Identification of a Ctcf Cofactor, Yy1, for the X Chromosome Binary Switch  Mary E. Donohoe, Li-Feng Zhang, Na Xu, Yang Shi, Jeannie T. Lee  Molecular Cell  Volume 25, Issue 1, Pages 43-56 (January 2007) DOI: 10.1016/j.molcel.2006.11.017 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Yy1 Protein Binds Tsix In Vitro (A) Map of Tsix, its previously defined Ctcf sites, and Yy1 sites identified here. The relative orientation of Yy1 sites is indicated by the triangle. DXPas34 is a tandem repeat with 40 potential Ctcf and 20 potential Yy1 sites. The Tsix bipartite enhancer is indicated in green. (B) Yy1 and Ctcf sites are paired at Tsix. All sites shown are mouse, except where noted. Yy1 and Ctcf sites are boxed in red and green, respectively. Consensus sequences are shown below. (C) Yy1 specifically binds Tsix DNA elements in vitro. By EMSA, in vitro-synthesized Yy1 protein (Yy1) or d0 ES nuclear extract (NE) shifts 32P-labeled sites A, C, or E. Unprogrammed reticulocyte lysate (lane 1 of each panel) does not produce a specific shift. Comp, unlabeled competitors at 100× molar excess. WT, wild-type probe. Mut, mutated Yy1 probe. Arrows, Yy1-DNA shift. Asterisks, Yy1 supershift. Molecular Cell 2007 25, 43-56DOI: (10.1016/j.molcel.2006.11.017) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 Yy1 Protein Interacts with Tsix In Vivo (A) ChIP analysis of Ctcf sites A, C, and F of Tsix in male and female ES cells on d0 and d12. Mock, control ChIP with no chromatin. Chic1, a control locus flanking the Xic. (B) Real-time PCR quantitation of Ctcf and Yy1 binding at site E in differentiating wild-type female ES cells. All y values (in arbitrary units) are normalized to input after subtracting out the background from no-antibody controls. Error bars represent one standard deviation from the mean. (C) The allele-specific ChIP assay is based on a polymorphic AseI site in site E. PCR primers, green. Probe, blue. Expected fragment sizes are shown below the map. (D) Allele-specific ChIP analysis for Ctcf and Yy1 binding in differentiating wild-type and TsixΔCpG/+ female ES cells. The percent 129 PCR product is indicated. Molecular Cell 2007 25, 43-56DOI: (10.1016/j.molcel.2006.11.017) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 Yy1 Directly Interacts with Ctcf (A) Ctcf coimmunoprecipitates with Yy1 when overexpressed in HEK cells. HEK cells were cotransfected with Ctcf and/or HA-tagged Yy1 constructs, the proteins immunoprecipitated (IP) from whole-cell extracts with α-HA, α-Yy1, or control IgG antibodies, and then western blotted and detected by α-Ctcf to look for Ctcf among Yy1 immunoprecipitants. Arrow, Ctcf. Asterisk, immunoglobulin heavy chains. An α-HA western blot confirmed equal starting materials. (B) Endogenous Ctcf also coimmunoprecipitates with endogenous Yy1 in ES cells. CoIP of endogenous Yy1 and Ctcf using α-Yy1 antibodies from d0 ES nuclear extracts. α-control, normal rabbit serum. The IP was subjected to α-Ctcf western analysis. Arrow, Ctcf. (C) Endogenous Yy1 also coimmunoprecipitates with endogenous Ctcf in ES cells. IP with α-Ctcf antibodies from d0 female ES cells. α-control, normal rabbit serum. The immunoprecipitate was subjected to α-Yy1 western analysis. Arrow, Yy1. (D) Yy1 binds Ctcf through its zinc finger (Zn) and HDAC (H) regions. Purified GST-Yy1 fusion proteins (20 μg each) were tested for binding to 35S-labeled Ctcf protein. GST fusions to various Yy1 domains are noted by amino acid residues above lanes. GST pull-downs were performed in 150 mM or 300 mM NaCl. A summary of domain interactions is presented below. (E) Ctcf binds the Yy1 through multiple domains but interacts with the Yy1 zinc finger region only through its N terminus. Purified GST-Ctcf fusion proteins (20 μg each) were tested for binding to either 35S-labeled full-length Yy1 protein or its zinc finger domain. Fusions to various Ctcf domains are as indicated above lanes. Pull-downs were performed in 300 mM NaCl. A summary of domain interactions is presented below. Molecular Cell 2007 25, 43-56DOI: (10.1016/j.molcel.2006.11.017) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Yy1+/− Mice and Transmission Ratio