Interaction of HIPPI with putative promoter sequence of caspase-1 in vitro and in vivo.
To investigate the mechanism of increased expression of caspase-1 in Hippi expressing HeLa and Neuro 2A cells, reported earlier, we report here that HIPPI directly interacted with upstream sequence of caspase-1 gene (-700 to +17, 717 bp). Deletion of this 717 bp sequence and further analysis by electrophoretic mobility shift assay and fluorescence quenching revealed that HIPPI interacted with 60 bp (-151 to -92) upstream sequence of caspase-1. We also observed by chromatin immunoprecipitation assay that HIPPI interacted with the 717 bp sequence in vivo. In luciferase assay, when expression of the reporter gene was driven by either 717 bp or 60 bp caspase-1 upstream sequences, luciferase activity was increased in GFP-Hippi expressing HeLa cells in comparison to that obtained with parental HeLa cells with the same constructs. Similar result was obtained in Neuro2A cells with 717 bp caspase-1 upstream sequence. In summary, we showed that HIPPI could interact with the putative promoter sequence of caspase-1 and increased the expression of the downstream gene suggesting that HIPPI could act as transcription regulator.
Majumder
P
P
Structural Genomics Section and Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, 1/AF Bidhan Nagar, Kolkata 700 064, India.
Chattopadhyay
B
B
Sukanya
S
S
Ray
T
T
Banerjee
M
M
Mukhopadhyay
D
D
Bhattacharyya
N P
NP
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites; D020170:Caspase 1 / Q000235:genetics* / Q000378:metabolism*; D002460:Cell Line; D006367:HeLa Cells; D006801:Humans; D009474:Neurons / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D015533:Transcriptional Activation / Q000502:physiology*
D048868:Adaptor Proteins, Signal Transducing; C499179:IFT57 protein, human; D020170:Caspase 1