top of page

Feedback

Öffentlich·10 Mitglieder

@19 43 Rar ##BEST##


Retinoic acid receptor (RAR) signaling is important for regulating transcriptional activity of genes involved in growth, differentiation, metabolism and reproduction. Defects in RAR signaling have been implicated in cancer. TEL, a member of the ETS family of transcription factors, is a DNA-binding transcriptional repressor. Here, we identify TEL as a transcriptional repressor of RAR signaling by its direct binding to both RAR and its dimerisation partner, the retinoid x receptor (RXR) in a ligand-independent fashion. TEL is found in two isoforms, created by the use of an alternative startcodon at amino acid 43. Although both isoforms bind to RAR and RXR in vitro and in vivo, the shorter form of TEL represses RAR signaling much more efficiently. Binding studies revealed that TEL binds closely to the DNA binding domain of RAR and that both Helix Loop Helix (HLH) and DNA binding domains of TEL are mandatory for interaction. We have shown that repression by TEL does not involve recruitment of histone deacetylases and suggest that polycomb group proteins participate in the process.




@19 43 rar


Download Zip: https://www.google.com/url?q=https%3A%2F%2Fgohhs.com%2F2uf8ey&sa=D&sntz=1&usg=AOvVaw1v1HO7Qih3q2CSP_ZgEwn0



Citation: Meester-Smoor MA, Janssen MJFW, ter Haar WM, van Wely KHM, Aarnoudse A-JLHJ, van Oord G, et al. (2011) The ETS Family Member TEL Binds to Nuclear Receptors RAR and RXR and Represses Gene Activation. PLoS ONE 6(9): e23620.


Copyright: 2011 Meester et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Funding: This project was funded by the Dutch Cancer Society, grant DDHK 2003-2869 (www.kwf.nl). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


Nuclear receptors (NR) belong to the large family of well studied transcription factors that are important for growth, differentiation, metabolism and reproduction in higher organisms. Small molecules, such as steroids, thyroid hormones and retinoids serve as ligands and bind to the ligand binding domains (LBDs). NRs bind DNA of target promotors as hetero or homo dimers using their highly homologous DNA binding domain (DBD) [1]. One of these NR, the retinoic acid receptor (RAR) has several isoforms, forms a heterodimer with retinoic-x-receptor (RXR), and binds the ligand all-trans retinoic acid (ATRA). RAR-signaling induces differentiation and apoptosis in a wide variety of cells. Furthermore, retinoic acid has tumor-suppressive activity and defects in RAR signaling are implicated in cancers [2], [3].


The regulation of gene expression by NR involves the release and binding of co-repressor and co-activator complexes. Nuclear receptor co-repressor (N-CoR) and silencing mediator of retinoid and thyroid hormone receptors (SMRT) are co-repressors that associate with RAR and recruit complexes with histone deacetylase (HDAC) activity [4], [5]. Activation of RAR target genes involves the binding of co-activators of the p160 family (SRC1, SRC2 also known as Tif2, and SRC3 also known as RAC3) of which most of these have intrinsic histone acetylase (HAT) activity. In addition, HAT activity-containing p300/CBP proteins are recruited by these co-activator complexes [6], [7]. Recent studies have shown that both active and repressed RAR-regulated genes continuously exchange co-activator and co-repressor complexes [8], [9]. This dynamic and cyclic process cause a continuous recruitment of both HAT and HDAC activity to the promoters and the balance between these complexes finally results in either activation or repression of gene expression. In addition, various other processes including ubiquitination, sumoylation, methylation and phosphorylation have been implicated in regulation of NR activity [9], [10], [11], [12].


Here we describe a novel mode of repression of RAR signaling. The repression involves the binding of the transcriptional repressor TEL to RARα and RXRα. TEL (ETV6) is a member of the ETS family of transcription factors. TEL is expressed throughout the body including the hematopoietic system and is crucial for hematopoietic stem cell maintenance, as has been shown in a Tel knockout mice model [13]. These mice have been shown to die due deficient yolk sac angiogenesis. The classical mode of repression by TEL has been studied extensively and involves the binding of TEL to DNA-responsive elements within promoters with its DBD domain. The helix-loop-helix (HLH) domain, also called Pointed (PNT) or SAM domain is important for polymerization of TEL [14], [15]. Repression involves either the recruitment of co-repressor complexes and HDACs [16], [17], or the recruitment of L(3)MBT-containing polycomb group-complexes [18] that facilitate long-term repression by chromatin remodeling other than deacetylase activity. Here we show that the interaction between TEL, RARα and RXR involves both DBD and HLH domains of TEL and the DBD domain of RARα. Furthermore, we show that both isoforms of TEL, generated by the use of an alternative start codon, influence RAR signaling, that this repression is HDAC-independent, and that the shorter isoform is a much more efficient repressor compared to the larger isoform.


