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Nuclear receptor corepressor N-CoR: role in transcriptional repression
Linköping University, Department of Biomedicine and Surgery, Cell biology. Linköping University, Faculty of Health Sciences.
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.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 869
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-24064Local ID: 3623ISBN: 91-7373-846-8 (print)OAI: oai:DiVA.org:liu-24064DiVA: diva2:244380
Public defence
2004-07-01, Berzelius-salen, Hälsouniversitetet, Linköping, 09:00 (Swedish)
Opponent
Supervisors
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2014-06-13Bibliographically approved
List of papers
1. The nuclear receptor corepressor (N-CoR) modulates basal and activated transcription of genes controlled by retinoic acid
Open this publication in new window or tab >>The nuclear receptor corepressor (N-CoR) modulates basal and activated transcription of genes controlled by retinoic acid
2003 (English)In: Journal of Steroid Biochemistry and Molecular Biology, ISSN 0960-0760, E-ISSN 1879-1220, Vol. 84, no 1, 15-21 p.Article in journal (Refereed) Published
Abstract [en]

Variation in cell morphology and function is caused by differentiation. In myeloid differentiation, retinoid signaling, acting through heterodimers consisting of retinoic acid receptor and retinoid X receptor (RAR/RXR) plays a crucial part. The RAR/RXR heterodimers bind to naturally occurring response elements in the promoter regions of target genes, deciding whether the gene is to be transcribed or not. In the absence of the RAR-specific ligand all trans retinoic acid, RAR/RXR heterodimers are associated with the nuclear receptor corepressor N-CoR or the related SMRT.

Here we show, using Western, far-Western and Northern blot techniques, that when the human monocytic cell line THP-1 is allowed to differentiate into macrophage-like cells the expression of N-CoR is down-regulated both at the protein and at the mRNA level. To investigate how this affects the transcriptional activity of retinoic acid response element (RARE)-controlled genes, we performed transient transfection experiments in THP-1 and CV-1 cells. The results indicate that N-CoR functions not merely as a repressor of basal transcription, but rather as a modulator of both basal and ligand-activated transcription of genes controlled by RAR/RXR heterodimers in a dose-dependent manner.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-25258 (URN)10.1016/S0960-0760(03)00007-4 (DOI)000182278400003 ()9698 (Local ID)9698 (Archive number)9698 (OAI)
Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2017-12-13Bibliographically approved
2. Functional analyses of an LXXLL motif in nuclear receptor corepressor (N-CoR)
Open this publication in new window or tab >>Functional analyses of an LXXLL motif in nuclear receptor corepressor (N-CoR)
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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-28799 (URN)10.1016/j.jsbmb.2004.04.006 (DOI)000224043500001 ()13987 (Local ID)13987 (Archive number)13987 (OAI)
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2017-12-13
3. An LXXLL motif in nuclear receptor corepressor mediates ligand-induced repression of the thyroid stimulating hormone-β gene
Open this publication in new window or tab >>An LXXLL motif in nuclear receptor corepressor mediates ligand-induced repression of the thyroid stimulating hormone-β gene
2005 (English)In: Journal of Steroid Biochemistry and Molecular Biology, ISSN 0960-0760, E-ISSN 1879-1220, Vol. 97, no 4, 322-327 p.Article in journal (Refereed) Published
Abstract [en]

Nuclear receptor corepressor (N-CoR) regulates gene expression through interaction with DNA-bound nuclear receptors, recruiting multicomponent repressor complexes to the sites of target genes. We recently reported the presence of an LXXLL motif in N-CoR, and showed that this motif interacts in vitro and in vivo with retinoic acid receptor α (RARα) and thyroid hormone receptor β (TRβ). Transient transfection experiments now suggest that TRβ and N-CoR act synergistically and may both be required for ligand-induced repression from the negative TR response element in the thyroid stimulating hormone-β (TSHβ) gene promoter. Mutation of the LXXLL motif in N-CoR abolished ligand-induced repression at this response element. Furthermore, in vitro binding of N-CoR to a complex between TRβ and the negative TR response element was strictly ligand-dependent. We conclude that N-CoR and TRβ cooperate in the regulation of the TSHβ gene and that the ligand-dependent repression is mediated by the LXXLL motif in N-CoR.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-33337 (URN)10.1016/j.jsbmb.2005.06.031 (DOI)000233855300002 ()19348 (Local ID)19348 (Archive number)19348 (OAI)
Available from: 2009-10-09 Created: 2009-10-09 Last updated: 2017-12-13Bibliographically approved
4. N-CoR interacts directly with the DNA sequence ATNNTNCTC
Open this publication in new window or tab >>N-CoR interacts directly with the DNA sequence ATNNTNCTC
(English)Manuscript (preprint) (Other academic)
Abstract [en]

N-CoR (nuclear receptor corepressor) and SMRT (silencing mediator of thyroid and retinoid hormone receptors) are large proteins which are constituent parts of numerous multicomponent repressor complexes. They function by recruiting repressors to the sites of DNA-bound transcription factors, thereby modifying chromatin condensation to restrict access for general transcription factors to a regulated gene. Both N-CoR and SMRT contain two highly conserved SANT (SWI3, ADA2, N-CoR, TFIIIB) domains which, by homology, have been suggested to confer DNA-binding properties. To date however, there have been no data published that support a DNA-binding function for N-CoR (or SMRT). To investigate if N-CoR is capable of binding to DNA, we used stably transfected S2 cells expressing FLAGtagged N-CoR We then allowed N-CoR to interact with double stranded degenerated oligonucleotides. Our results showed that oligonucleotides were retained by N-CoR, and that these contained the consensus sequence ATNNTNCTC.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-84853 (URN)
Available from: 2012-10-25 Created: 2012-10-25 Last updated: 2013-10-23Bibliographically approved

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