DNA-Transcription regulation

HIPPI (HIP-1 protein interactor), also known as ESRRBL1 (estrogen-related receptor beta like 1), a homolog of Chlamydomonas intraflagellar transport 57 (IFT57), does not have any known domain except a ‘pseudo death effector domain’ (pDED) and a myosin like domain (MLD). Interaction of HIPPI with HIP-1 is through the pDED, specifically through 409 K, present in the putative helix5 of HIPPI-pDED, although other regions might have influence on such interactions . HIPPI-HIP-1 heterodimer recruits procaspase-8 and activates the initiator caspase and its downstream apoptotic cascades . It has been shown earlier that the strength of interaction of HIP-1 with Huntingtin (HTT) protein, whose mutation causes Huntington’s disease (HD), is inversely correlated with the number of glutamines (Q) at the N-terminal region of HTT . It is proposed that weaker interaction of HIP-1 with mutated HTT in HD might increase the freely available pool of HIP-1 and might, in turn, enhance the propensity of hetero-dimerization of HIP-1 with HIPPI. The elevated pool of HIPPI-HIP-1 complex may then recruit procaspase-8 and lead to increased cell death as observed in HD .

In addition to increased apoptosis by the activation of different caspases, truncation of Bid, release of AIF from the mitochondria, endogenous expressions of caspase-1, -3, -7 and -8 are also increased in GFP-Hippi expressing Neuro2A and HeLa cells, whereas mitochondrial genes ND1, ND4 and anti-apoptotic gene Bcl-2 are down regulated (2). We have subsequently shown that HIPPI can directly interact both in vitro and in vivo with a 60 bp sequence (–151 to –92) upstream of the caspase-1 gene. HIPPI, especially its C-terminal pDED, interacts with the specific sequence motif AAAGACATG (–101 to –93) present at the promoter sequence of caspase-1 . Similar motifs are also present at the putative promoter sequences of caspase-8 and caspase-10. HIPPI interacts with these promoters and increases the expression of these genes . This result indicates that HIPPI, without having any known DNA-binding domain, interacts with DNA and regulates transcription. Specific amino acid(s) that interact with the DNA sequence still remains unknown. Besides, the question of nuclear translocation of cytoplasmic HIPPI for transcription regulation, without having classical nuclear localization signal (NLS) is yet to be resolved.

HIP-1, the molecular partner of HIPPI interacts with membranes, traffics endocytic vesicles and translocates into the nucleus using its own NLS at the C-terminus and has been implicated in cancer . HIP-1 interacts directly with androgen receptor (AR), accumulates in the nucleus upon androgen stimulation and recruits to DNA elements regulated by AR. AR also translocates to the nucleus in response to androgen and the process is facilitated by HIP-1 . HIP-1 thus regulates the transcription of AR responsive genes through its interaction with AR. Given that HIP-1 can act as the nuclear transporter for AR and regulate the expressions of its target genes, we tested the hypothesis that HIPPI might also be translocated to the nucleus assisted by HIP-1 and could regulate the expression of caspase-1 gene.


Antibodies and other reagents
RNase A, BSA, Geniticin, Hygromycin, DAPI, Hoechst, nuclei isolation kit, anti-Beta-actin (A2228, clone AC-74, Lot number: 107K4791) antibody and Protein G were obtained from Sigma Chemicals (MO, USA). Assay kit for detection of caspase-8 activation was obtained from Alexis Biochemicals, Switzerland. The anti-mouse and anti-rabbit secondary antibodies conjugated with horseradish peroxidase, TRITC and FITC conjugated antibodies were purchased from Bangalore Genei, India; anti-GFP antibody was purchased from BD Biosciences, USA (632375, Lot number: B7040316); anti-histone 2B (H2B) antibody (IMG-359 Lot number: 073101A) and anti-caspase-1 antibody (IMG-804-4, Lot number: AB093004A) were from Imgenex, USA; anti-HIP-1 was purchased from Novus Biologicals (NB300-204, 1B11, Lot number: A); and anti-HIPPI antibody (ab5205-100, Lot number: 63362) and anti-LaminB antibody (ab16048-25, Lot Number 393854) were purchased from Abcam, USA. Immobilon-P Transfer membrane was from Millipore, USA, Chemilumiscence kit from Pierce, USA, Taq polymerase from Bioline, USA, and restriction enzymes (BamHI, SalI, SmaI, XhoI and HindIII) were from Promega, USA. Protease inhibitor cocktail was purchased from Roche, USA. Other molecular biology grade fine chemicals were procured locally.

