Recruitment of CREB1 and histone deacetylase 2 (HDAC2) to the mouse Ltbp-1 promoter regulates its constitutive expression in a dioxin receptor-dependent manner.
Latent TGFbeta-binding protein 1 (LTBP-1) is a key regulator of TGFbeta targeting and activation in the extracellular matrix. LTBP-1 is recognized as a major docking molecule to localize, and possibly to activate, TGFbeta in the extracellular matrix. Despite this relevant function, the molecular mechanisms regulating Ltbp-1 transcription remain largely unknown. Previous results from our laboratory revealed that mouse embryonic fibroblasts (MEF) lacking dioxin receptor (AhR) had increased Ltbp-1 mRNA expression and elevated TGFbeta activity, suggesting that AhR repressed Ltbp-1 transcription. Here, we have cloned the mouse Ltbp-1 gene promoter and analysed its mechanism of transcriptional repression by AhR. Reporter gene assays, AhR over-expression and site-directed mutagenesis showed that basal Ltbp-1 transcription is AhR-dependent. Chromatin immunoprecipitation (ChIP) and RNA interference (RNAi) revealed that AhR regulates Ltbp-1 transcription by a mechanism involving recruitment of co-activators such as CREB1 and co-repressors such as HDAC2 to the Ltbp-1 promoter. In AhR-expressing (AhR+/+) MEF cells, the recruitment of HDAC1, 2 and 4 correlated with decreased K8H4 acetylation and impaired binding of pCREB(Ser133) to the Ltbp-1 promoter, likely maintaining a constitutive repressed state. AhR-/- MEF cells had the opposite pattern of HDACs and pCREB1(Ser133) binding to Ltbp-1 promoter, and therefore, over-expressed Ltbp-1 mRNA. In agreement, siRNA for HDAC2 increased Ltbp-1 expression and K8H4 acetylation in AhR+/+ but not in AhR-/- MEF cells. We suggest that HDAC2 binding keeps Ltbp-1 promoter repressed in AhR+/+ MEF cells, whereas in AhR-null MEF cells the absence of HDAC2 and the binding of pCREB(Ser133) allow Ltbp-1 transcription. Thus, epigenetics can contribute to constitutive Ltbp-1 repression by a mechanism requiring AhR activity.
Gomez-Duran
Aurea
A
Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias, Universidad de Extremadura, Avenida de Elvas s/n, 06071-Badajoz, Spain.
Ballestar
Esteban
E
Carvajal-Gonzalez
Jose M
JM
Marlowe
Jennifer L
JL
Puga
Alvaro
A
Esteller
Manel
M
Fernandez-Salguero
Pedro M
PM
D000107:Acetylation; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D019175:DNA Methylation; D005786:Gene Expression Regulation; D005838:Genotype; D056464:Histone Deacetylase 2; D006655:Histone Deacetylases / Q000235:genetics / Q000378:metabolism*; D006657:Histones / Q000378:metabolism; D053779:Latent TGF-beta Binding Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D034622:RNA Interference; D018336:Receptors, Aryl Hydrocarbon / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D020218:Response Elements / Q000235:genetics
C492004:Creb1 protein, mouse; D017362:Cyclic AMP Response Element-Binding Protein; D006657:Histones; D053779:Latent TGF-beta Binding Proteins; C509033:Ltbp1 protein, mouse; D018336:Receptors, Aryl Hydrocarbon; D012097:Repressor Proteins; C533424:Hdac2 protein, mouse; D056464:Histone Deacetylase 2; D006655:Histone Deacetylases