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Functional analyses of an LXXLL motif in nuclear receptor corepressor (N-CoR)
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.ORCID iD: 0000-0003-3927-4394
2004 (English)In: Journal of Steroid Biochemistry and Molecular Biology, ISSN 0960-0760, E-ISSN 1879-1220, Vol. 91, no 4-5, 191-196 p.Article in journal (Refereed) Published
Abstract [en]

Transcriptional repression is a major regulatory mechanism in cell differentiation, organogenesis, and oncogenesis. Two repressors of ligand-dependent transcription factors, nuclear receptor corepressor (N-CoR) and the related protein SMRT were identified as a silencing mediator for thyroid hormone receptor β and as a silencing mediator for retinoic acid and thyroid hormone receptors, respectively. Nuclear receptor coactivators such as steroid receptor coactivator-1 (SRC-1) contain multiple LXXLL motifs, which are essential and sufficient for its ligand-dependent interaction with nuclear receptors. N-CoR also has an LXXLL motif, located between repressor domains 1 and 2, and conserved between mouse and man. In contrast, SMRT lacks this motif.

This paper describes functional implications of the LXXLL motif in N-CoR. A 57-amino acid portion of N-CoR containing the LDNLL sequence (N-CoRLDNLL) fused to GST interacted with retinoic acid receptor α (RARα) and thyroid hormone receptor β (TRβ) in vitro. Similarly, [35S-methionine]N-CoRLDNLL interacted with a RARα fusion protein. N-CoRLDNLL also bound to RARα in vivo as determined in mammalian one-hybrid system in transfected CV-1 cells and by two-hybrid assays in bacteria. The interaction with RARα in vitro and in vivo was specific as determined by mutation of the sequence LDNLL to LDNAA. Our data suggest that the LDNLL motif in N-CoR has functional significance because it mediates interaction with nuclear receptors such as RARα and TRβ.

Place, publisher, year, edition, pages
2004. Vol. 91, no 4-5, 191-196 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:liu:diva-28799DOI: 10.1016/j.jsbmb.2004.04.006ISI: 000224043500001Local ID: 13987OAI: diva2:249611
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2013-10-22
In thesis
1. Nuclear receptor corepressor N-CoR: role in transcriptional repression
Open this publication in new window or tab >>Nuclear receptor corepressor N-CoR: role in transcriptional repression
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The human body consists of a multitude of cells of varying appearance and function. With a few exceptions they are genetically identical, and the key to their divergence lies in their different specific patterns of gene expression. Gene expression may be regulated at the level of transcription, in two opposing directions; either activation or repression. Gene transcription is controlled by transcription factors, which bind to regulatory DNA sequences, and direct gene expression in concert with auxiliary proteins. Among these the nuclear receptor corepressor N-CoR holds a central position. It serves as a docking unit between many different DNA-bound transcription factors, such as nuclear receptors, and large complexes of repressor proteins. Many repressor complexes of distinct compositions have been shown to contain N-CoR.

N-CoR plays a vital part in normal fetal development, and its involvement has been implicated in several pathological conditions. It has been shown to interact with unliganded nuclear receptors via CoRNR-box motifs in the C-terminal half of the protein. We have identified an NR-box motif, typical of coactivators, in the N-terminal part of N-CoR, which we have shown to be capable of interacting with the nuclear receptors RARα and TRß both in vitro and in vivo. A mutated NR-box motif did not interact accordingly. We discovered that the NR-box motif found in N-CoR displayed a ligand-dependent interaction with TRß in GST pulldown experiments, and that the immediate NR-box environment in N-CoR resembles NRbox environments in the coactivator CBP. We investigated a possible role for theN-CoR NRbox motif in regulation of the TSHß gene from a negative TR response element found in its promoter. In transient transfectiqns of GH3 cells, we found that both TRß3 and N-CoR are necessary for ligand-induced repression from this response element to occur. Mutating the NR-box abolished the repressive potential of N-CoR. The results were corroborated by results from transient transfections of HEK293/T cells, where siRNA-targeted degradation of endogenous N-CoR mRNA annihilated the ligand-induced repression, and where wild type mouse N-CoR but not mutated N-CoR restored the repression. In vitro binding assays also showed that TR, bound to its negative response element in the TSHß gene promoter, displayed an obligate ligand dependence in its interaction with N-CoR.

In several different leukemias N-CoR holds a key role. Abberant transcription factors bind stronger toN-CoR than their normal counterparts, leading to constitutive repression of key genes in hematopoiesis. Retinoid signaling, mediated by RARs plays a central part in differentiation of myeloid cells. We therefore investigated the extent of N-CoR expression in the myeloid cell line THP-1 during differentiation. Analyses both at mRNA- and at protein level showed that N-CoR expression was down-regulated as the myeloid cells differentiated. Exploring the effects of this on genes controlled by retinoic acid, we found in transient transfections of THP-1 cells that N-CoR modulated the expression level both at basal and at ligand-activated level. Several reports by others have also emphasized the importance of relative levels of different coregulatory proteins for determining the amplitude of the transcriptional response.

N-CoR binds both to transcription factors and to repressor complexes, but so far no report has been published regarding its possible DNA-binding capacity, anticipated by analysis of its amino acid sequence. Employing the selected and amplified binding sites (SAAB) assay we showed that N-CoR bound to DNA. Sequence determination resulted in the identification of a DNA sequence, ATNNTNCTC, which binds specifically toN-CoR. This finding adds another variable in the spectrum of N-CoR interactions.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet, 2004. 72 p.
Linköping University Medical Dissertations, ISSN 0345-0082 ; 869
National Category
Medical and Health Sciences
urn:nbn:se:liu:diva-24064 (URN)3623 (Local ID)91-7373-846-8 (ISBN)3623 (Archive number)3623 (OAI)
Public defence
2004-07-01, Berzelius-salen, Hälsouniversitetet, Linköping, 09:00 (Swedish)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2014-06-13Bibliographically approved

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Loinder, KristinaSöderström, Mats
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