Methods for modeling and prediction for DNA-binding property
Comparative structure-based modeling was done using MODELLER 8v1 with a multiple structural alignment template, generated by CEMC using 3D co-ordinates of all DED containing proteins (CATH homologous super family 1.10.533.10 [EC] ) from PDB . Out of twenty energy-minimized models, one with the minimum objective function was chosen for generating accessible molecular surface with electrostatic potential map using GRASP . Prediction of DNA-binding property was made using PreDs, by submitting the coordinates of modeled pDED of HIPPI and its mutant protein via the online tool using default parameters (http://pre-s.protein.osaka-u.ac.jp/~preds/). The tool makes prediction of dsDNA-binding site on protein surfaces. The prediction for the query protein is made based on the value of the prediction score, Pscore, which is a vectorial property of the surface and an indicator of the ratio of the predicted area (Parea) to the whole area on the protein surface. The protein with a higher Pscore than 0.12 is considered as a dsDNA-binding protein.

Construction of clones
Constructions of GFP-Hippi and GFP-pDED (coding for 335 to 429 amino acids of HIPPI) have been described earlier . The HIP-1 clone in pcDNA3 (pcDNA3 Hip-1) was kindly provided by Prof. T.S Ross, University of Michigan Medical School, USA. The mutant HIP-1 containing a glutamic acid (E) at the position 58 and 1005 (designated as Hip-1 58E and Hip-1 1005E) clones in pcDNA3 were kindly provided by Dr. Ian Mills, CRUK Uro-Oncology Research Group, Cambridge, CB2 0RE, UK. Full length Hip-1, N terminus Hip-1 (Hip-1N, 1–258aa) and pDED of Hip-1 (Hip-1P, 410–491aa) were sub-cloned in DsRed C1 vector. The details of the primer sequence, PCR condition and restriction enzymes used .




Site directed mutagenesis
A mutation [replacing the Arginin (R) residue at 393 position of HIPPI (59th position of pDED of HIPPI), by Aspartic acid (E) residue (AGA to GGA)] was introduced in GFP-pDED of HIPPI by PCR directed site-specific mutagenesis with GFP-pDED as a template. The recombinant GFP-pDED was subjected to PCR-directed mutagenesis using mutagenic oligonucleotide primers containing mismatch bases. The mutagenic PCR involved the generation of two PCR products that overlap in sequence containing the same mutation introduced as part of the PCR primers. A subsequent re-amplification of these fragments with cloning primers as described earlier resulted in the enrichment of the full-length pDED-HIPPI.

MutR59EF: 5'-GACATTgaAATTGGCATTGTGG-3'

MutR59ER: 5'-GCCAATTtcAATGTCCATCT-3'

The mutation thus introduced was confirmed by sequencing. The plasmid harboring the mutated gene was designated as GFP-mpDED.

Cell culture and transfection
HeLa and Neuro2A cells were routinely grown in MEM (HIMEDIA, India) while K562 cells were grown in RPMI (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 µg (60 mm plate) or 5 µg (100 mm plate) of DNA constructs as well as 7 or 15 µl of Lipofectamine 2000 respectively were used. For co-transfection, equal amount of DNA constructs (5 µg each for 100 mm plate) were used. After 24 h, transiently transfected cells were checked for transfection efficiency by monitoring GFP or DsRed expression under fluorescence microscope and were used for experiments. Transfection efficiency varied from 70 to 90%.

To generate cell lines that stably express wild type and mutant HIP-1, Neuro2A cells were grown in 100 mm plates to ~30% confluency. Cells were then transfected using 5 µl of Lipofectamine 2000 and 2 µg of the required plasmid [pcDNA3Hip-1, Hip-1 1005E, Hip-1 58E]. Transfected cells were then selected using G418 (final concentration 0.4 mg/ml). After 10–15 days, clones were pooled and grown in the presence of G418. Before the experiment selection was withdrawn and used for the studies.