GST fusion constructs were expressed in E. coli and the fusion proteins were loaded onto glutathion sepharose beads (GE Healthcare, Uppsala, Sweden) according to the manufacturer's recommendations. The amount of GST protein (µg) used for loading was similar for the different fusion proteins in each experiment. When necessary, because of varying levels of GST fusion protein expression, untransformed E. coli lysate was added during loading of the beads, to keep also the total amount of lysate (µg protein) at similar levels. Binding of proteins to the immobilized GST fusion proteins was carried out as described by Van Wely et al [25] with the exception that instead of using columns, the beads were incubated and washed in microfuge tubes and collected by centrifugation at maximal speed. Beads containing non-fused GST were used as controls. In vitro transcription/translation products labeled with 35S-methionine were used for the binding experiments. The bound translation products were eluted from the beads by boiling in SDS-PAGE sample buffer, separated by SDS-PAGE, and visualized by autoradiography.


2106 Hep3B cells were transfected with pcDNA3, pcDNA3 TEL-HA M1C or pcDNA3 TEL-HA M43C in combination with pcDNA3 BioV5-RARN3 and BirA plasmids using Fugene 6 transfection reagent (Roche) according to manufacturer's protocols. 48 hours after transfection, cells were harvested and subsequently lysed using a buffer containing 20 mM Tris pH 8.0, 137 mM NaCl, 10% glycerol, 1% NP-40, 2 mM EDTA (pH 8.0) supplemented with Complete Mini protease inhibitor cocktail 9 (Roche). BioV5 RAR-N3 proteins were precipitated using streptavidin-coated Dynabeads (Invitrogen Dynal AS, Oslo, Norway). Precipitated proteins were subjected to SDS PAGE and Western blotting according to standard protocols. Antibodies: a-TEL, clone 3B10 (Abnova, Taipei, Taiwan); a-HA, biotin-tagged, clone 3F10 (Roche); streptadivin-HRP, (BioGenex Laboratories Inc., San Ramon, CA, USA)


The transcriptional cofactor MN1 stimulates and inhibits RAR/RXR-mediated transcription [26]. In order to investigate if MN1 bound to RARα and/or RXRα we performed GST pulldown experiment using GST-fused RARα (GST-RARα) and GST-RXRα together with in vitro produced and 35S-labeled proteins. MN1 was unable to bind to GST-RARα and RXRα (Figure 1A). Other cofactors are thus involved in this transcription regulation. In this experiment we also tested the MN1-TEL protein. This leukemogenic protein is formed by an AML-causing translocation (12;22) between MN1 and TEL genes. To our surprise, MN1-TEL bound very strongly to RARα and RXRα. In order to locate the domain that binds RARα and RXR we investigated whether binding was a property of the TEL moiety in the MN1-TEL protein. This indeed appeared to be the case as is shown in figure 1A, right panel. The presence of an alternative start methionine at aa 43 of the coding region of the TEL gene gives rise to two isoforms of TEL [27], [28]. Both isoforms of TEL bound GST-RARα and GST-RXRα equally efficiently in this GST pulldown assay.


(A) TEL and MN1-TEL bind RAR and RXR whereas MN1 does not. In vitro generated, 35S-labeled proteins, as shown in the input lanes, were used for GST pulldowns with GST-RAR, GST-RXR and GST (as control). (B) TEL binds the DBD domain of RARα. Upper panel: Schematic overview of the RARα and RXRα constructs. The domains within RARα and RXRα are indicated with letters commenly used to indicate the domains within the nuclear receptor family. At the bottom the different regions are indicated with respect to their function. AF1, Activator function 1; DBD, DNA binding domain; CTE, C-terminal extension; LBD, ligand-binding domain; AF2, activator function 2. Lower panel: GST pulldowns with in vitro transcribed/translated TEL and TIF2. Binding of TEL to RAR and RXR differs from the binding of TIF2 to RARα and RXRα. Binding of TIF2 was detected for LB-containing RARα constructs whereas TEL bound to the DBD region of RARα. No binding of TEL to the AF1 and first part of DBD was detected (GST-RAR-N2). (C) HLH and DBD domains of TEL are crucial for binding to RARα. Upper panel: Schematic overview of deletion constructs of TEL. Numbers indicate the amino acid positions. Middle left: ITT products were used for GST pulldown assays with GST-RAR-N3. Middle right: Only deletion constructs that contain both HLH and DBD domains were detected in the in vitro binding assay. Lower panel: WT TEL showed binding to GST-RARα, whereas HLH, DBD or double mutant TEL proteins failed to bind GST-RARα. (D) bio-precipitation of bioV5-tagged RAR-N3 proteins. Only in the presence of bioV5-tagged RARN3, TEL-HA proteins were detectable in the precipitations, showing the in vivo binding between TEL and RAR. Right panel shows input lysates. TEL proteins were detectable with both a-TEL and a-HA antibodies. 041b061a72


  • Info

    Willkommen in der Feedback Gruppe! Hier dürft Ihr alles über...

    Gruppenseite: Groups_SingleGroup
    bottom of page