Báo cáo khoa học: Interactions of HIPPI, a molecular partner of Huntingtin interacting protein HIP1, with the speciﬁc motif present at the putative promoter sequence of the caspase-1, caspase-8 and caspase-10 genes

Interactions of HIPPI, a molecular partner of Huntingtin interacting protein HIP1, with the speciﬁc motif present at the putative promoter sequence of the caspase-1, caspase-8 and caspase-10 genes P. Majumder1, A. Choudhury2, M. Banerjee1, A. Lahiri2 and N. P. Bhattacharyya1

1 Structural Genomics Section, Saha Institute of Nuclear Physics, Bidhan Nagar, Kolkata, India 2 Department of Biophysics, Molecular Biology and Genetics, University of Calcutta, Kolkata, India

Keywords caspase; HIPPI; motif; pDED; transcription regulation

Correspondence N. P. Bhattacharyya, Structural Genomics Section, Saha Institute of Nuclear Physics, 1 ⁄ AF Bidhan Nagar, Kolkata 700 064, India Fax: +91 033 2337 4637 Tel: +91 033 2337 5345 E-mail: nitai_sinp@yahoo.com

the C-terminal

(Received 11 April 2007, revised 1 June 2007, accepted 5 June 2007)

doi:10.1111/j.1742-4658.2007.05922.x

To investigate the mechanism of increased expression of caspase-1 caused by exogenous Hippi, observed earlier in HeLa and Neuro2A cells, in this work we identiﬁed a speciﬁc motif AAAGACATG () 101 to ) 93) at the caspase-1 gene upstream sequence where HIPPI could bind. Various muta- tions in this speciﬁc sequence compromised the interaction, showing the speciﬁcity of the interactions. In the luciferase reporter assay, when the reporter gene was driven by caspase-1 gene upstream sequences () 151 to ) 92) with the mutation G to T at position ) 98, luciferase activity was decreased signiﬁcantly in green ﬂuorescent protein–Hippi-expressing HeLa cells in comparison to that obtained with the wild-type caspase-1 gene indicating the biological signiﬁcance of such 60 bp upstream sequence, binding. It was observed that ‘pseudo’ death effector domain of HIPPI interacted with the 60 bp () 151 to ) 92) upstream sequence of the caspase-1 gene containing the motif. We further observed that expression of caspase-8 and caspase-10 was increased in green ﬂuores- cent protein–Hippi-expressing HeLa cells. In addition, HIPPI interacted in vitro with putative promoter sequences of these genes, containing a sim- ilar motif. In summary, we identiﬁed a novel function of HIPPI; it binds to speciﬁc upstream sequences of the caspase-1, caspase-8 and caspase-10 genes and alters the expression of the genes. This result showed the motif- speciﬁc interaction of HIPPI with DNA, and indicates that it could act as transcription regulator.

It has been known for more than 13 years that increased CAG repeats beyond position 36 in exon1 of the Huntingtin (Htt) gene causes Huntington’s disease [1], resulting in increased apoptosis in a speciﬁc region of the brain [2]. Among various interacting partners of the protein Htt [3–5], Huntingtin interacting protein 1 (HIP1), identiﬁed in the yeast two-hybrid assay [6] and subsequently characterized as an endocytic adaptor to protein with clatharin assembly activity, binds

various cytoskeleton proteins [7]. In the search for interacting partners of HIP1, a novel protein HIPPI (HIP1 protein interactor) has recently been identiﬁed. HIPPI does not have any known domains except for a ‘pseudo’ death effector domain (pDED) and a myosin- like domain. Interaction of HIPPI with HIP1 takes place through the pDED present in both proteins. The HIPPI–HIP1 heterodimer recruits procaspase-8, and activates the initiator caspase and its downstream

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Abbreviations DED, death effector domain; EMSA, electrophoretic mobility shift assay; GFP, green ﬂuorescent protein; GST, glutathione S-transferase; HD, Huntington’s disease; HIP1, Huntingtin interacting protein 1; HIPPI, Huntingtin interacting protein 1 protein interactor; Htt, Huntingtin; IOD, integrated optical density; pDED, ‘pseudo’ death effector domain; TSS, transcription start site.

P. Majumder et al. Role of HIPPI as a transcription regulator

In vitro

cells.

could bind speciﬁcally. In addition, we observed that a similar motif was present at the putative promoter sequences of the caspase-8 and caspase-10 genes; the expression of these genes was also increased in GFP– Hippi-expressing HeLa experiments showed that HIPPI also interacted with the promoter sequences of these genes.

Results

Speciﬁc motif at the upstream sequences of the caspase-1 gene

apoptotic cascades [8,9]. It has been shown earlier that the interaction of HIP1 with normal Htt (a protein with fewer than 36 Gln) is stronger than that observed with the mutated Htt (a protein with more than 36 Gln resi- dues) [10]. On the basis of this observation, it has been proposed that the weaker interaction of HIP1 with mutated Htt (HD) may in Huntington’s disease increase the amount of freely available HIP1 and enhance the propensity for the HIP1–HIPPI heterodi- mer to form. The increased amount of HIP1–HIPPI may in turn lead to the increase in cell death observed in HD [8]. A role of HIPPI in apoptosis regulation has also been inferred from other studies. Apoptin, a chicken anemia virus-encoded protein, has been shown to colocalize with HIPPI in the cytoplasm of normal cells, whereas in tumor cells the two proteins localize separately in the nucleus and cytoplasm. It has been proposed that the HIPPI–apoptin interaction may suppress apoptosis [11]. The bifunctional apoptosis inhibitor, which regulates neuronal apoptosis, also interacts with HIPPI, although the functional relevance of this interaction remains unknown [12]. Very recently, it has been reported that HIPPI interacts with the postsynaptic scaffold protein Homer1c and regulates apoptosis in striatal neurons [13]. All these studies through its interacting partner, show that HIPPI, regulates apoptosis. Even though the exact function of HIPPI remains unknown, it has been shown, using knockout mouse (Hippi– ⁄ –), that HIPPI is involved the Sonic hedgehog signaling pathway [14].

the hypothesis that

caspase-1,

caspase-3,

the

Interactions of several transcription factors with Htt and alterations of a large number of genes observed in microarray studies support the pathology of HD is mediated through alterations in transcription [15]. In several studies using cellular and animal models of HD (where the mutated full-length Htt gene or exon1 are expressed by knockin), the caspase-2, expression of caspase-6 and caspase-7 genes is increased [16,17]. How the expression of these genes is altered is not known.

To search for the speciﬁc DNA sequence motif where HIPPI might interact, we analyzed 1 kb upstream regions of the caspase-1, caspase-3 and caspase-7 genes i.e. using four different motif prediction algorithms, meme, alignace, bioprospector and mdscan. The motifs predicted using the different methods, parame- ters and sequence sets (masked ⁄ unmasked) were then assembled and compared, and the redundant motifs were discarded (data not shown). The motif pre- dicted using the methods mentioned was 5¢-AA AGA[CG]A[TA][GT]-3¢. We investigated whether any similar motif was present within the 60 bp stretch of the caspase-1 gene upstream sequence where HIPPI actually interacted [18]. It was observed that the motif 5¢-AAAGACATG-3¢ () 101 to ) 93) was present in the positive strand of the caspase-1 gene upstream sequence. This motif was conserved in promoters of caspase-1 orthologs from Pan troglodytes (DOOP ID: 83123145, ) 245 to ) 253) and Macaca mulatta (DOOP ID: 94252893, ) 245 to ) 253). The motif sequences of the caspase-3 gene (5¢-AAAGAGATG-3¢, ) 828 to ) 820) and the caspase-7 gene (5¢-AAAGACATA-3¢, ) 245 to ) 253) were present in the positive strand. In subsequent studies, we tested whether HIPPI could interact with the 5¢-AAAGACATG-3¢ () 101 to ) 93) motif present in the putative promoter of the caspase-1 gene.