Knockdown of HIP-1 in HeLa and Neuro2A cells by siRNA
DNA sequences 779-ACCGCTTCATGGAGCAGTTTA-799 and 1394-ACAGCGATATAGCAAGCTAAA-1415 of human Hip-1 (gi|38045918|ref|NM_005338.4|) were designed for the siRNAs using the online software from GenScript (https://www.genscript.com/ssl-bin/app/rnai). The scrambled sequence (5'-TAGTCGCATACGGAACATTCG-3') for the first siRNA was also designed using GenScript sequence scrambler tool. The complete sequence which was 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). Similarly for a second siRNA to target the human Hip-1 the complete sequence 5'-TTTAGCTTGCTATATCGCTGTTTGATATCCGACAGCGATATAGCAAGCTAAATTTTTTCCAA-3' (designated as Hip1Si1) was cloned as above. The entire sequence for the scramble siRNA was 5'-CGAATGTTCCGTATGCGACTATTGATATCCGTAGTCGCATACGGAACATTCGTTTTTTCCAA-3' (designated as Hip1Scr). The cloned fragments were sequenced and confirmed. The cloned DNA fragments (Hip1Si and Hip1Si1) were purchased from GeneSript, USA and the Hip1Scr was cloned in our laboratory using the restriction enzymes BamH1 and HindIII. The purchased clones were further checked with the recommended restriction enzymes (BamH1 and HindIII) digestion and used for our experiments. Mouse HIP-1 (gi|22122460|NM_146001) sequences contain a single mismatch (410-ACCGCTTCATGGAGCAGTTCA-430 of mouse HIP-1) compared with that of in human (779-ACCGCTTCATGGAGCAGTTTA-799).

HIP-1 siRNA clones and the scramble siRNA were transfected in HeLa cells using Lipofectamin2000 (Invitrogen, USA) using protocol provided by the manufacturer. Transfected cells were selected for Hygromycin resistance. Colonies grown in presence of Hygromycin were pooled and grown to sufficient numbers for protein isolation and other experiments. Protein was isolated and western blot analysis was carried out using anti HIP-1 antibody. Similarly for Neuro2A cells, Hip1Si was transfected to knock down HIP-1. Similar down regulation of HIP-1 was observed in spite of a single mismatch in siRNA sequence. In parallel, HeLa cells were also transfected with the empty vector (pRNATin-H1.2/Hygro, without the insert) and selected for Hygromycin resistance and used to check the expression of HIP-1. No change in the expression of HIP-1 was observed with cells expressing the empty vector. In all our experiments, we have used HeLa cells or Neuro2A cells as the control, while studying the effect of HIP-1 (in knocked down cells).

Detection of apoptosis by nuclear fragmentation and caspase-8 activation
Nuclear fragmentation and caspase-8 activation were detected using the methods described earlier (2). In brief, cells were grown on cover slips, washed thrice with PBS and fixed with 1:1 mixture of methanol and acetone (1 h at 4°C). Cells were then stained with 1 mM Hoechst in phosphate buffer saline (PBS) in the dark at room temperature for 5 min and observed under a fluorescence microscope (Olympus BX60 with appropriate attachment, Japan). Cells with intact nuclear morphology (normal) and fragmented nuclei (apoptotic cells) were determined and the percentage of cells with apoptotic nuclei was calculated. About 200–500 cells were counted for each experiment. Activation of caspase-8 was determined according to the protocols provided by the manufacturers of the kit. Exponentially growing 2 million HeLa cells expressing GFP–wpDED and GFP-mpDED were collected and processed as mentioned earlier . For the caspase-8 detection, the fluorescence of liberated AFC was measured at its emission maxima ({lambda}max 505 nm) with the excitation at the 400 nm.

Confocal microscopy
Cells were grown on cover slips overnight and were washed with PBS, fixed with 1:1 mixture of methanol and acetone, stained with 4',6-diamino-2-phenylindole (DAPI; final concentration 10 µg/ml) and mounted on clean glass slides using 1–2 µl of glycerol. The cells were visualized under confocal microscope (Zeiss LSM 510) and the localization of GFP tagged and DsRed tagged proteins were observed by exciting at 488 and 543 nm, respectively. Subsequent fluorescence was monitored at 503 nm and 583 nm respectively using LSM 510 software.

