Genome wide gene expression regulation by HIP1 Protein Interactor, HIPPI: Prediction and validation
© Datta et al; licensee BioMed Central Ltd. 2011
Received: 18 January 2011
Accepted: 26 September 2011
Published: 26 September 2011
HIP1 Protein Interactor (HIPPI) is a pro-apoptotic protein that induces Caspase8 mediated apoptosis in cell. We have shown earlier that HIPPI could interact with a specific 9 bp sequence motif, defined as the HIPPI binding site (HBS), present in the upstream promoter of Caspase1 gene and regulate its expression. We also have shown that HIPPI, without any known nuclear localization signal, could be transported to the nucleus by HIP1, a NLS containing nucleo-cytoplasmic shuttling protein. Thus our present work aims at the investigation of the role of HIPPI as a global transcription regulator.
We carried out genome wide search for the presence of HBS in the upstream sequences of genes. Our result suggests that HBS was predominantly located within 2 Kb upstream from transcription start site. Transcription factors like CREBP1, TBP, OCT1, EVI1 and P53 half site were significantly enriched in the 100 bp vicinity of HBS indicating that they might co-operate with HIPPI for transcription regulation. To illustrate the role of HIPPI on transcriptome, we performed gene expression profiling by microarray. Exogenous expression of HIPPI in HeLa cells resulted in up-regulation of 580 genes (p < 0.05) while 457 genes were down-regulated. Several transcription factors including CBP, REST, C/EBP beta were altered by HIPPI in this study. HIPPI also interacted with P53 in the protein level. This interaction occurred exclusively in the nuclear compartment and was absent in cells where HIP1 was knocked down. HIPPI-P53 interaction was necessary for HIPPI mediated up-regulation of Caspase1 gene. Finally, we analyzed published microarray data obtained with post mortem brains of Huntington's disease (HD) patients to investigate the possible involvement of HIPPI in HD pathogenesis. We observed that along with the transcription factors like CREB, P300, SREBP1, Sp1 etc. which are already known to be involved in HD, HIPPI binding site was also significantly over-represented in the upstream sequences of genes altered in HD.
Taken together, the results suggest that HIPPI could act as an important transcription regulator in cell regulating a vast array of genes, particularly transcription factors and at least, in part, play a role in transcription deregulation observed in HD.
HIPPI (HIP1 Protein Interactor) was identified by Gervais and co-workers as an interacting partner of Huntingtin Interacting Protein 1 (HIP1). HIPPI interacts with HIP1 through its pseudo death effector domain (pDED) and the resulting heterodimer recruits Procaspase8 and activates it thereby inducing Caspase8-mediated apoptosis . Subsequently we have shown in detail the downstream pathways of Caspase8 activation leading to cell death in neuronal and non-neuronal cells . Such non-receptor mediated induction of apoptosis may play a role in Huntington's disease (HD) pathogenesis. HD is an autosomal dominant neurodegenerative disease caused by the expansion of poly glutamine (Q) stretch at the N-terminus of the protein Huntingtin (HTT) . HIP1 interacts strongly with wild type HTT but the interaction is feeble with mutant HTT . Thus, in the diseased condition where one of the HTT allele is mutated, the freely available HIP1 in the cytoplasm may undergo heterodimerization with HIPPI, which in turn activate Caspase8 . In addition, exogenous expression of HIPPI also increases the expression of Caspase1, Caspase3, Caspase7 and Caspase8 in cells as well as it induces truncation of Bid and release of AIF from mitochondria . Although the protein lacks any conventional DNA binding domain, it is shown to interact in vitro and in vivo with a specific 9 bp DNA sequence 5'-AAAGACATG-3' present at the putative promoter of Caspase1 gene and positively regulate its transcription. Using various variants of the motif, we observed that HIPPI binds with the motif (AAAGA[G/C]A[A/C/T][T/G]) [5–7].
Structural analysis of HIPPI failed to detect any known protein domain except for a pseudo death effector domain (pDED) and a myosin like domain (MLD). The protein does not contain nuclear localization signal (NLS) and therefore is expected to translocate to nucleus via some carrier protein. Earlier study from our lab demonstrates that HIP1 acts as the carrier for HIPPI. The HIPPI-HIP1 heterodimeric protein complex formed in cytoplasm enters the nucleus through the NLS present at the C terminus of HIP1  and assembles on the putative promoter of Caspase1 gene to regulate its transcription .