Interactions of HIPPI with AAAGACATG and various mutants of this sequence at the caspase-1 gene upstream sequence

We have previously shown that exogenous expres- sion of Hippi increases various apoptotic markers. In the course of this study, it was also observed that the endogenous expression of caspase-1, caspase-3 and caspase-7 is upregulated in green ﬂuorescent protein (GFP)–Hippi-expressing cells, whereas the mitochond- rial genes ND1 and ND4 and the antiapoptotic gene Bcl-2 are downregulated [9]. Recently, we have also shown that HIPPI can directly interact with the ca- spase-1 gene upstream 60 bp sequence () 151 to ) 92) in vitro and in vivo [18]. In the present investigation, we identiﬁed and characterized a motif within this 60 bp sequence of the caspase-1 gene where HIPPI

The speciﬁc sequence AAAGACATG identiﬁed within the 60 bp upstream sequence was used to test whether HIPPI interacted with this motif. The results of a typical electrophoretic mobility shift assay (EMSA) experiment carried out using the above-mentioned sequence and its mutants (mutations at the fourth, ﬁfth and sixth positions) are shown in Fig. 1A. A mobility shift of the band corresponding to [32P]ATP[cP]-labeled

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P. Majumder et al. Role of HIPPI as a transcription regulator

in the presence of gluta- dsDNA, AAAGACATG, thione S-transferase (GST)–HIPPI (Fig. 1A, panel I, lane 3) indicated interaction of the puriﬁed protein with the motif. No shift was observed in the presence of GST protein only (lane 2).

escence intensities of GST–HIPPI increased, indicating a lesser extent of interactions of GST–HIPPI with AA AGACATG. This result indicated that the interaction of GST–HIPPI with AAAGACATG was electrostatic in nature, although other possibilities cannot be ruled out.

EMSA with mutants of the 9 bp motif AAAGA CATG indicated that AAAGAGATG (mutation of the sixth nucleotide, C to G) interacted with the GST– HIPPI, as is evident from the mobility shift of the band corresponding to radiolabeled dsDNA in the presence of puriﬁed protein (Fig. 1A, panel II, lanes 3 and 4). However, mutation at the fourth nucleotide (G to T) and ﬁfth nucleotide (A to C) affected the interaction. In both cases, there was no shift of the probe, as shown in lane 2 and lane 6, indicating that GST–HIPPI did not interact with these mutated motifs.

The above results showed that puriﬁed GST–HIPPI interacted with the 9 bp motif AAAGACATG present at the upstream sequence () 101 to ) 93) of the cas- pase-1 gene, and that mutation at the sixth and eighth positions of the motif did not affect this binding, as is evident from the signiﬁcant quenching of GST–HIPPI respective protein ﬂuorescence observed with the sequences (Fig. 1B, panels I and II). A summary of the results is shown in Table 1. From the experimental studies described above with the various mutant motifs and their interactions in vitro with HIPPI, the consen- sus HIPPI-binding motif AAAGASAHK, i.e. AAAG A[GC]A[ATC][TG], was derived.

of

amounts

Reduction of the promoter activity of the 60 bp ()151 to ) 92) caspase-1 gene upstream sequence by mutation at position ) 98 (G to T) to the speciﬁc motif AAAGACATG ()101 to ) 93) in GFP–Hippi-expressing cells

A similar result was also obtained in the ﬂuores- cence quenching study (Fig. 1B, panel I). With increas- ing dsDNA (AAAGACATG and AAAGAGATG), the ﬂuorescence (kemission ¼ 340 nm, protein was kexcitation ¼ 295 nm) of GST–HIPPI reduced and reached a plateau. The value of the dissociation constant, determined from the plateau region, obtained with AAAGACATG was calculated to be 1.2 nm (Fig. 1C, panel I). A similar result was obtained with AAAGAGATG, with a dissociation constant of 0.3 nm (Fig. 1C, panel II). A ﬂuorescence quenching assay with the DNA AAAGACACG (point mutation at the eighth position T to C of the predicted motif mentioned above) revealed a decrease in the intrinsic ﬂuorescence of GST–HIPPI protein, indica- ting binding of the protein with this mutated motif (Fig. 1B, panel II). The apparent dissociation constant (Kd) of this binding was 4 nm (Fig. 1C, panel III). However, a similar assay with AAATACATG and AAAGCCATG did not alter the GST–HIPPI ﬂuores- cence signiﬁcantly (Fig. 1B, panel I), which further supported the results of EMSA with the same DNA sequences, discussed before (Fig. 1A, panel II). Further point mutations at the second (A to G), third (A to G), seventh (A to C) and ninth (G to A) nucleotides of the 9 bp motif AAAGACATG and a subsequent ﬂuorescence quenching study indicated no signiﬁcant quenching of ﬂuorescence of GST–HIPPI in the pres- ence of these mutants. This result revealed that GST– HIPPI did not interact with these mutated sequences of the 9 bp motif (Fig. 1B, panel II).

the putative promoter

sequence of

To explore the nature of the interactions of GST– HIPPI with AAAGACATG, we increased the concen- tration of NaCl from 50 mm (normally used in all binding assays) to 1000 mm. As is evident from Fig. 2, with the increasing concentrations of NaCl, the ﬂuor-

the 717 bp () 700 to We have earlier shown that + 17) and 60 bp () 151 to ) 92) sequences can act as the promoter in the luciferase reporter assay in HeLa as well as in Neuro2A cells. It has been shown that the luciferase activity of pGL3 when driven by the 717 bp caspase-1 gene upstream sequence is higher than that obtained with the 60 bp-driven construct [18]. This has been attributed to the presence of binding sites for other factors within these ﬂanking sequences [19]. As shown above, the 60 bp upstream sequence contains the motif AAAGACATG () 101 to ) 93), and muta- tion at position ) 98 (G to T) abolished the interaction of HIPPI. To check whether this mutation also decrea- ses the expression of the reporter gene driven by this mutated 60 bp caspase-1 gene upstream sequence in vivo, we carried out the luciferase assay after cloning both the wild-type 60 bp sequence and the mutated 60 bp sequence in pGL3. The luciferase activity, seen in GFP–Hippi-expressing HeLa cells when the lucif- erase gene was driven by the 60 bp region with a mutation at position ) 98 (G to T), was decreased (Fig. 3) signiﬁcantly (P ¼ 0.01) in comparison with that obtained with the wild-type 60 bp sequence. The result of this experiment is shown in Fig. 3, and indi- cates that mutation of the speciﬁc site of the binding motif at the caspase-1 gene, where HIPPI can bind, decreased the

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1

2

3 A

1

2

3

4

5

6 shifted band

Probe

Probe (9 bp)

