A DNA Sequence Directed Mutual Transcription Regulation of HSF1 and NFIX Involves Novel Heat Sensitive Protein Interactions

Westermark B (2009) A DNA Sequence Directed Mutual Transcription Regulation of HSF1 and NFIX Involves Novel Heat Sensitive Protein Interactions. PLoS ONE 4(4): e5050. doi:10.1371/journal.pone.0005050 A DNA Sequence Directed Mutual Transcription Regulation of HSF1 and NFIX Involves Novel Heat Sensitive Protein Interactions Umashankar Singh 0 Erik Bongcam-Rudloff 0 Bengt Westermark 0 Alfredo Herrera-Estrella, Cinvestav, Mexico 0 1 Department of Genetics and Pathology, Uppsala University , Uppsala , Sweden , 2 Linnaeus Center for Bioinformatics , Uppsala University , Uppsala , Sweden Background: Though the Nuclear factor 1 family member NFIX has been strongly implicated in PDGFB-induced glioblastoma, its molecular mechanisms of action remain unknown. HSF1, a heat shock-related transcription factor is also a powerful modifier of carcinogenesis by several factors, including PDGFB. How HSF1 transcription is controlled has remained largely elusive. Methodology/Principal Findings: By combining microarray expression profiling and a yeast-two-hybrid screen, we identified that NFIX and its interactions with CGGBP1 and HMGN1 regulate expression of HSF1. We found that CGGBP1 organizes a bifunctional transcriptional complex at small CGG repeats in the HSF1 promoter. Under chronic heat shock, NFIX uses CGGBP1 and HMGN1 to get recruited to this promoter and in turn affects their binding to DNA. Results show that the interactions of NFIX with CGGBP1 and HMGN1 in the soluble fraction are heat shock sensitive due to preferential localization of CGGBP1 to heterochromatin after heat shock. HSF1 in turn was found to bind to the NFIX promoter and repress its expression in a heat shock sensitive manner. Conclusions/Significance: NFIX and HSF1 exert a mutual transcriptional repressive effect on each other which requires CGG repeat in HSF1 promoter and HSF1 binding site in NFIX promoter. We unravel a unique mechanism of heat shock sensitive DNA sequence-directed reciprocal transcriptional regulation between NFIX and HSF1. Our findings provide new insights into mechanisms of transcription regulation under stress. - Nuclear factor 1 family of genes codes for site-specific DNAbinding proteins known to have multiple roles in replication, signal transduction and transcription [1]. Four known members of the family, NFIA, NFIB, NFIC and NFIX in higher eukaryotes, are evolutionarily highly conserved. No NFI gene is known in unicellular organisms indicating the importance of NFI genes in complex metazoan biology [2]. NFI proteins contain an Nterminal MH-1 DNA-binding domain and a C-terminal CTF-1 transcription modulation domain, which allows them to interact with other proteins [3,4]. They bind as homodimers or heterodimers to TTGGC(N5)GCCAA sites and can also bind to either half of the palindrome as monomers [57]. The NFI proteins have highly similar peptide sequences and might have some redundant functions. Mouse knockout studies show that NFIA mutation results in hydrocephalus and abnormal brain development [8], NFIB mutation causes retarded lung development [9] and NFIC mutation causes abnormal tooth development [10], with very little overlap in phenotypes. NFIX knockouts generated by different groups have resulted in different phenotypes; hydrocephalus and abnormal ossification in one case [11] and defects in hippocampal development, neural stem cell differentiation, and weight-loss in the other [12]. Thus, other members of the family do not compensate for the functions of individual NFI genes. There are very few reports on the mechanisms of action of human NFI proteins. NFI protein overexpression results in resistance of chicken cells to transformation by qin, jun and fos oncogenes [13]. NFIC interacts with histone H1 [14], PIRIN [15] and TAFII55 [16] proteins and activates transcription at specific loci, such as glucocorticoidresponsive MMTV promoter [17,18]. NFIX is important for activation of GFAP transcription in astrocytes [19] and provides resistance against TGFB-induced apoptosis in mink epithelial cells [20]. At the CDKN1A promoter, all different NFI members exhibit different levels of transcriptional repression [21]. This and different knockout phenotypes of NFI genes indicate the existence of mechanisms specific for each member. Differential interactions with other proteins, conferred by different posttranscriptional modifications and subtle differences in peptide sequences might result in functional specificities of different NFI members. Retroviral tagging using MMULV expressing PDGFB to identify novel glioma-causing genes gave one integration in NFIA, NFIB and NFIC each and five integrations in NFIX [22]. Despite such strong indications of involvement of NFIX in PDGF-induced tumorigenesis, no systematic study has been undertaken to address molecular mechanism of action of NFIX. Heat shock factor 1 (HSF1), a key regulator of heat shockinduced transcription [23,24] is a potent modifier of carcinogenesis induced by a wide range of factors, including PDGFB [25]. Heat shock-induced misfolding of proteins leads to induction of chaperone activity and expression, which tends to rectify the errors in protein folding [26,27]. Following heat shock HSF1 gets recruited to heat shock elements in the promoters of its target genes, and activates transcription [28,29]. Unlike normal cells, tumor cells have higher proteotoxic stress and require higher levels of chaperones to survive [23]. While it is known that heat shock and protein denaturation induces HSF1 expression, the exact molecular mechanisms behind HSF1 transcriptional regulation are not known. In this study, we describe NFIX peptides for the first time and report that NFIX regulates expression of stress related genes including HSF1. We identify that NFIX exists in a heat sensitive complex with CGGBP1 and HMGN1. CGGBP1 organizes a transcription regulatory complex comprising of NFIX and HMGN1 at a small CGG repeat element in the HSF1 promoter and suppresses its expression. HSF1 was also found to repress NFIX expression by binding to a potential HSF1-binding site in the NFIX promoter. We report a unique DNA sequence-directed reciprocal transcription regulatory mechanism between NFIX and HSF1 involving heat shock-sensitive protein interactions. NFIX regulates genes involved in stress response To identify the functions of NFIX, we suppressed it by transient siRNA transfections. By real time quantitative RTPCR (qRTPCR) we confirmed that NFIA, NFIB and NFIC were not affected (not shown). As an NFIX peptide has never been demonstrated before, we also characterized the NFIX peptide and its intracellular localization. We also found that tyrosine phosphorylation affects intracellular localization of NFIX (Figure S1, Figure S2, Figure S3 and supplementary results in file Results S1). Thus we identified a 60 KDa nuclear peptide which could be immunoprecipitated and was down-regulated by NFIX-siRNA. To detect transcriptional targets of NFIX, (...truncated)