title: 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.
abstract: 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).
  
  
  CONCLUSIONS
  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.
authors: Departamento de Genética y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, México.
authors: M Y
authors: MY
authors: Díaz-Gallardo
authors: A
authors: A
authors: Cote-Vélez
authors: A
authors: A
authors: Carreón-Rodríguez
authors: J L
authors: JL
authors: Charli
authors: P
authors: P
authors: Joseph-Bravo
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D007031:Hypothalamus / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D011988:Receptors, Thyroid Hormone / Q000378:metabolism*; D037042:Thyroid Hormone Receptors beta / Q000378:metabolism; D013973:Thyrotropin-Releasing Hormone / Q000235:genetics* / Q000378:metabolism*; D014285:Triiodothyronine, Reverse / Q000378:metabolism
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D012333:RNA, Messenger; D011988:Receptors, Thyroid Hormone; D037042:Thyroid Hormone Receptors beta; D014285:Triiodothyronine, Reverse; D013973:Thyrotropin-Releasing Hormone; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D003850:Deoxyribonuclease I