AAAGACATG

I

II

B

16 1.00E+008

14

AAAGAGATG AAAGACATG AAAGCCATG AAATACATG

8.00E+007

12 6.00E+007

10 4.00E+007

AAGGACATG AGAGACATG AAAGACCTG AAAGACACG AAAGACATA

8 l

0 4 3 e c n e c s i r o u F

2.00E+007

6 0.00E+000

0.06 4 0.00

0.08

0.02

0.00

0.01

0.02

0.04

0.05 0.03 [DNA]µM I

0.04 [DNA]µM II

C

0.110 1.50E-008

AAAGAGATG Kd=0.29 nM

1.32E-008

AAAGACACG Kd=4 nM

0.105 1.40E-008

AAAGACATG Kd=1.2 nM

0.100 1.30E-008

0.095 /

F Δ 1

/

1.20E-008

1.28E-008

0 4 3 F Δ 1

0.090 1.10E-008

0.085 1.26E-008

1.00E-008

0.080

0

75

100

0

11

22

33

44

55 20 25 30 35 40 45 50

25 50 1/[DNA]µM

1/[DNA]µM

I

II

III

Fig. 1. In vitro binding assay of putative HIPPI-binding motif AAAGACATG and its mutant. (A) EMSA of binding of the end-labeled 9 bp motif AAAGACATG and two of its mutants with puriﬁed GST–HIPPI protein. Panel I. Lane1: probe (200 nM of [32P]ATP[cP]-labeled 9 bp motif AAAGACATG) only. Lane 2: probe + 4 lM GST protein. Lane 3: probe + 1.7 lM GST–HIPPI. Panel II. Typical results of similar analysis with the same 9 bp DNA with mutation at the fourth, ﬁfth or sixth base and GST–HIPPI protein are shown. Lane 1: 200 nM [32P]ATP[cP]-labeled AAATACATG (probe only). Lane 2: 200 nM same probe + 3.8 lM GST–HIPPI protein. Lane 3: band corresponding to 200 nM [32P]ATP[cP]- labeled AAAGAGATG (probe only). Lane4: 200 nM probe + 3.8 lM GST–HIPPI protein. Lane 5: AAAGCCATG (probe only). Lane 6: result obtained with 200 nM same probe + 3.8 lM GST–HIPPI protein. (B) Quenching of intrinsic ﬂuorescence of GST–HIPPI (0.8 lM) at 340 nm (kexc ¼ 295 nm) in the presence of the 9 bp putative motif sequence and six of its mutants. Panel I. Fluorescence quenching of GST–HIPPI protein due to addition of AAAGACATG (red line) and its mutants: AAAGAGATG (black line), AAAGCCATG (green line), AAATACATG (blue line). Inset: Curves representing wavelength scan of each point of quenching experiment with the 9 bp motif sequence AAAGACATG. Fluor- escence intensities were measured in a Fluoromax 3 spectroﬂuoremeter. Panel II. Study of any quenching of intrinsic ﬂuorescence of GST– HIPPI due to addition of point-mutated sequences of the 9 bp potent HIPPI-binding motif, namely: AAGGACATG (black line), AGAGACATG (red line), AAAGACCTG (green line), AAAGACACG (blue line) and AAAGACATA (sky blue line). Positions of point mutations are indicated by underlines. (C) Linear plot of 1 ⁄ DF versus 1 ⁄ c, where as DF is the change in ﬂuorescence with respect to the intrinsic ﬂuorescence of GST– HIPPI due to addition of DNA with concentration c (lM). Kd values were calculated from such plots. In panel I, DF is of GST–HIPPI versus c of 9 bp motif sequence AAAGACATG. In panel II and panel III, DF is of GST–HIPPI versus c of mutated products of 9 bp sequence: AAAGA GATG and AAAGACACG, respectively.

tional transcription regulator-binding sites within the ﬂanking sequence of the motif. It has been shown that p53 can bind within this region [19]. The luciferase activity of the pGL3 driven by the mutated 60 bp caspase-1 gene upstream sequence in GFP–Hippi- expressing HeLa cells was similar (3.0 ± 1.1) to that

promoter activity of the 60 bp upstream sequences sig- niﬁcantly. As shown above, the interaction of HIPPI with the mutated 9 bp motif (G to T at the fourth the motif) was abolished, whereas the position of 60 bp sequence with the mutated motif exhibited sub- stantial promoter activity. This could be due to addi-

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GST-Hippi 25µg + AAAGACATG 0.05 µM R2=0.92422

16000000

p=0.01

14000000

12000000

10000000

e c n e c s n m u

l i

8000000

6000000

0 4 3 e c n e c s e r o u F

4000000

2000000

600 800 1000

200 400

[NaCI]mM

m e h c n i e s a e r c n i d o F

60_HI

60M_HI

P. Majumder et al. Role of HIPPI as a transcription regulator

Fig. 2. (A) Sigmoidal curve (R2 ¼ 0.924) showing gradual increase in ﬂuorescence intensity of GST–HIPPI (0.8 lM) at 340 nm (kexc ¼ 295 nm) at saturation level of binding with the 9 bp motif AAAGA- CATG with increasing salt (NaCl) concentrations ranging from 50 mM to 1 M.

Table 1. Summary of binding study with the putative HIPPI-binding motif and its mutants. ND, not determined.

Fluorescence quenching

Result Sequence EMSA Average Kd (nM)

0.25

Fig. 3. The caspase-1 gene upstream 60 bp and a point mutation (G to T) incorporated in the same sequence at position ) 98, cloned in the pGL3 enhancer plasmid (60 M) and transfected (4 lg each) in GFP–Hippi-expressing cells. Cells expressing GFP–Hippi were mon- itored by the presence of GFP under a ﬂuorescence microscope. About 80–90% of cells expressed GFP after 20 h of transfection with GFP–Hippi. Transfected cells are denoted as 60_Hi and 60 M_Hi, respectively. The corresponding average fold increase (n ¼ 3) in luciferase activities compared to control (cell expressing only pGL3 without any insert) are given in bar diagrams. P-values of signiﬁcance are mentioned above the bar diagram in each of the cases studied.

1.5

caspase-1 in GFP–Hippi-expressing cells was due to interaction of HIPPI with this motif.

4 + ND ND – – + ND ND ND + – – – – + – + – AAAGACATG AGAGACATG AAGGACATG AAATACATG AAAGCCATG AAAGAGATG AAAGACCTG AAAGACACG AAAGACATA

Interaction of pDED of HIPPI with upstream sequences of the caspase-1 gene

the

60 bp wild-type

To check which portion of HIPPI was responsible for this interaction, a cDNA portion corresponding to the two termini of HIPPI, i.e. the N-terminal portion com- prising amino acid residues 10–334 (NCBI protein ID NP_060480) and the C-terminal pDED region (amino acids 335–429), were cloned and expressed in bacteria, and the proteins were puriﬁed. Interactions of the puri- ﬁed 6X(HN)-pDED and the N-terminal domains of HIPPI [also tagged with 6X(HN)] were studied in vitro by EMSA and ﬂuorescence quenching. The results revealed that the 6X(HN)-pDED domain of HIPPI interacted with the 60 bp upstream sequence of the caspase-1 gene (Fig. 4A, panel II, lanes 1 and 3). In contrast, the N-terminal region of HIPPI did not inter- act with the upstream sequence of the caspase-1 gene (Fig. 4A, panel I, lane 3). This result showed that the C-terminal end containing the pDED domain of HIPPI could interact with the upstream sequence of the caspase-1 gene.

