Functional interactions with Pit-1 reorganize co-repressor complexes in the living cell nucleus

Abstract

The co-repressor proteins SMRT and NCoR concentrate in specific subnuclear compartments and function with DNAbinding factors to inhibit transcription. To provide detailed mechanistic understanding of these activities, this study tested the hypothesis that functional interactions with transcription factors, such as the pituitary-gland-specific Pit-1 homeodomain protein, direct the subnuclear organization and activity of co-repressor complexes. Both SMRT and NCoR repressed Pit-1-dependent transcription, and NCoR was co-immunoprecipitated with Pit-1. Immunofluorescence experiments confirmed that endogenous NCoR is concentrated in small focal bodies and that incremental increases in fluorescent-protein-tagged NCoR expression lead to progressive increases in the size of these structures. In pituitary cells, the endogenous NCoR localized with endogenous Pit-1 and the co-expression of a fluorescent-protein-labeled Pit-1 redistributed both NCoR and SMRT into diffuse nucleoplasmic compartments that also contained histone deacetylase and chromatin. Automated image-analysis methods were applied to cell populations to characterize the reorganization of corepressor proteins by Pit-1 and mutation analysis showed that Pit-1 DNA-binding activity was necessary for the reorganization of co-repressor proteins. These data support the hypothesis that spherical foci serve as co-repressor storage compartments, whereas Pit-1/co-repressor complexes interact with target genes in more widely dispersed subnuclear domains. The redistribution of corepressor complexes by Pit-1 might represent an important mechanism by which transcription factors direct changes in cell-specific gene expression.

A Nuclear Receptor Corepressor–Dependent Pathway Mediates Suppression of Cytokine-Induced C-Reactive Protein Gene Expression by Liver X Receptor

“…Nuclear localization of overexpressed NCoR was confirmed by indirect immunofluorescence ( Figure 5A). In accordance with previous observations, 46 NCoR-transfected cells displayed a characteristic nuclear speckle staining ( Figure 5A), whereas an IgG control antibody exhibited only background staining (data not shown). As shown in Figure 5B, cotransfection of the NCoR expression vector resulted in a dose-dependent inhibition of IL-1␤/IL-6 -induced CRP promoter activity, indicating that NCoR participates in regulation of CRP transcriptional activity.…”

Energy homeostasis targets chromosomal reconfiguration of the human GH1 locus

“…PIT-1 binds to sequences in HS I/II within the GH1 LCR as well as the proximal GH1 promoter region and is essential for activation of the GH1 locus during somatotroph development and for ongoing efficient postnatal expression (28,34,35,37). An interaction between NCOR and PIT-1 was previously suggested as the mechanism by which GH1 is silenced in PIT-1-producing lactotrophs during embryonic differentiation of the anterior pituitary (40,41). Our current observations suggest that NCOR may also act as a corepressor in the postnatal dynamic and the transient transcriptional suppression of GH1 in somatotrophs.…”

“…The failure to detect a similarly increased association of NCOR with the murine Gh promoter is consistent with the lack of negative response to excess caloric intake. NCOR is known to interact with the pituitary transcription factor PIT-1 (40,41). PIT-1 binds to sequences in HS I/II within the GH1 LCR as well as the proximal GH1 promoter region and is essential for activation of the GH1 locus during somatotroph development and for ongoing efficient postnatal expression (28,34,35,37).…”

“…RNA polymerase (pol) II interaction with the GH1 promoter region was examined as an indication of corresponding changes in promoter accessibility and potential activity (42). Chromatin isolated from the pituitaries of 171hGH/CS-TG mice fed HFD or LFD for 3 days with and without prescribed physical activity was assessed by ChIPincluding GH1, in pituitary lactotrophs (38)(39)(40)(41). HFD challenge resulted in a 4-fold increase in association of NCOR with the human GH1 promoter region in the pituitary, as assessed by ChIP assay (P < 0.01, n = 3; Figure 4A).…”

EBV nuclear antigen EBNALP dismisses transcription repressors NCoR and RBPJ from enhancers and EBNA2 increases NCoR-deficient RBPJ DNA binding

“…NCoR recruits class I and II histone deacetylases (HDAC), including HDAC3, which repress transcription and associate with matrix-associated deacetylase (MAD) bodies, subnuclear sites of repressed transcription (24,25). NCoR overexpression represses EBNA2 up-regulation of promoter constructs and sequesters RBPJ into MAD bodies (21,26).…”