Characterization of Rex1 Role in Tsix Regulation Abstract Tsix activation and the subsequent silencing of Xist that occurs as a result of Tsix expression is a crucial part of the proper inactivation of a single X chromosome. The transcription of Tsix, antisense transcript of Xist, is regulated by factors Rex1, Klf4, and c-Myc. Previous studies have shown that Rex1 (Zfp42) is critical for expression of Tsix. As cells that exhibit decreased levels of Tsix transcript lack the binding of Rex1 to DXPas34. In addition studies have shown that knockdown of Rex1 results in decreased levels of Tsix. These studies have shown the importance of Rex1 but have yet to characterize the function of Rex1 in the Tsix regulatory pathway. This study seeks to clarify Rex1 function in the context of Tsix expression, by identifying which proteins Rex1 interacts with when Tsix expression is observed. In addition we will explore the importance of Rex1 binding to the DXpas34 region. The results of this study will shed light on the regulatory pathway of Tsix. Introduction X-inactivation is essential for the proper development of organisms. Improper inactivation typically is lethal as this leads to over expression of genes located on the X chromosome. The mechanism by which a single X chromosome is silenced or inactivated is a complicated one. Research currently is focused on the expression of Xist, a noncoding RNA that surrounds the inactivated X (Xi) chromosome and recruits polycomb and other proteins to facilitate silencing by converting the future Xi into its heterochromatic state (Navarro et al. 2010) Initially Xist is expressed by both X chromosomes; although Xist is eventually only expressed on the future inactive X. Similarly the antisense transcript of Xist, Tsix, is initially also expressed by both X chromosomes at low levels, but will only be continue to be expressed on the active X chromosome (Xa). Tsix is able to repress Xist transcription in cis by recruiting RNAi machinery to interfere with Xist RNA as well as inducing heterochromatin formation in Xist promoter region (Navarro et al. 2010). Thus for a single X to be inactivated and for the other to remain active proper transcription of Xist as well as Tsix must occur. The DXPas34 region located in the 5’ untranslated region of Tsix specifically located between the major promoter of Tsix and Xist, and is essential for their expression. Deletion of DXPas34 results in neither Tsix nor Xist being expressed (Vigneau et al. 2006). DXPas34 is a tandem repeat that is bound by CTCF, Yy1, and Rex1. CTCF and Yy1 have been shown to be involved in the regulation of both Tsix and Xist (Cohen et al. 2007). The function of Rex1 on the other hand is largely unknown. The DXPas34 region must be bound by Rex1 in order for Tsix to be fully expressed; Rex1 binds both ends of the tandem repeat. Only Rex 1 binding is not sufficient for Tsix expression; other elements that need to be bound include Klf4 and c-Myc (Stavrpoulos et al. 2005). Rex1 is an intriguing element within the Tsix activation mechanism. Cells with reduced Tsix expression were found to lack Rex1 binding to DXPas34 but other elements, Klf4 and c- Myc, were still able to bind normally. The exact role of Rex1 is unknown, though it has been suggested that Rex1 may be required for elongation of Tsix transcript (Navarro et al. 2010). Rex1 is a zinc finger protein that has two sequences to which it typically binds (5- GGCAGCCATTA-3 and 5-GGCCATTA-3) (Do Kim et al. 2007). Interestingly Rex1 is a member of the Yy1 sub family of zinc finger proteins. Yy1 is known to interact with subunits of histones deacetylase complexes, RYBP (a repressor protein), and c-Myc (Do Kim et al. 2007). The interactions of Yy1 with regulatory elements and the similarities with Rex1 suggest that Rex1 may also be involved gene regulation specifically regulation of Tsix. In order to characterize the role of Rex1 in Tsix regulation this study will seek to determine the proteins that Rex1 interacts with and determine the functional significance of Rex1 binding to the DXPas34 region. Specific Aims 1. Determine which proteins Rex1 interact with in Tsix activation. 