observed in HeLa cells (without any detectable HIPPI expression) when the luciferase gene was driven by the 60 bp wild-type sequence (1.2 ± 1.3). The difference was not statistically signiﬁcant (P ¼ 0.4). Furthermore, there was no signiﬁcant difference (P ¼ 0.6) between the luciferase activities in HeLa cells expressing pGL3 driven either by sequence (1.2 ± 1.3) or the 60 bp mutated (1.7 ± 0.9) sequence. Thus, these luciferase activities could be due to the presence of promoter-binding site(s) within the 60 bp caspase-1 gene upstream sequence other than for HIPPI. This indicated that, due to point mutation at position 98 (G to T), HIPPI could not bind to the caspase-1 gene upstream to transcribe the downstream gene; this was manifested by about a two-fold decrease in luciferase acitivity. Thus, the results of promoter assay experiments further conﬁrmed the in vitro result that mutation of the motif abolished the binding of HIPPI to the speciﬁc sequence of the caspase-1 gene upstream sequence, and the increased expression of

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A

shifted band

is

shown

in Fig. 4B,

labeled Casp1 ups 717 bp

labeled Casp1ups 60 bp

B

C1ups 717bp (HIPPI-pDED) C1ups 717bp (HIPPI-Nterm) C1ups 60bp (HIPPI-pDED)

0.21

C1ups 60bp (6XHN-HIPPI_pDED) Kd=0.34nM

5 0 3

0.20

0.19

F Δ / 1

0.18

e c n e c s e r o u F

0.17

0 20 40 60 80100 120 140

9 8 7 6 5 4 3 2 1 0 0.00

0.01

0.02

0.03

0.04

caspase-1 gene upstream 60 bp sequence could also quench the intrinsic ﬂuorescence of 6X(HN)-pDED of HIPPI from 6.997 to 1.802 (Fig. 4B, panel I) with an (Kd) 0.34 nm; a double apparent binding constant panel II. plot reciprocal However, addition of the upstream sequences of the caspase-1 gene (717 bp) to the N-terminal domain (without the pDED domain) of HIPPI did not decrease the ﬂuorescence intensities determined by exciting either at 295 nm (kem ¼ 340 nm; ﬂuorescence intensity changed from 15.62 to 14.42 due to addition of 0.5 lm DNA) or 280 nm (kem ¼ 305 m; ﬂuores- cence intensity changed from 8.77 to 7.59). This result also showed that pDED of HIPPI actually interacted with the caspase-1 gene upstream sequences. We recently observed that pDED of HIPPI could also in vivo with the caspase-1 gene upstream interact sequence (data not shown).

Increase in caspase-1 gene expression and induction of apoptosis by C-terminal pDED of HIPPI

The role played by pDED of HIPPI in alteration of caspase-1 gene expression in HeLa cells was monitored by western blot analysis using antibody to caspase-1 (Fig. 5, middle panel). The band intensities were measured using image master vds software. The aver- age integrated optical density (IOD) of three different experiments is shown in Table 2. The results indicated that caspase-1 expression, as detected by western blot

Fig. 4. (A) In vitro binding assay of pDED of HIPPI with the ca- spase-1 gene upstream sequences. In panel I, typical results of EMSA with the 717 bp caspase-1 probe and 6X(HN)-pDED of Hippi and 6X(HN)-tagged N-terminal domain of Hippi protein are shown. Lane 1 shows the result with the probe only (400 nM). Lanes 2 and 3 show results obtained when 400 nM probe was allowed to inter- act with 2.7 lM 6X(HN)-pDED of HIPPI and 5.2 lM 6X(HN)-tagged N-terminal domain of HIPPI, respectively. In panel II, 500 nM 60 bp caspase-1 gene upstream sequence () 151 to ) 92) labeled with [32P]dCTP[aP] was used as probe. Lanes 1 and 2 show results obtained with probe only and probe + 4.8 lM BSA (nonspeciﬁc pro- tein), respectively. Lane 3 shows the result when 500 nM probe was allowed to react with 1.6 lM 6X(HN)-pDED of HIPPI, and lane 4 shows the result obtained when 500 nM labeled 60 bp probe was allowed to react with 1.6 lM 6X(HN)-pDED protein in the presence of 500 nM unlabeled 60 bp caspase-1 gene upstream sequence. (B) Panel I, quenching of intrinsic ﬂuorescence of 6x(HN)-tagged pDED of HIPPI (pDED-HIPPI, 2 lM) at 305 nm in presence of the 717 bp (squares) and 60 bp (triangles) upstream sequences of the ca- spase-1 gene (denoted as C1ups 717 bp and C1ups 60 bp, respect- ively) and any change in intrinsic ﬂuorescence of 6x(HN)-tagged N- terminal domain of HIPPI (HIPPI-Nterm, 1 lM) at 305 nm in the presence of 717 bp upstream sequence of the caspase-1 gene, denoted by circles. Panel II shows a linear plot of 1 ⁄ DF versus 1 ⁄ c where, DF represents the decrease in intrinsic ﬂuorescence of 6x(HN)-tagged HIPPI-pDED protein in the presence of the 60 bp upstream sequence of the caspase-1 gene; c, concentration (lM). The apparent binding constant (Kd) was calculated from this plot and is given with the graph.

Fig. 5. Role of exogenous pDED of HIPPI in alteration of caspase-8 and caspase-1 activation in HeLa cells. Western blot analysis using antibodies to caspase-8 (upper panel) and caspase-1 (middle panel) with total protein isolated from HeLa cells (H), HeLa cells expres- sing GFP-tagged N-terminus of HIPPI containing Myosin-like domain (HiN), and that expressing GFP-tagged pDED of HIPPI (HiD). The upper bands of the upper panel correspond to proca- spase-8 (57 kDa), and the lower bands represent the 12 kDa activa- ted caspase-8; the upper bands of the middle panel correspond to procaspase-1 (45 kDa), and the lower bands correspond to the 20 kDa activated caspase-1. The lowermost panel shows the level of b-actin (14.4 kDa).

As 6X(HN)-pDED of HIPPI does not contain any tryptophan, a 280 nm excitation ﬁlter was used, and the ﬂuorescence (characteristics of tyrosine and phe- nylalanine) was measured at 305 nm. A decrease in the ﬂuorescence intensity of 6X(HN)-pDED due to the addition of the 60 bp region of the caspase-1 gene the upstream sequence was observed. Addition of

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Table 2. Comparison of apoptosis induction and alteration in caspase-1 gene expression in GFP-tagged pDED of HIPPI and N-terminal domain of HIPPI-expressing HeLa cells.

