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Faith Group

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In vertebrates, retinoic acid (RA) is an important morphogenetic signal that controls embryonic development, as well as organ homeostasis in adults. RA action depends on the function of the RA-genetic machinery, which includes a metabolic module and a signaling module. The metabolic module regulates the spatiotemporal distribution of RA by the combined action of the RA-synthesizing Aldh1a enzymes, and the RA-degrading Cyp26 enzymes. The signaling module includes members of the nuclear hormone receptors family RAR and RXR, and controls the transcriptional state of RA-target genes. RA-signaling has been described primarily in chordates, but the recent finding of elements of the RA-genetic machinery in non-chordate deuterostomes has changed our perspective on the evolutionary origin of this morphogenetic signal, challenging previous assumptions that related the invention of the RA-genetic machinery with the origin of the chordate body plan. To illuminate the evolutionary origin of the RA machinery we have conducted an extensive survey of Aldh1a, Cyp26 and RAR orthologs in genomic databases of 13 non-deuterostome metazoans. Our results show for the first time the presence of Aldh1a, Cyp26 and RAR in protostomes, which implies that the components of the RA machinery may be ancient elements of animal genomes, already present in the last common ancestor of bilaterians. Interestingly, our data also reveal that independent losses of the RA toolkit have occurred multiple times during animal evolution, which may have been relevant for the evolution and developmental diversity of the current metazoan lineages.

Family time.rar

For a family with four kids, two dogs, assorted cats, and a constant stream of family and friends dropping by, life in the Austin family home has always been remarkably steady and contented. When a family friend suddenly dies in a plane crash, the Austins open their home to an orphaned girl, Maggy Hamilton. And that's when it all changes.

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Thank you to Jerald and Heather for making this a much easier experience than anticipated. Our family felt like we were working with friends. Thank you for making mom and dads memorial an amazing tribute to them.

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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.

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.

(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.

The TEL protein is member of the ETS family of transcription factors that have several domains in common. We have generated deletion constructs to investigate which domains of the protein are crucial for binding to RARα. A schematic overview is shown in Figure 1C. All constructs were efficiently produced in the in vitro transcription/translation system (middle left panel of Figure 1C) and were used in pulldown experiments (middle right panel of Figure 1C). All constructs that contain both the HLH and DBD domains of TEL were binding to RARα whereas other constructs failed to bind. Both HLH and DBD domains of TEL are thus crucial for binding to RARα. It has also been described that the HLH domain of TEL causes aggregation of TEL in vitro and this could result in entrapping of other proteins. Such a a-specific entrapping of proteins does not explain the interactions shown in Figure 1 since (1) we do not see interaction with GST, and (2) we also do not see any interaction with two TEL mutants in which the polymerization domain is still intact, i.e. the TEL-DBDmutant and the deletion mutant HCE.To investigate whether also small point mutations within the DBD and HLH domains of TEL can abrogate the interaction between TEL and RARα we generated two mutants of TEL. The mutations within the HLH domain of TEL (V112A/L113A) are located in the binding surface of the HLH structure important for polymerization. This mutant is impaired in polymerization (data not shown). The DBD mutant of TEL (R396L, R399L) was described previously [21], [29] and fails to bind ETS responsive elements. In our in vitro binding assay with GST-RARα both TEL mutants were tested for interaction with RARα. Figure 1C, lower panel shows that these TEL mutants failed to bind RARα. From this we conclude that both HLH and DBD domains are crucial for binding to RARα and that mutations that impair the function of these domains also disrupt binding of TEL to RARα. 041b061a72


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