2. Determine if binding of Rex1 to DXPas34 affects binding of other proteins. Experiments Specific aim 1 Experiment 1: GST pull-down assay In order to determine what proteins Rex1 interacts with a GST pull-down will be performed similar to the pull-down described in “Detection of Protein-protein Interactions Using the GST Fusion Protein Pull-down Technique”. Rex1 will be cloned in to an inducible expression vector with isopropyl-D-thiogalactoside. The resulting fusion protein will be expressed in bacteria. Using affinity chromatography on glutathione-agarose beads the fusion protein with be purified. At the onset of X-inactivation cells from female ES cell lines will be lysed, using liquid homogenization. The cell lysate will be added to the purified fusion protein, and incubated with glutathione-agarose beads. The Rex1 fusion protein will now be bound to proteins to which Rex1 interacts; the fusion protein will also be bound to the glutathione-agarose beads. The lysate fusion protein glutathione-agarose bead mixture will then be centrifuged to separate proteins not bound to Rex1. Once separated the proteins bound to the Rex1 fusion protein can be washed then separated by SDS-PAGE. The proteins that interact with Rex1 can then be determined using Matrix-assisted laser desorption/ionization (MALDI). Experiment 2: Yeast two hybrid In order to assess the validity of the results from the GST pull-down assay a yeast 2 hybrid assays will be performed. These assays will look for interactions between Rex1 and proteins known to be involved in Tsix regulation, specifically CTCF, Yy1, Klf4, c-Myc, Sox2, Oct 4, and Nanog, HDAC2, HDAC3, DNMT1, DNMT3A and B. Depending on the results of the GST pull-down more proteins may be chosen for inclusion in Yeast two hybrid experimentation. The proposed Yeast two hybrid assay as describe in Gietz et al. 1997. The coding sequence on Rex1 will be inserted into a binding domain plasmid of Gal4. The coding sequence of the protein of interest (proteins identified by GST pull-down assay) will be fused with the activating domain plasmid of Gal4. Using a lithium acetate method the newly created plasmids will be inserted into yeast. To test for activation the yeast strains will be grown on a rich medium until significant cultures are present. Samples of the cultures will then be transferred to SC-His + 3-AT plates. If activation does occur the cultures will grow on SC-His + 3-AT plates. Experiment 3: Fluorescence resonance energy transfer A fluorescence resonance energy transfer (FRET) experiment will be used to evaluate the in vivo interactions of Rex1 with proteins that were identified in both the yeast two hybrid assay and GST pull-down. The construction of GFP-Rex1, BFP-protein of interest constructs as wells as transfection of female ES cells and image analysis will follow the procedure as described by (Mahajan et al. 1998). Specific aim 2 Experiment 4: Does Rex1 affect CTCF and YY1 binding in DXPas34 To test if Rex1 binding interferes with the binding of CTCF and Yy1 to the DXPas34 tandem repeat I propose an experiment similar to a yeast two hybrid assay as describe in experiment 2. The main difference being the Gal4 promoter will be replaced with the DXPas34 tandem repeat in a yeast strain. Like a yeast two hybrid assay the activating domain of Gal4will be fused to the protein of interest; CTCF and Yy1. In addition to the CTCF and Yy1 constructs being inserted in to the separate yeast strains that have the Gal4 binding domain replaced with DXPas34. A copy of WT Rex1 DNA will also be inserted into each strain. Following the same procedure as described in experiment to the remainder of the experiment will be the same. Yeast strains will be grown on rich medium then transferred to SC-His + 3-AT plates. If either strain exhibits growth on the SC-His + 3-AT plates, then Rex1 binding will be assumed not to effect CTCF or Yy1 binding to DXPas34. Experiment 5: Can a single copy of Rex1 induce Tsix expression Targeted deletion of DXPas34 region will remove one of the two binding regions for Rex1. The ES cells will then be assessed on their ability to fully develop. Also cells will be tested for levels of Tsix mRNA throughout the X-inactivation process. If Tsix mRNA levels are normal then it can be assumed that only a single Rex1 unit is needed to induce Tsix expression. If the cell experiences improper X-inactivation but normal Tsix mRNA levels this could implicate Rex1 directly in the Xist regulatory pathways this would only be expected in experiment 4 showed that Rex1 did not affect the binding of CTCF or Yy1. Discussion of expected results To begin the characterization of Rex1 in the regulation of Tsix we looked to find which proteins Rex1 interacted with at the time of Tsix activation. We begin with a genome wide search with a GST pull-down assay. From this assay we expect to find that Rex1 interacts with other factors that regulate Tsix expression including Klf4, Sox2, and c-Myc. While less likely we may see Rex1 interacts with CTCF and Yy1. Results from the yeast two hybrid assay should mimic the results of the GST pull-down. The assay should simply provide more evidence in support of GST pull-down results as it is less likely to receive two false positives from two different assays.