Endpoints HeLa GFP–pDED of HIPPI (fold) P-values GFP–N-terminus of HIPPI (fold) P-values Fold increase: pDED versus N-terminal domain (P-values)

8.9 ± 2.6 0.0001 12.5 ± 3.9 0.3 4.4-fold (0.0005) 2.4 ± 0.9 0.0001 0.0002

9.2 ± 2.0 0.0005 17.6 ± 1.9 (7.3-fold) 14.8 ± 4.9 0.1

40.4 ± 3.6 0.0002 57.2 ± 10.4 0.06 54.9 ± 2.1 (6.2-fold) 32.5 ± 1.8 (13.5-fold) 23.1 ± 1.3 (2.5-fold) 90.2 ± 11.1 (2.2-fold) 1.5 ± 0.3 10.8 ± 2 0.001 0.048 Caspase-1 expression Nuclear fragmentation Caspase-8 activation Caspase-1 activation Caspase-3 activation (7.2-fold) 5.1 ± 2.2 (3.4-fold) 1.8-fold 0.0006 1.6-fold (0.047) 1.6-fold (0.02) 2.1-fold (0.03)

analysis, was increased in GFP–pDED-expressing cells by 4.4 ± 0.7-fold as compared to that in parental HeLa cells. However, this increase in the N-terminal part of HIPPI-expressing cells was only 1.4-fold. The increase in caspase-1 expression was again 6.2 ± 1.1- fold in pDED HIPPI-expressing cells as compared to the N-terminal part of HIPPI-expressing cells.

expression in HeLa cells induced cleavage of proca- spase-8 (Fig. 5, upper panel) and procaspase-1 (Fig. 5, middle panel) proteins more efﬁciently as compared to that of the N-terminal domain of HIPPI. A similar higher activation (2.1-fold, P ¼ 0.03) of caspase-3 was observed in GFP–pDED of HIPPI-expressing cells in comparison to that observed in GFP-–N-terminal HIPPI-expressing HeLa cells. These results are shown in Table 2.

Presence of the motif and the putative promoter sequences of caspase-8 and caspase-10 increased expression of the genes in GFP–Hippi-expressing HeLa cells

indicated that,

The derived motif AAAGASAHK, i.e. AAAGA[GC] A[ATC][TG], was used to search for the presence of the motif at the 1000 bp upstream sequences of the caspase-8 and caspase-10 genes using motiflocator (http://www.esat.kuleuven.ac.be/(cid:2)dna/BioI/Software. html). These genes are supposed to be involved in HD. The results for similar motifs identiﬁed in the caspase-1 (for reference) caspase-3, caspase-7, caspase-8 and caspase-10 genes are shown in the Table 3. In Table 3, the start and end positions of the motifs are indicated by distance from the transcription start site (TSS). The position upstream of the TSS of any gene is denoted by ‘–’ followed by the distance from the TSS. In the caspase-8 gene, the putative upstream sequence motifs AAAGAGAAC () 955 to ) 963) in the positive strand, and AAAGAAAAG () 418 to ) 410) and AA AGACATA () 800 to ) 808) in the negative strand, were observed (variants are underlined). As shown above, mutation at the last base, G to A, abolished the interaction of HIPPI, so the last motif would not interact with HIPPI. The other two motifs might be the target of HIPPI. In the upstream sequence of the

To test whether the C-terminal pDED of HIPPI could induce apoptosis more efﬁciently than the N-ter- minal domain in our system, these two domains cloned in pEGFP C1 vectors were transfected into HeLa cells. After 32 h, when 80–90% of cells were expressing GFP-tagged protein, we determined the nuclear frag- mentation as an indication of apoptosis induction and caspase activation. GFP–pDED-expressing cells exhib- ited nuclear fragmentation in 32.5 ± 1.8% of the total cell population, whereas this value in the GFP-tagged N-terminal domain of HIPPI-expressing cells was only 17.6 ± 1.9%. This difference was statistically signiﬁ- cant (P ¼ 0.0006). Thus, GFP–pDED of HIPPI was more effective in inducing apoptosis in HeLa cells. Fluorometric determination of caspase-1 activity by a commercially available kit in GFP– pDED of HIPPI-expressing cells, caspase-1 activity was 1.6-fold higher (P ¼ 0.02) than that observed in the GFP–N-terminal domain of HIPPI-expressing HeLa cells. Fluorometric determination of caspase-8 activity indicated that in HeLa cells expressing GFP– pDED of HIPPI, caspase-8 activation was also 1.6-fold higher in comparison to that obtained in the GFP–N- terminal domain of HIPPI-expressing cells. This value (P ¼ 0.047, n ¼ 3). was also statistically signiﬁcant Activation of caspase-1 and caspase-8 in GFP–pDED- expressing cells was further supported by western blot analysis (Fig. 5) using total protein isolated from HeLa cells expressing pDED and the N-terminal domain of HIPPI. It is evident from Fig. 5 that ectopic pDED

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Table 3. Summary of the presence of similar motifs in caspase-1, caspase-3, caspase-7, caspase-8 and caspase-10 gene upstream sequences.

Sequence name Strand Match type Motif sequence Start End

ENSG00000137752 Caspase-1 ENSG00000164305 Caspase-3 ENSG00000165806 Caspase-7 ENSG00000064012 Caspase-8

ENSG00000003400 Caspase-10

+ – – – – – + – – + – – – ) 101 ) 675 ) 814 ) 801 ) 355 ) 315 ) 963 ) 808 ) 418 ) 262 ) 651 ) 725 ) 857 ) 93 ) 667 ) 806 ) 793 ) 347 ) 307 ) 955 ) 800 ) 410 ) 254 ) 643 ) 717 ) 849 Consensus Allowing one substitution Consensus Consensus Allowing one substitution Allowing one substitution Allowing two substitutions Allowing one substitution Allowing two substitutions Allowing one substitution Allowing two substitutions Allowing two substitutions Allowing one substitution AAAGACATG AAAGACAGG AAAGAGATT AAAGAGATG AAAGACATA AAAGACATA AAAGAGAAC AAAGACATA AAAGAAAAG AAACAGATG AAAGAAAAG AAAGAAAAG GAAGACATT

caspase-10 gene, four variant motifs were identiﬁed. Among them, AAACAGATG () 254 to ) 262) is present in the positive strand, and the sequences AA AGAAAAG () 651 to ) 643), AAAGAAAAG () 725 to ) 717) and GAAGACATT () 849 to ) 857) are pre- sent in the negative strand.

Given that the caspase-8 and caspase-10 genes har- bor similar motifs as that in the caspase-1 gene and increase caspase-1 expression, we ﬁrst tested the expression of the caspase-8 and caspase-10 genes in GFP–Hippi-expressing cells by the semiquantitative RT-PCR described previously [18]. The numbers of PCR cycles and the amount of total RNA were chosen

so that the yield of RT-PCR products was in the linear range. The IOD value of the RT-PCR product obtained with RNA isolated from GFP–Hippi-expres- sing HeLa cells was increased 2.5-fold in comparison to the value obtained when RNA from the HeLa cells was used. This increase was statistically signiﬁcant (P ¼ 0.0004). A similar signiﬁcant increase in the IOD value of the RT-PCR products for the caspase-10 gene (1.8-fold, P ¼ 0.0002) was detected. A bar diagram showing the mean IOD of bands corresponding to caspase-8 and caspase-10 gene-speciﬁc products run on 1.5% agarose gel is shown in Fig. 6A). A representa- the RT-PCR products run on tive photograph of

A

B

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Fig. 6. (A) Bar diagrams showing mean of IOD of bands obtained by RT-PCR at caspase-8 and caspase-10 loci, along with error bars calcula- ted on the basis of three independent experiments using mRNA isolated from GFP–Hippi-expressing (gray bar) and parental (white bars) HeLa cells. Levels of signiﬁcance (P-values) are shown on the top of the bars. (B) Picture of RT-PCR products run on 1.5% agarose gel and stained with ethidium bromide, representing PCR ampliﬁcation with RNA isolated from HeLa cells (lane 1:H), GFP–Hippi-expressing HeLa cells (lane 2:Hi), and reaction carried out where no RNA was added (lane 3:ve). The uppermost panel shows the PCR reaction carried out with caspase-8 (164 bp)-speciﬁc primers; the middle panel represents PCR ampliﬁcation using primers speciﬁc for caspase-10 (178 bp) genes; and the lowermost panel shows bands (315 bp) corresponding to b-actin (loading control).