The role of HIPPI as a transcription regulator of Caspase1 thus imparts a new function to the protein. It is, therefore, important to identify other genes that could be regulated by HIPPI and the downstream effect of such regulation in cells. Gene expression regulation is a complex phenomenon in which several transcription factors work in concert to bring about the alteration. Thus, it is also important to look for the involvement of other cellular transcription factors in HIPPI mediated transcription regulation. In an attempt to decipher HIPPI's role as a general transcription regulator, in the present communication, we carried out genome wide search for the presence of HIPPI binding sites in the upstream sequences of genes coded by the human genome. Using our in-house search tool, we also predicted other transcription factors that might co-operate with HIPPI. Finally, to study the global changes in gene expression by HIPPI in cell, microarray experiment was carried out. One of our aims was to investigate the role of HIPPI in Huntington's disease pathogenesis. For this, we analyzed the gene expression data obtained by Hodges et al., . Subsequent analysis of all the data could establish HIPPI's role as a global transcription regulator as well as its involvement in the deregulation of genes in HD.
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.
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'-TAAACTGCTCCATGAAGCGGTTTGATATCCG ACCGCTTCATGGAGCAGTTTATTTTTTCCAA-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
Genome wide search for the presence of HIPPI binding sites in the upstream of coding genes
GO Molecular Function and Biological Process analysis of the genes having HBS within 2 Kb upstream sequence
Data set frequency (%)
Background frequency (%)
p value (unadjusted)
p value (corrected)
Transforming growth factor, beta receptor 1, glutamate receptor, ionotropic, N-methyl D-aspartate 2D, gamma-aminobutyric acid (GABA) B receptor, 2
Sequence-specific DNA binding
cAMP responsive element binding protein 1, Activating transcription factor 2, CCAAT/enhancer binding protein (C/EBP), gamma, POU class 4 homeobox 2
Transcription factor activity
CREB/ATF bZIP transcription factor, cAMP responsive element binding protein 1, activating transcription factor 2, Signal transducer and activator of transcription 4, Transcription factor AP-2 beta
Transcription regulator activity
Inhibitor of DNA binding 1, dominant negative helix-loop-helix protein, POU class 4 homeobox 2, HMG-box transcription factor 1
CREB binding protein, RE1-silencing transcription factor, polymerase (DNA directed), alpha 1, catalytic subunit, SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily e, member 1
Protein kinase, cAMP-dependent, regulatory, type II, beta, CREB binding protein, cAMP responsive element modulator, Transforming growth factor, beta receptor 1, cAMP responsive element binding protein 1, Caspase 1, apoptosis-related cysteine peptidase
Regulation of transcription, DNA-dependent
CREB binding protein, Nuclear transcription factor Y, alpha, Nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 4, cAMP responsive element binding protein 1, Cyclin-dependent kinase inhibitor 2A
Eukaryotic translation initiation factor 6, WD repeat-containing protein 46
Synapsin I, Neurexin 2, Amyloid beta (A4) precursor protein-binding, family A, member 1, Glutamate decarboxylase 1 (brain, 67 kDa), Gamma-aminobutyric acid (GABA) B receptor, 2
Positive regulation of transcription from RNA polymerase II promoter
Nuclear transcription factor Y, alpha, Transforming growth factor, beta 3, CREB binding protein, SMAD family member 4, Thyroid hormone receptor, beta,
DNA damage response, signal transduction resulting in induction of apoptosis
Methyl-CpG binding domain protein 4. Ataxia telangiectasia mutated. CHK2 checkpoint homolog (S. pombe)
Enrichment of other transcription factor binding sites in the vicinity of HIPPI binding site
Alteration of gene expression by HIPPI: microarray study
Altered genes in Microarray Study
No. of probes altered
No. of probes up-regulated
No. of probes down-regulated
