Development of search tools
In our study, the computational analysis of transcription factor binding site (TFBS) enrichment was performed using an in house custom made tool. A set of 245 position weight matrices (PWMs), corresponding to known vertebrate TFBSs were obtained from the publicly available TRANSFAC database . Binding sites of two other TFs, HIPPI  and NRSF/REST [12, 13] were also included in this list. 10 Kb upstream sequences (5' of TSS) of all Human (version GrCh37) and Mouse (version NCBIM37) protein-coding genes were retrieved using the Biomart utility provided by Ensembl web server (http://www.ensembl.org/biomart/martview). The Human-mouse homolog genes were also determined using the same Biomart utility of the Ensembl web server.
PWM search (including both strands) with various similarity cutoff levels were performed to identify the location of each putative TFBS in all human and mouse 10 Kb upstream sequences. Enrichment of TFBS was analyzed among a target and a background set of upstream sequences using two different statistical tests. Depending on the source organism of the target genes (human or mouse) the entire collection of non-redundant human/mouse upstream sequences was used as the background set. The binding sites for which the cumulative hypergeometric P-value or the Chi square test P-values was less than 0.05 were considered to be enriched in the target upstream sequence set.
Transcription factor binding site analysis
To identify the genes that harbor the 9 bp HIPPI binding motif (AAAGA[G/C]A[A/C/T][T/G]) in their upstream promoter region, we searched the 10 Kb upstream sequences of all the genes in the human genome using the in-house matrix search tool (MST). The Matrix combination search tool (MCST) was used to identify co-occurrence of HIPPI binding sites and other transcription factor binding sites in the gene promoters within a defined distance (100 bp). Functional classification of selected genes was performed by data retrieval tool. Functional classes having hypergeometric p value (corrected using Benjamini and Hochberg method) less than 0.05 were selected.
Additionally, TF binding site analysis and GO analysis on subset of genes were carried out using online tool Genecodis v2.0 [14, 15].
Dataset preparation for Huntington's disease microarray analysis
Microarray data obtained from HD patients' brain sample  were analyzed to study the involvement of HIPPI in HD pathogenesis. In the original study, Hodges et al., analyzed the mRNA expression level in 44 HD patients (Vonsattel Grades 0-4) and 36 age and sex matched controls using Affimetrix HG-U133A and HG-U133B arrays. Expression profiling was done for caudate, cerebellum and two cortical areas, BA4 (motor cortex) and BA9 (prefrontal association cortex). A statistical criterion p < 0.001 was used to obtain the differentially expressed genes. For our analysis, we downloaded the gene expression data for caudate, the brain region mostly affected in HD and sorted them based on the statistical significance (p < 0.05). We used false discovery rate (FDR, Benjamini and Hochberg method) for multiple testing of the array data and genes having corrected p value less than 0.05 were selected for analysis. The gene ids of the differentially regulated genes were converted to their corresponding Ensembl ids using Ensembl Biomart and analyzed using the in house search tools as described above.
Antibodies and other reagents
Geniticin, Hygromycin, and anti Beta-actin (A2228, clone AC-74, Lot number: 107K4791) antibody were obtained from Sigma Chemicals (MO, USA). The anti-mouse and anti-rabbit secondary antibodies conjugated with horseradish peroxidase and protein A agarose beads were purchased from Bangalore Genei, India. Anti HIPPI antibody (ab5205-100, Lot number: 63362) was purchased from Abcam, USA. Anti P53 (IMG 80061) and anti Caspase1 antibodies (IMG-804-4, Lot number: AB093004A) were purchased from Imgenex, USA. Anti HIP1 antibody (NB300-204, 1B11, Lot number: A) was purchased from Novus Biologicals. Immobilon-P Transfer membrane was from Millipore, USA, Taq polymerase from Bioline, USA, and restriction enzymes (BamHI, SalI, and HindIII) were from Promega, USA. Protease inhibitor cocktail was purchased from Roche, USA. TRIZOL reagent was obtained from Invitrogen, USA. Microarray labeling kit was from GE Healthcare. Other molecular biology grade fine chemicals were procured locally.