P. Majumder et al. Role of HIPPI as a transcription regulator

A

Caspase10 ups + GST-Hippi Caspase8 ups + GST-Hippi

agarose gel is shown in Fig. 6B. Equally intense signals for internal control (b-actin gene-speciﬁc primers) were obtained in all the cases (Fig. 6B, lowermost panel).

0 4 3

Fluorescence quenching assay to measure the interactions of GST–HIPPI with the caspase-8 and caspase-10 gene upstream sequences

0.03 0.04 0.05

11 10 9 8 7 6F 5 4 3 2 0.00 0.01 0.02

[DNA]µM

B

Casp8ups (GST-HIPPI) Kd=0.33 nM

0.25

Casp10ups (GST-HIPPI) Kd=15 nM

0.145

0.20

0.140

0.135

F 0.15 Δ / 1

F Δ / 1

0.10

0.130

0.05

0.125

0.00

30 40 50 60 70

10 20

30 40 50

0 10 20

1/[DNA]µM I

1/[DNA]µM II

Expression of caspase-8 and caspase-10 increased in GFP–Hippi-expressing cells, as described above, and DNA sequences similar to the putative HIPPI-binding motif were present within the 1000 bp upstream the caspase-8 and caspase-10 genes sequences of (Table 3). To check interactions of GST–HIPPI with these upstream regions containing the motifs, the caspase-8 gene upstream 710 bp () 991 to ) 282) and caspase-10 gene upstream 768 bp () 914 to ) 147) regions were PCR-ampliﬁed. The results, shown in Fig. 7A, indicated quenching of GST–HIPPI intrinsic ﬂuorescence at 340 nm, due to addition of increasing concentrations (0.001 lm to 0.05 lm) of the caspase-8 and caspase-10 gene upstream sequences.

caspase-10 gene

Average (n ¼ 2) Kd values for binding of puriﬁed GST–HIPPI with the caspase-8 gene (0.32 ± 0.13 nm) (11 ± 3.8 nm) upstream and the sequences were calculated from reciprocal plots as des- cribed previously [18], and typical cases are shown in Fig. 7B, panel I and panel II, respectively.

Discussion

Fig. 7. (A) In vitro binding study of GST–HIPPI with caspase-8 gene () 991 to ) 282) and caspase-10 gene () 914 to ) 147) upstream sequences. The upper line (squares) represents a gradual decrease in the intrinsic ﬂuorescence of GST–HIPPI protein (0.8 M) at 340 nm (kex ¼ 295 nm) due to addition of the caspase-10 gene upstream sequence. The lower line in the graph (triangles) repre- sents a similar alteration in ﬂuorescence intensity of GST–HIPPI when the caspase-8 gene upstream sequence was added gradually to it. (B) Linear plot of 1 ⁄ DF versus 1 ⁄ c, where DF represents change in intrinsic ﬂuorescence of GST–HIPPI due to addition of upstream sequences from the caspase-8 (panel I) and caspase-10 (panel II) genes, and c represents ﬁnal concentration (lM) of DNA allowed to bind with the protein.

In the present work, we have shown that HIPPI inter- acted speciﬁcally with the motif AAAGACATG () 101 to ) 93) present in the upstream region of the caspase-1 gene in vitro. Decreased expression of the reporter gene luciferase when driven by the 60 bp caspase-1 upstream sequence () 151 to ) 92) containing this motif with a mutation at position 98 position (G to T) in compar- ison with the wild-type 60 bp upstream sequence was observed. The same mutation in the motif also abol- ished the interactions of HIPPI in vitro. In addition, we observed that HIPPI could interact with the putative promoter sequences of the caspase-8 and caspase-10 genes. Expression of caspase-8 and caspase-10, as detected by semiquantitative RT-PCR, was also increased in the GFP–Hippi-expressing HeLa cells.

the change of C at the sixth position to G, and of T to C at the eighth position, did not decrease the interac- tion, whereas a change from G to A at the ninth posi- tion compromised the interactions (Fig. 1B, panel I and panel II). The change at the ninth position of the motif in the caspase-8 gene upstream sequence is G to C. As the caspase-8 gene 710 bp upstream sequence () 991 to ) 282) was shown to interact with HIPPI, we speculated that HIPPI interacted with the motif AAAGAGAAC () 963 to ) 955). We could not exclude the possibility that the other motif AAAG AAAAG () 418 to ) 410) present in the negative strand of the caspase-8 gene promoter interacted with HIPPI. Further experiments are necessary to establish this. Interaction of HIPPI with the motif AAAGA CATA () 808 to 800) at the positive strand (the vari- ant substitution G to A is underlined) of the caspase-8 gene was not possible, as we showed above that this particular motif did not interact with HIPPI (Fig. 1B). The putative HIPPI-binding motifs at the caspase-10

The motif derived from bioinformatics analysis and interaction studies of HIPPI with various mutants of AAAGACATG present in the caspase-1 gene was AAAGASAHK, i.e. AAAGA[GC]A[ATC][TG]. For the caspase-8 gene, there are three variations in the motif from that of the motif in the caspase-1 gene upstream region. Our experimental data suggest that

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sufﬁcient

gene upstream sequences are AAACAGATG () 254 to ) 262) in the positive strand, and AAAGAAAAG () 651 to 643), AAAGAAAAG () 725 to ) 717) and GAAGACATT () 849 to ) 857) in the negative strand. We were unable to exclude any of the motifs as the target of HIPPI, as we observed that HIPPI interacted with the putative promoter 768 bp () 914 to ) 147) sequence of the caspase-10 gene (Fig. 7B, panel II). Further experiments are necessary to deter- mine the speciﬁc sequences where HIPPI could interact at the upstream sequence of this gene.

thus regulate the transcription of the target genes has been also provided [23]. We hypothesized that the pDED of HIPPI might have similar DNA-binding ability to that of its distant relative DEDD.Our ﬁnd- ings that the puriﬁed C-terminal pDED of HIPPI was able to interact with the caspase-1 gene upstream sequence (Fig. 4A,B), similar to what was observed with full-length HIPPI [18], and that the exogenous expression of cDNA corresponding to the pDED of HIPPI alone was to increase caspase-1 expression and apoptosis (Fig. 5, Table 2) showed that the C-terminal pDED of HIPPI contributed to the increased expression of caspase-1 and apoptosis.

HIPPI generally resides in the cytoplasm. How this cytoplasmic protein is transported to the nucleus remains unknown. In an earlier study, we showed that exogenously expressed Hippi in HeLa cells can be detected in the nuclear fraction [Fig. 2(b) in Majumder et al. [9]]. It can be seen that there was no detectable endogenous expression of Hippi in HeLa cells, whereas the expression of HIP1 was detected in HeLa cells [Fig. 2(c), III, in Majumder et al. [9]]. Recently, trans- portation of androgen receptor to the nucleus has been reported to be mediated through HIP1 [24]. On the basis of this observation, we hypothesized that HIP1 might play similar role in the transport of HIPPI into the nucleus. We are presently testing this hypothesis.