Sub-cellular fractionation, immunoprecipitation and western blot analysis
Cells grown in 100 mm Petri dishes were washed with ice cold PBS and harvested at 300g for 3 min at 4°C. The pellet was suspended in cytosol extraction buffer (50 mM Tris–Cl pH 7.5, 10 mM NaCl, 2 mM EDTA, 1 mM PMSF and 1x protease inhibitor cocktail) and kept on ice for 15–20 min. Cells were then lysed by adding 0.25% NP-40 and centrifuged immediately at 800g for 5 min at 4°C. The supernatant was kept as cytosolic extract. The pellet was then suspended in nuclear extraction buffer (50 mM Tris–Cl pH 7.5, 400 mM NaCl, 2 mM EDTA, 1 mM PMSF and 1x protease inhibitor cocktail) and kept on ice for 40 min followed by centrifugation at 13 000g for 20 min at 4°C. The supernatant was kept as nuclear extract. For immunoprecipitation assay with cytosolic and nuclear extract, cytosolic extract was prepared as described above. Nucleus was isolated by resuspending the nuclear pellet in nuclear IP buffer (50 mM Tris–HCl 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 000g for 20 min at 4°C. The extracts are then incubated with anti HIP-1 antibody (1:200 dilutions) for 2 h at 4°C. Next BSA soaked protein-G agarose was added to the reaction mix and incubated over night at 4°C under continuous rotating condition. Next day the immunoprecipitated complex was collected by centrifugation at 1000g for 2 min at 4°C. Beads were washed twice with nuclear IP buffer, once with wash buffer I (50 mM Tris–Cl pH 7.5, 400 mM NaCl, 2 mM EDTA, 1 mM PMSF and 1x protease inhibitor cocktail) and finally with wash buffer II (50 mM Tris–Cl pH 7.5, 400 mM NaCl, 2 mM EDTA, 0.1% Triton X-100, 1 mM PMSF and 1x protease inhibitor cocktail). The bound proteins were extracted from beads by boiling with SDS gel loading buffer and were subjected to western blot using anti HIPPI and anti HIP-1 antibody. The methods used for western blot analysis was essentially the same as described earlier (2).

For immunoprecipitation assay using whole cell extract, cells grown in 10 cm Petri dishes were washed in ice cold PBS and harvested at 300g for 3 min at 4°C. The pellet was suspended in 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), lysed by freezing and thawing and centrifuged at 13 000g for 15 min. The supernatant was collected and protein was estimated. To detect GFP tagged proteins, immunoprecipitation with anti-GFP antibody (1:500 dilutions) was carried out using the protocol described above. The beads were washed several times with co-immunoprecipitation buffer and the precipitated complex was extracted from beads by boiling with SDS gel loading dye. Western blot analysis was performed using anti-GFP, anti-caspase-8 and anti-HIP-1 antibodies. For other proteins, western blot analysis was carried out using anti HIP-1; anti HIPPI, anti-GFP, anti-H2B, anti Lamin B and anti Beta actin antibodies after standardizing for appropriate dilutions. Beta actin was used as internal control for cytoplasmic extract while H2B or lamin B was used for loading controls for proteins in the nuclear fractions. Each experiment was repeated 2–3 times. Integrated optical density (IOD) of each band was calculated using Image Master VDS software (Amarsham Biosciences, UK). When ever necessary, IOD was normalized with that of the loading control.

Immunocytochemistry
Cells were grown on cover slips and transfected with respective constructs whenever necessary as described above. Cells (after 24 h of transfection, for transfected cells) were fixed with 3.7% freshly prepared Paraformaldehyde for 20 min at room temperature, washed thrice with PBS, and permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature. It was then rinsed thrice with PBS and blocked in 2% BSA for 1 h. The coverslips containing cells were then incubated for 1 h at 37°C with primary antibodies (anti HIP-1, anti HIPPI) with (1:50) dilutions. Cells were then washed thrice with PBS and incubated with fluorophore-conjugated (FITC or TRITC) secondary antibodies at 37°C in dark. Cover slips were again washed with PBS and then mounted on slides. Photographs were taken using (ZEISS LSM 510) confocal microscope.