Functional classification of the altered genes in microarray study by Gene Ontology
Data set frequency (%)
Background frequency (%)
p value (unadjusted)
p value (corrected)
Transcription factor activity
Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2, NK2 transcription factor related, locus 5 (Drosophila), CCAAT/enhancer binding protein (C/EBP), beta, CREB binding protein
RNA polymerase II transcription factor activity
Forkhead box E1 (thyroid transcription factor 2), Transcription elongation regulator 1
Transcription repressor activity
Peroxisome proliferator-activated receptor delta, RE1-silencing transcription factor, NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 9, 39 kDa
NK2 transcription factor related, locus 5 (Drosophila), CREB binding protein, RE1-silencing transcription factor
Transcription co-activator activity
Transcription elongation regulator 1, junction mediating and regulatory protein, p53 cofactor, CREB binding protein
Sequence-specific DNA binding
v-rel reticuloendotheliosis viral oncogene homolog A (avian), CCAAT/enhancer binding protein (C/EBP), beta, NK2 transcription factor related, locus 5 (Drosophila)
Negative regulation of transcription from RNA polymerase II promoter
SIN3 homolog B, transcription regulator (yeast), RE1-silencing transcription factor
Regulation of transcription, DNA-dependent
Y box binding protein 1, POU class 2 homeobox 2, NK2 transcription factor related, locus 5 (Drosophila), CCAAT/enhancer binding protein (C/EBP), beta
Positive regulation of transcription, DNA-dependent
Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 2, Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1B
Positive regulation of transcription from RNA polymerase II promoter
Zinc finger and BTB domain containing 38, CCAAT/enhancer binding protein (C/EBP), beta, CREB binding protein, Tumor necrosis factor receptor superfamily, member 1A
Eukaryotic translation initiation factor 1B, Eukaryotic translation initiation factor 6
Transmembrane receptor activity
G protein-coupled receptor 123, Toll-like receptor 6
Transcription repressor activity
MYB binding protein (P160) 1a, Runt-related transcription factor 1; translocated to, 1 (cyclin D-related)
Transcription co-repressor activity
Nuclear receptor interacting protein 1, Histone deacetylase 9
Ataxia telangiectasia mutated, Somatostatin receptor 2, Protein kinase, cAMP-dependent, regulatory, type II, alpha, Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, beta
Neurexin 2, Potassium channel, subfamily K, member 3, Somatostatin
negative regulation of transcription from RNA polymerase II promoter
Nuclear receptor interacting protein 1, SP100 nuclear antigen, POU class 3 homeobox 3
Since nuclear localization of HIPPI is HIP1 mediated , we next carried out microarray experiment with HIP1 knocked down HeLa cells (designated as Hip1Si). Empty GFP vector transfected Hip1Si cells (designated as Hip1Si-Gfp) were used as controls while Hip1Si cells transfected with GFP-Hippi construct (designated as Hip1SiHippi) were used as test. Here, we observed alteration of 1652 genes of which 990 were up regulated and 662 were down regulated (p < 0.05, Table 2). The level of alteration of the genes in the two sets of microarray (HeLa-Gfp/HeLa-Hippi and Hip1Si-Gfp/Hip1SiHippi) was compared. It was expected that the genes that were regulated by HIPPI would show a diminished level of alteration in the second case as HIP1 knock down results in cytoplasmic accumulation of HIPPI . We arbitrarily chose 10% as the cut off values for the ratios of expression of genes altered by HIPPI in the presence or reduced expression of HIP1. Such comparisons revealed that 90 genes up-regulated by HIPPI in HeLa cells were decreased in cells where HIP1 was knocked down. Similarly 33 genes that were decreased by exogenous expression of HIPPI in HeLa cells with endogenous HIP1 were increased in HIP1 knocked down cells. We assigned these 123 genes as the potential targets of HIPPI-HIP1 mediated transcriptional regulation and termed them as 'common set'. The lists of these genes are shown in the Additional file 3.
