te stress-related genes including HSF1 and thus the interactions of CGGBP1and HMGN1 with NFIX have functional significance in this context. NFIX interacts with CGGBP1 and HMGN1 Since there are no known interacting partners of NFIX which could be used as candidates to address this issue, we performed a yeast-2-hybrid screen of a fetal human brain cDNA library. Using the CTF1 domain of NFIX as bait, we screened approximately 107 independent clones and identified two different prey clones corresponding to DNA-binding protein coding genes. These included a high mobility group protein and the CGG triplet repeat binding protein 1 . HMGN1, a sequence-non-specific DNA-binding protein replaces histone H1 from nucleosomes and establishes open chromatin conformation associated with transcriptional activation and the HSPA1A promoter is a proven target of HMGN1. CGGBP1 on the other hand is a transcriptional repressor binding to CGG triplet repeats. In vitro binding assays have shown that 8 units of CGG repeats, even with a G-A mismatch, constitute a CGGBP1 binding site. Naumann and coworkers found that CGGBP1 can bind to as small as five CGG repeats with one base mismatch. CGGBP1 binding to DNA in vivo has never been studied at loci other than the FMR1 gene which has long CGG repeats 12504917 in its 59-UTR. The HSF1 promoter region is associated with a CpG rich region and we found that there is a 6 CGG tandem repeat spanning from 211 to +7 nucleotide bases relative to HSF1 transcription start site. This raised a possibility that CGGBP1 and HMGN1 might mediate transcriptional regulation of HSF1 by NFIX. The deregulation of many stress-response genes by NFIX suppression could thus be routed through HSF1. Coregulation of HSF1 and NFIX in terms of gene expression in a manner as if they were subjected to heat shock. HMGN1-siRNA on the other hand resulted in enlarged morbid cells at both normal and heat shock conditions. Just like the effect on HSF1 expression, combining siRNAs of NFIX with CGGBP1 or HMGN1 did not show any additive effect on these Nutlin-3 web phenotypes. Overall, these results suggest that NFIX suppresses HSF1 transcription in a heat sensitive manner through pathways, which involve CGGBP1 and HMGN1. The soluble complex of NFIX, CGGBP1 and HMGN1 is heat shock sensitive The physical interactions of endogenously expressed NFIX with HMGN1 and CGGBP1 were confirmed by co-immunoprecipitation assays and the identities of HMGN1 and CGGBP1 bands were confirmed by using siRNA against them. We then asked if interactions of NFIX with CGGBP1 and HMGN1 are constitutive or affected by heat shock. Co-IPs were performed by using NFIX antibody, on lysates of U-2987 MG cells, which were either cultured at 37uC, acute heat shocked at 45uC for 10 minutes or chronically heat shocked at 39uC for 48 hours, and were probed on Western blots using antibodies against CGGBP1 or HMGN1. CGGBP1 precipitated with NFIX was greatly reduced after acute heat shock and 10604535 almost diminished after chronic heat shock. There was no detectable difference in HMGN1-NFIX interactions following acute heat shock. However, chronic heat shock diminished this interaction too. We then asked if NFIX interacts with CGGBP1 and HMGN1 
only separately or also together in one complex. CGGBP1 was immunoprecipitated by the HMGN1 antibody and this was reduced by NFIX-siRNA and chronic heat shock. Similarly, NFIX was immunoprecipitated by the CGGBP1 antibody and was sensitive to HMGN1-siRNA and chronic heat shock. Thus it w
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