Construction of clone
Construction of GFP-Hippi has been described earlier . In brief, full length human HIPPI was cloned in pEGFPC1 vector between SalI and BamHI sites. Full length p53 cloned in pCMV neo bam vector was kindly gifted by Dr. Susanta Roychoudhury, Indian Institute of Chemical Biology, Kolkata.
Cell culture and transfection
HeLa and Neuro2A cells were routinely grown in MEM (HIMEDIA, India) supplemented with 10% fetal bovine serum (Biowest, USA.) at 37°C in 5% CO2 atmosphere under humidified condition. Transfection of cells was performed using Lipofectamine 2000 (Invitrogen, USA). Unless otherwise mentioned, for single transfection experiment 2.5 μg (60 mm plate) or 5 μg (100 mm plate) of DNA constructs as well as 5 μl or 10 μl of Lipofectamine 2000 respectively were used. After 24 h, transiently transfected cells were checked for transfection efficiency by monitoring GFP expression under fluorescence microscope and were used for experiments. Transfection efficiency varied from 70-90%.
Knockdown of HIP1 and p53 in HeLa cells
Knock down of HIP1 in HeLa cells by sequence specific siRNA has been described earlier . Briefly, DNA sequences 779-ACCGCTTCATGGAGCAGTTTA-799 of human HIP1 (gi|38045918|ref|NM_005338.4|) were used for designing the siRNA using the online software from GenScript (https://www.genscript.com/ssl-bin/app/rnai). The complete sequence inserted into the expression vector pRNATin-H1.2/Hygro was 5'-TAAACTGCTCCATGAAGCGGTTTGATATCCGACCGCTTCATGGAGCAGTTTATTTTTTCCAA-3' (designated Hip1Si) with termination signal and appropriate restriction site linkers (BamH1 and HindIII, not shown) and an insert for loop formation (underlined). The clones were checked by restriction digestion. HIP1 siRNA clone was transfected in HeLa cells using Lipofectamine 2000 (Invitrogen, USA) following manufacturer's protocol. Stably transfected cells were selected by Hygromycin resistance. Knock down of HIP1 in these cells was confirmed by western blot analysis using anti HIP1 antibody.
For knock down of HIP1 in Neuro 2A cells, the same siRNA construct and protocol was used as described above.
For generating p53 knock down HeLa cell line, pSuppressorNeo p53 plasmid DNA containing p53 siRNA construct (Imgenex, USA, catalog no. IMG 803) was used.
For microarray study, total RNA from cell was extracted using RNeasy Mini Kit (Qiagen, USA) following manufacturer's protocol. RNA samples were quantified by measuring the absorbance at 260 nm and purity was determined using the OD260/OD280 ratio. For cDNA preparation and labeling of the cDNA with fluorescent dyes Cy3 and Cy5, 10 μg of total RNA was reverse transcribed and labeled using CyScribe Post-Labeling Kit (GE Healthcare). Equal concentration of differentially labeled control and test samples were mixed and hybridized to the whole genome human 40 K array (Ocimum Biosolutions, India). Hybridization was carried out over night at 42°C in Hybstation (Genomic Solution). Hybridized array was scanned using GenePix Pro 4200 A scanner. Each experiment was repeated four times, twice with swapping the dye.
Analysis of Microarray data
Analysis of microarray data was carried out by GenePix Pro 6.0 soft ware. The GenePix Result file (GPR) generated for each array were then transferred to Acuity 4.0 soft ware for statistical analysis. Significantly altered probes were identified by student's t test (p < 0.05). Using the same software, the correction for multiple testing was carried out. The detail protocol and array data has been submitted to Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo) GEO accession nos. GSE26115 and GSE26116).