The common set of genes was analyzed for the enrichment of transcription factor binding sites. Binding motifs of ZID (p = 0.02), ELK1 (p = 0.003), XFD1 (p = 0.02), NFKappaB p50 (p = 0.04), AP2 (p = 0.0009) CREB (p = 0.01), GC (p = 0.04), Sp1 (p = 0.02) and AREB6 (p = 0.007, Figure 3c) were enriched in the upstream of up-regulated genes while down-regulated genes contained binding sites for EGR3 (p = 0.02), EGR2 (p = 0.03), CREB (p = 0.03), EVI1/RunX1, (p = 0.02), SREBP1 (p = 0.03), GATA1 (p = 0.02), FOXJ2 (p = 0.04) and PAX4 (p = 0.003, Figure 3d). Here again HBS was not enriched. The reason for this could be the fact that HIPPI is a non conventional transcription factor and was also found to regulate several transcription factors like CREB binding protein (CBP), neuron restrictive silencing factor (NRSF) etc. Therefore it is expected that many genes altered in presence of HIPPI could be due to its secondary effect mediated through the regulation of some of the other crucial transcription factors.
Validation of subset of genes altered in microarray study
These genes along with some others were also tested for their expression in presence of HIPPI in a second cell line like human neuroblastoma cells SHSY5Y. A comprehensive list summarizing all the single gene expression data is given in Additional file 7.
Interaction of HIPPI with P53 in the nuclear compartment of cell and its effect on Caspase1 expression
The interaction of HIPPI and P53 in the nuclear compartment of cell might have important role in HIPPI mediated transcription regulation. From our microarray data we have seen that P53 binding site was enriched in the upstream of up-regulated genes. To elucidate the functional relevance of this interaction we determined the level of Caspase1 expression in presence and absence of these two transcription factors. Human Caspase1 promoter contains a consensus P53 binding site at the position -117 to -98 from TSS . It also contains a HIPPI binding site at the position -119 to -111 from TSS . Thus to see the combinatorial effect of HIPPI and P53 on Caspase1 gene expression we transfected GFP-Hippi exogenously in p53 knocked down HeLa cells (Figure 5d and 5e designated as 'p53SiHi'). Knock down of p53 in HeLa cell was confirmed by western blot analysis . These cells showed reduced expression of Caspase1 as compared to HeLa cells transfected with HIPPI (designated as 'Hippi', n = 3 p = 0.02) or P53 alone (designated as 'p53', p = 0.03). Co-transfection of HIPPI and P53 in HeLa cells (designated as 'p53+Hippi') resulted in even higher level of Caspase1 expression compared to the single transfected cells (p = 0.04). Thus in absence of P53, HIPPI alone could not increase Caspase1 gene expression; rather, the interaction with P53 appeared to be very important for HIPPI mediated up-regulation of the gene.
HIPPI binding sites at the deregulated genes in the caudate of HD patients: possible role of HIPPI in HD
To investigate the role of HIPPI as a novel transcription regulator, we first took bioinformatic approaches to predict the targets of HIPPI and then tried to experimentally validate the predictions. Generally, transcription factor binding sites are enriched near the TSS and the frequency of occurrence of TFBS decreases as we move further upstream. The predominant occurrence of 9 bp consensus HIPPI binding site within 2 Kb upstream sequences of genes was similar to that of known transcription factor CREB (Figure 1). This indicates that HBS might be functionally active and regulate the expression of genes harboring HBS. Next, this set of predicted HIPPI targets were classified functionally using Gene Ontology to understand which biological processes or functions could be regulated by HIPPI in cells. The genes showed significant enrichment for functional categories related to transcriptional regulation, chromatin binding, signal transduction, receptor activity and induction of apoptosis (Table 1 and Additional file 2). It should be mentioned that HIPPI's role in apoptosis induction and transcription regulation has already been reported [1, 2, 5–7, 9]. These functional categories were also found to be significantly over-represented when we analyzed our microarray data. Exogenous expression of HIPPI in HeLa cells resulted in alteration of ~ 1000 genes of which 580 genes were up-regulated and 457 genes were down-regulated (Table 2). Up-regulated genes were mainly enriched in the functional categories like transcription factor activity, transcription co-activator activity, chromatin binding, sequence specific DNA binding, positive regulation of transcription etc. (Table 3) while down-regulated genes were enriched in transcription repressor activity, transcription co-repressor activity, negative regulation of transcription, synaptic transmission etc. (Table 3). This suggests that HIPPI could participate in cellular transcription regulation machinery. To elucidate this property further, we carried out another set of microarray experiment where we blocked nuclear translocation of HIPPI by knocking down its transporter HIP1 . We then compared the genes altered in the two sets of microarray. It was expected that genes that were transcriptional targets of HIPPI would show a reduced level of alteration in HIP1 knocked down cells. Results showed that 90 genes that were up-regulated by HIPPI in HeLa cells (having endogenous HIP1) were up-regulated to a lesser extent in HIP1 knocked down cells (Table 2 and Additional file 3). Similar was the case for 33 down-regulated genes. This subset of altered genes was thus under control of HIPPI-HIP1 mediated transcription regulation.
