We and others have previously reported that the half-life of cirp mRNAs is only marginally influenced by temperature down-shift [2, 18]. Consistent with a notion that the transcription rate of cirp is increased at moderately low temperatures, we have herein identified a MCRE in the 5′ flanking region of the cirp gene that enhances gene expression at 32°C. We further demonstrated that Sp1 binds to MCRE, and that more Sp1 is localized in the nucleus at 32°C to bind to the genomic region containing MCRE. In addition, the reporter assays with mutant MCRE and modulation of the Sp1 expression level showed that the expression of cirp is dependent, at least partly, on Sp1 and MCRE and that Sp1 contributes to the cold shock response at 32°C.
Cis-regulatory elements like enhancers and promoters are usually characterized by reporter gene assays in cultured cell lines . We did the transient reporter gene assays using HEK293 cells, and identified the MCRE. Although present in two different genomic fragments of cirp with enhancer activity, mutagenesis experiments of the identified MCRE octanucleotide revealed that the first base could vary. Furthermore, the binding sequences of Sp1 predicted by TESS in the cirp genome did not include the first base of the MCRE, suggesting that the actual element might be the heptanucleotide 5′-CCCCGCC-3′.
The cold-responsive enhancer activity of MCRE was consistently observed when various cell lines including HEK293 and CHO-K1 cells were transiently transfected. In cells stably transfected with similar MCRE constructs, however, the cold-inducible enhancer activity was difficult to observe. This result may not be so surprising because transient reporter gene assays are often a poor proxy for the activities of regulatory elements integrated in the genome, which can be active in narrow windows of development, differentiation and cellular conditions . How the presence of many copies of MCRE seemingly circumvented the problem has yet to be clarified. Although many studies have suggested that recombinant protein production can be increased by culturing at sub-physiological temperatures , low temperature also causes growth arrest of cells and suppression of gene expression in general, resulting in variable yields. Thus, specific enhancement of the target gene transcription at 32°C by using MCRE is a promising method to increase the final yield of recombinant proteins in various cell lines. Interestingly, Thaisuchat et al.  have recently identified a novel temperature sensitive promoter (222 bp long) in the S100a6 (calcyclin) gene, which comprises two Sp1 sites.
Sp1 binds GC-rich motifs with high affinity and can regulate the expression of TATA-containing and TATA-less genes via multiple mechanisms [10, 11, 22]. Sp1 generally activates gene transcription, whereas Sp3 has both transcriptional repressor and activating properties. Discher et al.  showed by transient transfection that hypoxia enhances the expression of a reporter gene directed by the pyruvate kinase M promoter in myocytes. The hypoxia response was localized to a conserved GC-rich element that bound Sp1 and Sp3. Hypoxia induced gene expression because it caused depletion of Sp3, removing its transcriptional repression, whereas Sp1 levels remained unchanged. This is unlikely the case with the cirp gene, as the western blotting and ChIP assays demonstrated that more Sp1 bound to the chromatin region containing MCRE at 32°C than 37°C, whereas the level of chromatin-bound Sp3 did not differ between these temperatures.
Although Sp1 was once thought to serve mainly as a constitutive activator of housekeeping genes, growing evidence indicates that posttranslational modifications such as phosphorylation, acetylation, sumoylation, ubiquitylation and glycosylation can influence the transcriptional activity and stability of Sp1 [10, 22]. For example, Sp1 is phosphorylated at Ser101 by ataxia telangiectasia mutated kinase in response to DNA damage, and the proportion of chromatin-bound phosphorylated Sp1 rapidly increases . Thus, to clarify the molecular mechanisms underlying the observed increase in the amount of Sp1 in the nucleus at 32°C, modifications of Sp1 should be analyzed. Furthermore, Sp1 can regulate the expression of genes via interactions or interplay with other transcription factors such as Ets-1, c-myc, c-Jun and Egr-1, and/or components of the basal transcriptional machinery, and has been linked to chromatin remodeling through interactions with p300 and histone deacetylases . The factors collaborating or competing with ubiquitously expressed Sp1 should be identified to clarify the underlying mechanism of cirp induction at moderately low temperatures.