mass values with a sequence coverage of 44%). </p><p>Supplementary Figure 10</p><p>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</p><p>-p53 (DO-1) antibody. -ACTIN serves as an internal control.</p><p>Hublitz et al. 3 Supplementary Figure 11</p><p>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</p><p>NIR and myc-tagged p53 in BHK cells. IPs were performed with either -p53 specific antibodies</p><p>DO-1 and DO-7, or the -myc specific 9E10 antibody. Membranes were decorated with the -NIR</p><p>2719 or the rabbit -p53 CM1 antibody (Biocare Medical, 1:5000). 5 % of whole cell extract was used as input.</p><p>Supplementary Figure 12</p><p>Influence of NIR and p53 on reporter constructs lacking p53 binding sites. p53-deficient</p><p>Saos-2 and HCT116 cells were co-transfected with luciferase reporters containing mutated</p><p> p53 binding sites together with NIR and p53 expression plasmids as indicated. Bars represent</p><p> mean +SD (n>6).</p><p>Supplementary Figure 13</p><p>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</p><p>500ng reporter and the indicated amounts of expression plasmids. Error bars indicate the standard error of the mean (SEM).</p><p>Hublitz et al. 4 Supplementary Figure 14</p><p>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.</p><p>Supplementary Figure 15</p><p>Depletion of NIR does not increase expression of the p53 target genes BAX, NOXA, and PIG3.</p><p>HCT116 cells proficient or deficient in wildtype p53 were depleted for NIR by siRNA (NIR1)</p><p> and subsequently challenged with the genotoxic agent doxorubicin. RT-PCR analyses were</p><p> performed at day three post transfection with siRNA.</p><p>Supplementary Figure 16</p><p>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. </p><p>Supplementary Figure 17</p><p>NIR does not influence DNA binding of p53. HCT116/p53+/+cells were treated with siRNA</p><p> targeting NIR (NIR1) or with an unrelated control siRNA targeting firefly luciferase.</p><p>Occupancy of the endogenous p21 and PIG3 promoters by p53 in presence or absence of NIR</p><p> is demonstrated by ChIP using-p53 (DO-7). </p><p>Hublitz et al. 5</p>