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

Neuroendocrinology. 2010;91(1):64-76. doi: 10.1159/000228833. Epub 2009 Jul 14.


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 TRbeta2 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 TRbeta2 is bound; pCREB and TR do not present mutual interference on their binding sites.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Binding Sites / genetics
  • Cells, Cultured
  • Cyclic AMP Response Element-Binding Protein / metabolism*
  • Deoxyribonuclease I / metabolism
  • Glyceraldehyde-3-Phosphate Dehydrogenases / metabolism
  • Hypothalamus / metabolism*
  • Molecular Sequence Data
  • Phosphorylation
  • Promoter Regions, Genetic*
  • RNA, Messenger / metabolism
  • Rats
  • Rats, Wistar
  • Receptors, Thyroid Hormone / metabolism*
  • Thyroid Hormone Receptors beta / metabolism
  • Thyrotropin-Releasing Hormone / genetics*
  • Thyrotropin-Releasing Hormone / metabolism*
  • Triiodothyronine, Reverse / metabolism


  • Cyclic AMP Response Element-Binding Protein
  • RNA, Messenger
  • Receptors, Thyroid Hormone
  • Thyroid Hormone Receptors beta
  • Triiodothyronine, Reverse
  • Thyrotropin-Releasing Hormone
  • Glyceraldehyde-3-Phosphate Dehydrogenases
  • Deoxyribonuclease I