While analyzing our microarray data we found that multiple testing corrections yielded no statistically significant probes in the array. We, therefore, took the single gene validation approach. From our microarray data we chose 21 genes (unadjusted p value significant) and measured their expression in presence of HIPPI using a second method like semi quantitative RT-PCR or Real time PCR in HeLa (Figure 4) as well as in a second cell line (SHSY5Y, Additional file 7). Fifteen among the 21 genes (71%) showed similar trend as observed in microarray while one (NKX2-5) showed reverse trend. Five genes remained unaltered. Among these 16 validated genes, REST expression was found to be regulated by HIPPI in both neuronal and non-neuronal cells through interaction of HIPPI with REST promoter . Further analysis of these 16 genes using GO revealed that they were enriched in functions like transcription factor activity, transcription co-activator activity, chromatin binding, sequence specific DNA binding and processes like regulation of transcription DNA dependent, positive and negative regulation of transcription from RNA pol II promoter etc. (Additional file 7) similar to those obtained with genome wide analysis (Table 1) and microarray analysis (Table 3). Thus, even though multiple testing of array data indicated lack of statistical significance for the altered probes, our single gene validation experiments suggest that HIPPI indeed interferes with these processes in cell.
Gene expression regulation is a complex phenomenon involving participation of several transcription factors in a co-operative manner . To find out the transcription factor partners of HIPPI we carried out enrichment analysis for TF binding motifs within a fixed 100 bp distance from HBS (Figure 2a). Several transcription factor binding motifs, including NFAT, C/EBP beta, P53, TATA box binding protein, CREBP1 were over-represented in the 100 bp vicinity of HBS (Figure 2b). Presence of TBP, CREBP1 binding sites suggest that HIPPI may take part in basal transcription regulation machinery in cell. Among these TFs, C/EBP beta was found to be up-regulated by HIPPI (Figure 4 and Additional file 3). Binding motifs for GATA1, FOXJ2 and EVI1 have been enriched in the upstream of genes down-regulated by HIPPI (Figure 3c and 3d). Thus, interaction of HIPPI with these TFs may result in transcriptional repression. It was somewhat surprising that HBS was not enriched in the upstream of altered genes. One possible explanation for this could be the fact that HIPPI is not a conventional transcription factor (without DNA binding and transactivation domain). It is, therefore, possible that over-expression of HIPPI in cell increased its interaction with other cellular proteins rather than with DNA. Thus, the primary or direct targets of HIPPI constituted only a small fraction among the perturbed genes . Rest of the altered genes constituted the secondary target set that were differentially expressed in response to the primary set or the changes in cellular physiology brought about by the primary set. For example, GATA3 and EVI1 were down-regulated by HIPPI in microarray study (Additional file 3). Binding sites for GATA3 and EVI1 were enriched in the upstream of down-regulated genes (Figure 3b). Thus a subset of genes down-regulated by HIPPI was effect of GATA3 or EVI1 down-regulation. This was also true for REST. Transcription repressor REST was up-regulated by HIPPI and consequently REST binding site was enriched in the upstream of down-regulated genes (Additional file 4). Similarly increased expression of CBP by HIPPI may result in increased histone acetylation leading to up-regulation of certain genes. It seems therefore important to analyze the primary set (those harboring HBS) separately from the bulk to understand direct transcription regulatory role of HIPPI. The similarities in GO classes between the primary set (Additional file 6) and those predicted from genome wide analysis (Additional file 2) indicates that these cellular processes were indeed perturbed by HIPPI.