Distortion (A) Live mice born to Yy1+/+ × Yy1+/− crosses, with sex and genotype as shown. χ2 test applied to pairwise comparisons between wild-type versus Yy1+/− females (p < 0.01), or wild-type versus Yy1+/− males (p < 0.1). (B) Yy1+/− and Yy1−/− blastocysts (E3.5) from Yy1+/− intercrosses are morphologically normal. Troph, trophectoderm. ICM, inner cell mass. ZP, zona pellucida. (C) For processing of blastocysts derived from Yy1+/− intercrosses, nucleic acids for PCR genotyping and RT-PCR expression analysis are prepared as schematized. (D) Summary of Tsix and Xist expression patterns in 25 blastocysts obtained from Yy1+/− intercrosses. (E) For blastocysts from the Yy1+/− intercrosses, results from a single litter are shown. Negative controls were derived from PBS washes. Control, d0 male ES cells. Female and male controls for sex genotyping were prepared from tail biopsies. Minus-RT samples were all negative (data not shown). Molecular Cell 2007 25, 43-56DOI: (10.1016/j.molcel.2006.11.017) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Loss of Yy1 in Blastocyst Outgrowths Results in Aberrant Tsix and Xist Expression (A) Yy1+/+, Yy1+/−, and Yy1−/− blastocyst outgrowths. Troph, trophoblasts. ICM, inner cell mass. (B) Scheme for nucleic acid preparation from dissected ICM and trophoblast components. (C and D) Summary of Tsix and Xist expression patterns in ICM (C) and trophoblast (D) outgrowths from blastocysts of Yy1+/− intercrosses. Note: the number of samples in (C) and (D) do not agree because it was not always possible to isolate material for both components. (E) Results from a single litter are shown. PBS, negative control from the final PBS wash. Control, d0 male ES cells. Female and male controls for sex genotyping were prepared from tail biopsies. Molecular Cell 2007 25, 43-56DOI: (10.1016/j.molcel.2006.11.017) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 An ES Model for Yy1 Deficiency (A) Southern blot analysis of ES clones isolated from heterozygous Yy1 intercrosses, performed as previously described (Donohoe et al., 1999). (B) ES clone sex was determined by PCR genotyping with Zfy1 primers. (C) Quantitative RT-PCR analysis of Tsix and Xist expression in Yy1+/+ and Yy1+/− XY ES clones by real-time amplification (BioRad). ES cells were differentiated between 0 and 6 days as indicated. Error bars represent one standard deviation from the mean. (D) Tsix and Xist RNA FISH of male Yy1+/+ and Yy1+/− ES clones on d0 and d3 of differentiation. Transcripts are indicated by arrow. An Xist RNA coat was not observed in two independent differentiation experiments. n = 50–74 nuclei for each experiment. Because of contrast adjustments, the signal intensity shown does not reflect the absolute RNA levels. A female ES nucleus from d4 is shown as positive control for Xist RNA. (E) Cell death analysis of Yy1+/+ and Yy1+/− ES clones during cell differentiation by the trypan blue assay. Percent cell death = trypan blue-positive cells / total cells over each 3 day period. Error bars represent one standard deviation from the mean. Molecular Cell 2007 25, 43-56DOI: (10.1016/j.molcel.2006.11.017) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 7 Yy1 and Ctcf Directly Activate Tsix (A and B) Yy1 (A) or Ctcf (B) knockdown experiments in wild-type male ES cells. Western analysis confirms loss of Yy1 or Ctcf and is normalized to β-actin. Tsix and Xist levels are determined by real-time RT-PCR and normalized to β-actin. mut yy1, shRNA vector with mutated Yy1 sequence. d0 and d2 prenucleofection controls show wild-type RNA levels. gfp, control nucleofection using a GFP shRNA vector. Error bars represent one standard deviation from the mean. (C) Cotransfection of Yy1 and/or Ctcf transactivate the Tsix major promoter in d0 male wild-type ES cells. The minimal Tsix promoter is fused to a luciferase reporter gene in a standard luciferase assay. Overexpression of Yy1 and Ctcf was confirmed by western analysis in each case (data not shown). Error bars represent one standard deviation from the mean. Molecular Cell 2007 25, 43-56DOI: (10.1016/j.molcel.2006.11.017) Copyright © 2007 Elsevier Inc. Terms and Conditions