Phosphorylated Cyclic-AMP-Response Element-Binding Protein and Thyroid Hormone Receptor Have Independent Response Elements in the Rat Thyrotropin-Releasing Hormone Promoter: An Analysis in Hypothalamic Cells

Background: Thyrotropin-releasing hormone (TRH) from the hypothalamic paraventricular nucleus (PVN) controls the activity of the hypothalamus-pituitary-thyroid axis. TRH is expressed in other hypothalamic nuclei but is downregulated by 3,3′,5-L-triiodothyronine (T 3 ) exclusively in the PVN. Thyroid hormone receptors (TRs) bind TRH promoter at Site-4 (–59/–52), also proposed to bind phosphorylated cAMP response element-binding protein (pCREB). However, nuclear extracts from 8Br-cAMP-stimulated hypothalamic cells showed no binding to Site-4 and instead to cAMP response element (CRE)-2 (–101/–94). Methods: We characterized, by DNA footprinting and chromatin immunoprecipitation, the sites in the rat (–242/+34) TRH promoter that bind to nuclear factors of hypothalamic primary cultures incubated with 8Br-cAMP and/or T 3 . Results: In primary cultures of fetal hypothalamic cells, TRH mRNA levels rapidly diminished with 10 nM T 3 while they increased by 1 mM 8Br-cAMP (± T 3 ). Site-4 was protected from DNase I digestion with nuclear extracts from T 3 -incubated cells but not from controls or from those incubated with 8Br-cAMP, which protected CRE-2; T 3 + 8Br-cAMP coincubation caused no interference. The region protected by nuclear extracts from cAMP-stimulated cells included sequences adjacent to CRE-2-containing response elements of the SP/Krüppel family. A TRβ2 antibody immunoprecipitated chromatin containing Site-4 but not CRE-2, from cells incubated with T 3 . A pCREB antibody immunoprecipitated CRE-2 containing chromatin in controls and more in 8Br-cAMP-stimulated cells but none when cells were incubated only with T 3 . Recruitment of the 2 transcription factors was preserved in cells simultaneously exposed to 8Br-cAMP and T 3 . Discussion: These results show that pCREB binds to a response element in the TRH promoter (CRE-2) that is independent of Site-4 where TRβ2 is bound; pCREB and TR do not present mutual interference on their binding sites.