It is interesting to note that even though the initial motif search revealed that the upstream sequence of the caspase-7 gene contains the AAAGACATA sequence present in duplicate within the ) 355 to ) 347 and ) 315 to 307 regions, our experiments with this motif revealed that HIPPI did not interact with it (Fig. 1B, panel II). Thus, the increase in caspase-7 expression in GFP– Hippi cells [9] might not be due to the direct interaction of HIPPI with the promoter sequence. This was further supported by the observations that puriﬁed HIPPI did not interact with the caspase-7 gene upstream 592 bp sequence () 1080 to 489) in vitro (by EMSA and ﬂuor- escence quenching) or in vivo (chromatin immunopre- cipitation assay using antibody to HIPPI) (data not shown). We also failed to detect any interactions of puriﬁed HIPPI with the caspase-3 gene upstream 652 bp sequence () 997 to ) 346) (data not shown), even though the exact motifs with which HIPPI could inter- act were present in the negative strand (Table 3) of the gene. The reason behind this still remains obscure; whether strand bias or the neighboring nucleotides prevented the interaction remains to be determined.

the pDED at

the C-terminus

The role of HIPPI in HD, if any, remains unknown. Even though the caspase-8 and caspase-10 genes have been implicated in poly-Q-mediated toxicity [25,26], it is not known whether the expression of these genes is altered. The role of HIPPI in the increased expression of caspase-1 observed in various models and HD patients has to be established. We speculated that excess available HIP1 in HD, due to weaker interac- tion of HIP1 with the mutated Htt allele, leads to the formation of more HIPPI–HIP1 heterodimer, which in turn increases caspase-1 expression. We were able to immunoprecipitate the caspase-1 gene promoter by antibody to HIPPI after crosslinking DNA protein in vivo [18]. However, we failed to do the same thing with antibody to HIP1 (data not shown), indicating that HIP1 does not directly interact with the putative promoter of the caspase-1 gene. Thus, the role of HIP–HIPPI heterodimer formation in the increased expression of caspase-1, caspase-8 and caspase-10 is not clear. We speculated that it might be necessary for transporting HIPPI into the nucleus.

HIPPI does not have any similarity with known pro- it con- teins having DNA-binding motifs. However, tains (amino acid residues 335–426) and a myosin-like domain. The pDED of HIPPI shows only 34.9% similarity and 21% identity to other known death effector domains (DEDs), and 39.2% similarity and 26.7% identity with the pDED of HIP1. It has been shown that the inter- action of HIPPI with HIP1 is mediated through the pDED present at HIP1. The pDED differs from its conformational neighbor DED by the presence of charged residues at the interacting helices, as opposed to the hydrophobic ones in the later [8]. DED-contain- ing proteins are known to participate in diverse cellu- lar functions, including apoptosis through receptor signaling [20,21]. Other DED-containing proteins, such as DEDD, are known to bind DNA and inhibit RNA polymerase I activity in vivo [22]. Direct evidence that the DED-containing proteins DEDD and FLAME-3 interact with the transcription factor TFIIIC102 and

In summary, together with our earlier observations [9,18], we observed in the present work that HIPPI interacted with the speciﬁc motif present in the putative promoters of the caspase-1, caspase-8 and caspase-10 genes and altered the expression of these genes. The

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presence of this motif in the promoters of other genes and regulation by HIPPI is now actively being investi- gated. Even though the role of HIPPI in HD remains obscure, the protein takes part in the regulation of caspase-1, caspase-8 and caspase-10 gene expression.

(P8), and AAAGACATA (P9), and their complementary sequences CATGTCTCT (P2C), CATGTCCTT (P3C), CATGTATTT (P4C), CATGGCTTT (P5C), CATCTCTTT (P6C), CAGGTCTTT (P7C), CGTGTCTTT (P8C), and TATGTCTTT (P9C), were also synthesized chemically. The underlined sequences are changes from the original motif observed at the caspase-1 gene upstream sequence (P1).

Experimental procedures

Cell culture

HeLa cells were obtained from National Cell Science Cen- ter, Pune, India, and routinely grown in MEM medium (HIMEDIA, Mumbai, India) supplemented with 10% fetal bovine serum (Life Technology, Rockville, MD, USA) at 37 (cid:2)C in 5% CO2 atmosphere under humidiﬁed conditions.

Each single-stranded oligonucleotide was mixed with reverse oligonucleotide in a 1 : 1 molar ratio in sterile water; the mixture was then heated to 95 (cid:2)C for 15 min and slowly cooled to 4 (cid:2)C to allow perfect annealing. The kinase reaction was carried out with these double- stranded oligoneucleotides using polynucleotide kinase in the presence of 1 · polynucleotide kinase buffer and [32P]ATP[cP] (BRIT, Hyderabad, India). The radiolabeled oligonucleotides were used for EMSA as described previ- ously [18].

Computational analysis to identify the motif

Cloning of the Hippi pDED domain and N-terminal domain of Hippi in the bacterial expression plasmid pProTET and mammalian vector pEGFP

tool

Hi_pDEDR

induced by

One kilobase upstream sequences for genes (the caspase-1, caspase-3 and caspase-7 genes) whose expressions are increased by exogenous Hippi expression [9] were retrieved from the ENSEMBL database using the biomart data retrieval (http://www.ensembl.org/Multi/martview). Putative cis regulatory elements were searched in these upstream sequences using four widely used motif prediction programs: meme, alignace, bioprospector and mdscan [27–30]. The search was also carried out on a second sequence dataset, which was prepared by masking the pro- the upregulated genes using repeatmasker moters of (http://repeatmasker.org). The motif prediction process was repeated several times with different parameters and differ- ent motif lengths. The results were compiled, and motifs that occur within the 60 bp caspase-1 gene upstream sequence () 151 to ) 92) were selected out using a custom perl script. To test the phylogenetic conservation of the pre- dicted motifs, 1 kb upstream sequences of caspase-1 gene orthologs were downloaded from the DOOP database (http://doop.abc.hu) and were scanned for occurrence of the predicted motifs.

The pDED and N-terminal region of HIPPI encoded by the cDNA (gi|19923513) region (1003–1278 and 30–1003), respectively, were ampliﬁed by PCR using speciﬁc primer sets, namely, Hi_pDEDF (5¢-ACGCGTCGACGTCGGA (5¢-CG AATGGAGGAGTGACGG-3¢), GGATCCCGTTAATAAAAGCCTGTTGCTGGTT-3¢), Hi_Nterm F (5¢-ACGCGTCGACGTCATGACTGCTGCT CTGGCCGT-3¢), and Hi_Nterm R (5¢-CGGGATCCCGC TGCTGGTATCGCTCCTTTG-3¢), and cloned in pPRO- TET and pEGFP plasmids using methods essentially des- cribed previously [9]. pPROTET Clones were transformed in Escherichia coli strain BL21 Pro, and protein expression incubating with anhydrotetracycline was (90 ngÆmL)1) for 5 h. Proteins were isolated from cells by a freeze–fracture method, and puriﬁed by Ni2+–nitrilotriace- tic acid afﬁnity chromatography. Finally, the sizes of the pDED and N-terminal domain of HIPPI with the tag 6X(HN) were determined by 12.5% SDS ⁄ PAGE. The sizes were 13 kDa for pDED, and 40 kDa for the N-terminus of HIPPI. pEGFP clones were transfected in HeLa cells as described previously [9].

