Identification of a Thyroid Hormone Response Element in the Mouse Kruppel-Like Factor 9 Gene to Explain Its Postnatal Expression in the Brain

Brain development is critically dependent on thyroid hormone (T-3). Kruppel-like factor 9 (Klf9) is a T-3-inducible gene in developing rat brain, and several lines of evidence support that KLF9 plays a key role in neuronal morphogenesis. Here we extend our findings to the mouse and demonstrate the presence of a functional T-3 response element (T3RE) in the 5' flanking region of the mouse Klf9 gene. Klf9 mRNA is strongly induced in the mouse hippocampus and cerebellum in a developmental stage-and T-3-dependent manner. Computer analysis identified a near optimal direct repeat 4 (DR-4) T3RE 3.8 kb upstream of the Klf9 transcription start site, and EMSAs showed that T-3 receptor (TR)-retinoid X receptor heterodimers bound to the T3RE with high affinity. The T3RE acts as a strong positive response element in transfection assays using a minimal heterologous promoter. In the mouse neuroblastoma cell line N2a[TR beta 1], T-3 caused a dose-dependent up-regulation of Klf9 mRNA. Chromatin immunoprecipitation assays conducted with N2a[TR beta 1] cells showed that TRs associated with the Klf9 T3RE, and this association was promoted by T-3. Treatment of N2a[TR beta 1] cells with T-3 led to hyperacetylation of histones 3 and 4 at the T3RE site. Furthermore, TRs associated with the DR-4 T3RE in postnatal d 4 mouse brain, and histone 4 acetylation was greater at this site compared with other regions of the Klf9 gene. Our study identifies a functional DR-4 T3RE located in the mouse Klf9 gene to explain its regulation by T-3 during mammalian brain development. (Endocrinology 150: 3935-3943, 2009)