Semi quantitative RT-PCR
Total RNA was extracted from cells using TRIZOL reagent (Invitrogen, USA). Two μg RNA was reverse transcribed using random hexamer primer (Fermentas, USA) and MuLv-Reverse transcriptase (Fermentas, USA). Semi quantitative RT-PCR was carried out using Red Taq DNA polymerase (Bioline, USA). Expression of beta actin was taken as endogenous control. Densitometry of the bands was done using Image Master VDS software (Amarsham Biosciences, UK). The primer sequences used for the gene expression analysis are listed below.
CBP-F: 5' TTGCAGAGGTCTTTGAGCAGG 3'
CBP-R: 5' ATCGCGAGGAATGGTACACAG 3'
GNG10-F: 5' TGGTAGAGCAGCTCAAGTTGG 3'
GNG10-R: 5' TCAGAGTAAAGCACAGGATCTAGG 3'
CREB3L2-F: 5' CCCTTCACCCACATTACCAC 3'
CREB3L2-R: 5' TCATTTCCAGAGGAGGTTCC 3'
CACNG1-F: 5' TGTCCCTCGGGAAGAAGAG 3'
CACNG1-R: 5' CAGGCAAAGGACCAGGAGTA 3'
VTI1A-F: 5' GCAAATTGGTCAGGAGATGTT 3'
VTI1A-R: 5' GATGGTGATGACCACGATGA 3'
NKX2-5-F: 5' ACCCAGCCAAGGACCCTA 3'
NKX2-5-R: 5' GCGTGGACGTGAGTTTCAG 3'
C/EBPβ-F: 5' GAGCAAGGCCAAGAAGACC 3'
C/EBPβ-R: 5' AGCTGCTCCACCTTCTTCTG 3'
ID1-F: 5' GCTCTACGACATGAACGGCTGT 3'
ID1-R: 5' GTTCCAACTTCGGATTCCGAGT 3'
CCL5-F: 5' CTGCTGCTTTGCCTACATTGC 3'
CCL5-R: 5' CCGAACCCATTTCTTCTCTGG 3'
ITPR1-F: 5' ACCTGCTGGTGGCGTTTTT 3'
ITPR1-R: 5' TGAGAGGCAGGAAGAGCAGAGA 3'
Sub-cellular fractionation, Immunoprecipitation and Western Blot analysis
Methods for sub-cellular fractionation, immunoprecipitation and Western blot analysis were essentially the same as described earlier . Briefly, cells grown in 100 mm Petri dishes were washed with ice cold PBS and harvested at 300 g for 3 min at 4°C. Cytosol was extracted using cytosol extraction buffer (50 mM Tris-Cl pH 7.5, 10 mM NaCl, 2 mM EDTA, 1 mM PMSF and 1X protease inhibitor cocktail, 0.25% NP-40). The nuclear pellet was then suspended in nuclear IP buffer (50 mM Tris-Cl pH 7.5, 150 mM NaCl, 2 mM EDTA, 1 mM PMSF and 1X protease inhibitor cocktail) followed by repeated freezing and thawing and centrifugation at 13,000 g for 20 min at 4°C. The extracts were then incubated with anti P53 antibody (1:100 dilution) and BSA soaked protein-A agarose beads and kept overnight at 4°C under continuous rotating condition. Next day the immunoprecipitated complex was collected by centrifugation at 1000 g for 2 min at 4°C. Beads were washed and boiled with SDS gel loading buffer and were subjected to western blot using anti HIPPI and anti P53 antibodies.
For immunoprecipitation assay using whole cell extract, cell lysis was carried out using co-immunoprecipitation buffer (50 mM Tris-Cl pH 7.5, 15 mM EDTA, 100 mM NaCl, 0.1% Triton X-100 and PMSF with 100 μg/ml final concentrations). Beta actin was used as internal loading control. Integrated optical density (IOD) of each band was calculated using Image Master VDS software (Amarsham Biosciences, UK). Whenever necessary, IOD was normalized with that of the loading control.
All the experiments (except microarray experiment) were done for three times. Statistical analysis, mainly unpaired t test was carried out using the on-line software GraphPad QuickCalc available at http://www.graphpad.com/quickcalcs/ttest1.cfm