Chromatin immunoprecipitation (ChIP) and re-chromatin immunoprecipitation (reChIP) assay
Methods used for the ChIP experiments were adapted from the results published earlier (2). GFP-wpDED or GFP-mpDED transfected HeLa cells and K562 were grown on petri plates to 80–90% confluency as mentioned above. Cells were incubated with 2% formaldehyde for 2 min at room temperature to cross-link the proteins with the DNA. This cross-linking reaction was stopped using 150 mM Glycine. Cells were scraped and spun down at 300 g for 2 min, washed twice with PBS and the pellet was frozen in dry ice for 20 mins. Buffer C (20 mM HEPES (pH 7.9), 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA) with 1 mM PMSF was added to the pellet after thawing at 4°C to lyse the cells. Nuclei were spun down at 15 000 g for 10 min and the pellet was re-suspended in breaking buffer (50 mM Tris–HCl pH 8.0, 1 mM EDTA, 150 mM NaCl, 1% SDS and 2% Triton X-100) and sonicated twice (two pulses of 10 s each). Contents were then spun down. The pellet was discarded. Triton buffer (50 mM Tris–HCl pH 8.0, 1 mM EDTA, 150 mM NaCl and 0.1% Triton X-100) was added to the supernatant (nuclear extract). Anti-GFP (for GFP-pDED transfected HeLa cells) or anti-HIPPI (for K562 cell) antibody was added to a part of the nuclear extract (+Ab) and antibody reaction was carried out overnight at 4°C. Other part of the nuclear extract was kept at 4°C (–Ab). The next day Protein G Agarose beads were added to the +Ab and –Ab fractions and left on a shaker for 6 h at 4°C. After 6 h, beads were washed 4 times with Triton buffer and two times with Tris buffer [10 mM Tris–HCl (pH 8.0)].

Next SDS–NaCl–DTT buffer (62.5 mM Tris–HCl, pH 6.8, 200 mM NaCl, 2% SDS 10 mM DTT) was added to the beads and incubated at 65°C overnight for reverse cross-linking. Next day, all the fractions were subjected to phenol chloroform extraction and the aqueous layer was collected. Cross-linked DNA was precipitated with 3 M Sodium acetate and ethanol. DNA pellet was washed with 70% ethanol, dried and then dissolved in distilled water. The DNA so obtained was amplified by PCR using caspase-1 717 bp upstream sequences (4). The primer sequences (5'–3') were as follows:

Forward: 5'-GGAAGATCTGGCTTTTCTCTCTCCCTTC-3'

Reverse: 5'-CGGGGTACCAAGCCTAGGAAACACAAGGAGA-3'

For re-ChIP assay, HeLa cells grown in 10 cm Petri dishes to ~80–90% confluency were transfected with GFP-Hippi construct. After 24 h, transfection efficiency was monitored by fluorescence microscopy and was ~80%. The cells were then cross-linked and the first immunoprecipitation using anti HIPPI antibody was carried out as described above. The immunoprecipitated chromatin was then eluted from beads using ChIP elution buffer (50 mM Tris–HCl, pH 7.5, 10 mM EDTA, 1% SDS) and incubating at 68°C for 10 min. One part of the eluted chromatin was subjected to reverse cross-linking followed by phenol chloroform isolation of DNA as mentioned above to check the first immunoprecipitation. The other part of the eluted DNA-protein complexes was diluted with Triton buffer and incubated with anti HIP-1 antibody over night at 4°C. Next day, the DNA-protein-antibody complex was immunoprecipitated by adding Protein G Agarose beads and DNA was extracted by phenol chloroform isolation followed by ethanol precipitation. PCR amplification of the eluted DNA was carried out using caspase-1 upstream (300 bp) sequence specific primers. The primer sequences (5'–3') were as follows:

Forward: 5'AATGATTGAGAAACTCTTCACTGTGT-3'

Reverse: 5'-CGGGGTACCAAGCCTAGGAAACACAAGGAGA-3'

Real-time PCR
RNA (100 ng) was reverse transcribed as described earlier . Real-time RT–PCR reaction was carried out using Sybr green 2x Universal PCR Master Mix (Applied Biosystems, USA) in ABI Prism 7500 sequence detection system. Each reaction was performed in triplicate using primer sequences for caspase-1 . Non-template control reaction at the same condition was performed to ascertain the baseline and threshold value for the analysis. Ct value obtained for caspase-1 was normalized with respect to beta actin ({Delta}Ct). Differences between the {Delta}Ct values of test and control sets were determined ({Delta}{Delta}Ct). The fold change was determined using the formula 2–{Delta}{Delta}Ct.