HIPPI was found to interact with P53 in the protein level. This interaction took place in the nucleus (Figure 5b) and was dependent on the presence of HIP1, the nuclear transporter of HIPPI as HIPPI did not co-immunoprecipitate with P53 in the nuclear compartment of HIP1 knocked down cells (Figure 5c). This interaction was found to be necessary for HIPPI mediated up-regulation of Caspase1 gene expression. Human Caspase1 gene contains overlapping P53 binding site (position -117 to -98, ) and HIPPI binding site (position -119 to -111, ) in the promoter. It was observed that HIPPI could not induce Caspase1 expression in p53 knocked down HeLa cells (Figure 5d and 5e, comparing 'Hippi' with 'p53SiHi'). When both HIPPI and P53 were co-expressed, the expression of Caspase1 was strongly enhanced compared to the corresponding single transfected cells (Figure 5d and 5e, comparing 'Hippi' and 'p53' with 'p53+Hippi') indicating that both the transcription factors co-operated to regulate this gene expression. Such interaction may also play role in HD pathogenesis. It is observed that P53 protein level is increased in HD cell model . Also HIPPI-HIP1 interaction is more in the diseased condition  which in turn may increase nuclear pool of HIPPI in cell. Therefore, the combinatorial effect of these two regulators will be more in the diseased condition.
Deregulation of transcription and induction of neuronal apoptosis are two of the major contributors to the pathogenesis of HD. Gervais and co-workers identified a novel non receptor mediated apoptotic cascade involving HIPPI and HIP1 that could operate in HD . Work of Majumder et al., and Banerjee et al., imparted a transcription regulatory activity of HIPPI [2, 5, 6, 9]. We therefore, tried to analyze the role of HIPPI in the disease pathogenesis. Microarray data obtained from HD patients  was analyzed for the enrichment of HBS in the upstream of altered genes and we found that HIPPI binding sites were enriched in the set of genes that were down-regulated in HD (Figure 6c). Additionally, the functional categories in which the altered genes in HD belong, bared similarity with the functions played by the HIPPI regulated genes. This includes transcription factor activity (GO:0003700), chromatin binding (GO:0003682), transcription co-activator activity (GO:0003713) for the up-regulated genes and transcription co-repressor activity (GO:0003714) and synaptic transmission (GO:0007268) for the down-regulated genes. We have shown that transcription co-activator CBP was up-regulated by HIPPI which can lead to transcriptional activation of a vast array of genes. Similarly, expression of ID1, an inhibitor that prevents DNA binding of basic helix-loop-helix transcription factors was up-regulated in presence of HIPPI which may cause repression of genes. Recently we have reported that HIPPI mediated transcriptional induction of REST plays an important role in repressing essential neuronal genes such as BDNF in HD cell model . Thus HIPPI mediated transcriptional regulation may indeed contribute to the transcription deregulation observed in HD.
In summary, the manuscript addresses the potential transcription regulatory role played by HIPPI in cell. Using bioinformatic approach we identified a group of HIPPI regulated genes and their possible functions and subsequently validated them with microarray experiments. Although the lack of multiple testing corrections in array data is a limitation of this study, the similarities between functional classes of predicted and experimentally altered genes indicate perturbation of these functions by HIPPI in cells. We also predicted possible co-operativity of HIPPI with other transcription factors in the regulation of gene expression and have shown the synergistic role of HIPPI and P53 in regulation of Caspase1 expression. Finally, the involvement of HIPPI mediated transcription regulation is assessed in the context of Huntington's disease pathogenesis.
In conclusion, the work presented here identifies new transcriptional targets of HIPPI. It also reveals new interaction between HIPPI and P53 and the effect of such interaction in cell. Finally the work predicts the involvement of HIPPI in HD pathogenesis. It would be of immense interest to investigate in detail, the transcription regulatory role played by HIPPI in Huntington's disease.
Acknowledgement and Funding
We acknowledge Prof. Susanta Roychoudhury, Indian Institute of Chemical Biology, Kolkata, for providing the full length p53 clone in pCMV neo bam vector, Ms. Jayeeta Ghose, Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, for p53 knocked down HeLa cell and critical review of the manuscript and Dr. Debashis Mukhopadhyay and Dr. Mithu Raychaudhuri, Structural Genomics Section, Saha Institute of Nuclear Physics, for their help in microarray experiment. We also acknowledge Mr. Utpal Basu and Mr. Saikat Mukherjee, Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, for their technical support.
The work is funded by institutional grant given by Department of Atomic Energy, Govt. of India.
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