The in silico and experimentally derived motif consensus sequence AAAGASAHK, i.e. AAAGA[GC]A[ATC][TG], was searched for in upstream sequences of the caspase-8 and caspase-10 genes using patmatch [31].

EMSA

Motif sequences and methods for making dsDNA and labeling

2.5 mm)

glycerol,

Different concentrations of GST–HIPPI or 6X(HN)-pDED of HIPPI were added to the probe 9 bp motif sequences, and mutated forms (allowed to bind with GST–HIPPI) or caspase-1 gene upstream sequences [allowed to bind with 6X(HN)-pDED of HIPPI] in binding buffer (1·: Hepes, 12.5 mm; EDTA, 0.5 mm; dithiothreitol, 0.25 mm; KCl, 37.5 mm; containing 5%; MgCl2, 50 ngÆlL)1 poly(dI:dC) were incubated at room temperature

Motif sequence AAAGACATG (designated as P1) in the upstream region of the caspase-1 gene () 101 to ) 93) and its complementary sequence CATGTCTTT (P1C) were synthesized (IDT, Coralville, IA, USA). In addition, oligo- nucleotides, namely AGAGACATG (P2), AAGGACATG (P3), AAATACATG (P4), AAAGCCATG (P5), AA AGAGATG (P6), AAAGACCTG (P7), AAAGACACG

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Luciferase assay

for 40 min. At the end of incubation, products were loaded on 5% polyacrylamide gel and the gel was run at 200 V for 4.5 h at 4 (cid:2)C. The gel was then dried at 80 (cid:2)C for about 45 min. The dried gel was exposed to X-ray ﬁlm (Kodak, Mumbai, India) overnight at ) 80 (cid:2)C. After the ﬁlm had been developed, positions of bands on the ﬁlm were indicative of the positions of the probe. Cold 60 bp caspase-1 gene upstream sequence (without [32P]dCTP[aP] incorporation) in excess was used as competitive inhibitor of the reaction between the labeled 60 bp caspase-1 gene upstream sequence and 6X(HN)-pDED of HIPPI to determine the speciﬁcity of the interaction.

Fluorimetric quenching study

The puriﬁed GST–HIPPI protein was diluted (ﬁnal concen- tration varied from 0.8 lm to 4 lm) in reaction buffer (100 mm Tris ⁄ HCl, pH 8.0, 50 mm NaCl). The concentra- tion of DNA was increased gradually (3 nm to 200 nm) to the ﬁxed amount of the protein, and the ﬂuorescence intensi- ties were measured at 340 nm [305 nm for 6X(HN)-pDED of HIPPI], exciting at 295 nm [280 nm for 6X(HN)-pDED of HIPPI protein] in a Hitachi 4010 Spectroﬂuorimeter (Hita- chi, Tokyo, Japan) or Spex FluoroMax 3 Spectroﬂuorimeter (Edison, NJ, USA). Changes in ﬂuorescence intensities (DF) due to addition of upstream sequences of the caspase-1 gene were calculated. From double reciprocal plot of DF and con- centrations of DNA in the ranges where the decrease in ﬂuor- escence intensities reached saturation, apparent dissociation constants (Kd) for each DNA–protein binding reaction were calculated following the methods described by Sing & Rao [32].

Semiquantitative RT-PCR with gene-speciﬁc primers

The caspase-1 gene upstream 60 bp DNA () 151 to ) 92) was cloned in pGL3 as described previously [18]. A mutation (G to T) at position ) 98 of the 60 bp () 151 to ) 92) upstream sequence of the caspase-1 gene was introduced using speciﬁc primers (forward, 5¢-CCTGATGCAGGCTA CAGTTCT-3¢; and reverse, 5¢-GCATATGCATGTATT TATTTTTCTTC-3¢) and standard procedures. The speciﬁc mutation (G to T) was conﬁrmed by sequencing. HeLa cells were transfected with GFP–Hippi. Twenty-four hours after transfection, more than 90% of the transfected cells were expressing GFP-tagged protein (as visualized under a ﬂuor- [9]. The 60 bp or mutated 60 bp escence microscope) sequences (G to T at position ) 98) of the caspase-1 gene upstream sequence cloned in pGL3 vector and control pGL3 plasmid (without any insert) were transfected separately in HeLa cells expressing GFP–Hippi, and the cells were grown in the presence of geniticin (marker present at the pEGFP plasmid). Control pGl3 and 60 bp mutated 60 bp sequences of the caspase-1 gene upstream sequences cloned in pGL3 plasmid were also transfected into the parental HeLa cells. All these transfections were carried out using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA), following the procedure provided by the manufacturer. After 48 h of transfection of pGL3, cells were harvested and lysed, and luciferase substrate (Promega, Madison, WI, USA) was added. Luciferase activity was measured in a Sirius tube luminometer (Berthold Detection Systems, Pforzheim, Germany). In the same experiments, the transfection efﬁ- the ciencies of pGL3-containing upstream sequences of caspase-1 gene in both control and GFP–Hippi-transfected cells were monitored by cotransfecting the b-galactosidase gene containing vector pSV-b-galactosidase (Promega) and measuring the b-galactosidase activity along with lucif- erase activity. Appropriate correction was made for equal transfection, using results obtained with the b-galactosidase activity. The transfection efﬁciency of the Hippi construct cloned in pEGFP plasmid was monitored by assaying GFP–Hippi expression in those cells as described previously [9], and appropriate correction was incorporated on the basis of this.

Detection of nuclear fragmentation, and caspase-1, caspase-3 and caspase-8 activation

fragmentation, and caspase-1,

for

Nuclear caspase-3 and caspase-8 activation, were detected using methods described previously [9].

Western blot analysis

The methods of total protein isolation from exponentially growing cells and western blot analysis using antibodies to

Methods for RNA isolation, ﬁrst-strand DNA synthesis, etc. have been published previously [33]. After isolation, RNA was treated with RNase-free DNase (Sigma Chemi- cals, St Louis, USA) to remove possible genomic DNA contaminants. The ﬁrst strand of cDNA was synthesized as described before, and used to study the expression of ca- spase-8 and caspase-10 by PCR. The caspase-8 gene-speciﬁc primers were: forward, 5¢-AAGCAAACCTCGGGGATAC T-3¢; reverse, 5¢-GGGGCTTGATCTCAAAATGA-3¢. The caspase-10 gene-speciﬁc primers were: forward, 5¢-GA CGCCTTGATGCTTTCTTC-3¢; reverse, 5¢-ATGAAGGC these two GTTAACCACAGG-3¢. PCR conditions genes were similar, except for the annealing temperature. The common PCR conditions used for each of these loci were: initial denaturation at 94 (cid:2)C for 1 min, followed by 35 cycles each containing three steps, denaturation at 94 (cid:2)C for 15 s, annealing at 50 (cid:2)C (for caspase-8) or at 60 (cid:2)C (for caspase-10) for 30 s, and extension at 72 (cid:2)C for 1 min and ﬁnally extension for 10 min at 72 (cid:2)C.

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caspase-1 and caspase-8 were similar to those published previously [9].

inhibitor (BAR) protects neurons from diverse cell death pathways. Cell Death Differ 10, 1178–1187. 13 Sakamoto K, Yoshida S, Ikegami K, Minakami R,

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