Volume 43, Issue 3, Pages 359-371.e6 (November 2017) Genetic Intersection of Tsix and Hedgehog Signaling during the Initiation of X- Chromosome Inactivation  Brian C. Del Rosario, Amanda M. Del Rosario, Anthony Anselmo, Peggy I. Wang, Ruslan I. Sadreyev, Jeannie T. Lee  Developmental Cell  Volume 43, Issue 3, Pages 359-371.e6 (November 2017) DOI: 10.1016/j.devcel.2017.09.027 Copyright © 2017 Elsevier Inc. Terms and Conditions

Developmental Cell 2017 43, 359-371. e6DOI: (10. 1016/j. devcel. 2017 Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 Hedgehog and GLI Signaling in ESCs and the Early Embryo (A) Left: Map of the Xist/Tsix locus and the Tsix alleles used in this study. DXPas34 sequence with interspersed, underlined GLI (green, TGGGWGGTC), CTCF (red, CCNNNNGNNGGCR), and YY1/Rex1 motifs (blue, VKHCATNWB). Right top: DXPas34 single repeat logo. Right bottom: GLI motif and MEME discovered DXPas34 GLI motif. (B) Simplified, canonical HH-GLI signaling pathway. (C) Left: Two C57BL6/J E5.5 female embryos stained with HH and H3K27me3 primary antibodies (left, middle). The negative control embryo (right) is stained only with secondary antibodies. Nuclei labeled with DAPI. Yellow arrowheads and white arrows denote the visceral endoderm (VE) and epiblast (Epi), respectively. ExE, extraembryonic ectoderm. Single Z section at 40×. n = 23 E5.5 embryos (11 male, 9 female, 3 unknown sex) showed similar results. Right: Companion illustration of the stained embryo with labeled tissue layers. Red dots, H3K27me3 domains representing Xi. Orange arrows, visceral endoderm-secreted IHH diffuses to epiblast. (D) Epiblast staining of a fixed C57BL6/6J E5.5 embryo was confirmed by OCT4 immunostaining. A single 40× Z section is shown. (E) HH antibody staining of 293 and 293-SHH cells with two different secondary antibodies (green, red) confirmed that the primary antibody is HH specific. (F) RT-PCR using RNA from female ESCs (16.7) and C3H10T1/2 cells. See also Figure S1. Developmental Cell 2017 43, 359-371.e6DOI: (10.1016/j.devcel.2017.09.027) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 HH Signaling Blocks XCI through Tsix (A) Tsix (left) and Xist (right) RNA FISH (green) of female ESCs. Tsix genotypes are indicated. Nuclei stained with DAPI. % of nuclei (n, sample size) with observable Xist clouds or Tsix foci at day 8 (d8) indicated. (B) Tsix (left) and Xist (right) RNA FISH of male ESCs with indicated Tsix genotypes exhibited as in (A). See also Figures S2 and S3. Developmental Cell 2017 43, 359-371.e6DOI: (10.1016/j.devcel.2017.09.027) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 GLI1 and GLI2 Impede XCI (A) FLAG and OCT4 western blots of whole-cell lysates from undifferentiated male and female GLI1- and GLI2-3xFLAG ESCs grown for 48 hr under the indicated conditions. (B) qRT-PCR to determine GLI1 and GLI2 RNA levels from the cells in (A). Average relative fold change from two biological replicates with SDs. −dox samples set to 1. (C) Xist and Tsix RNA FISH on inducible male and female GLI1- or GLI2-3xFLAG ESCs differentiated for 8 days ± 1 μg/mL doxycycline. % of n nuclei with observable Xist clouds or Tsix foci indicated. (D) qRT-PCR to measure luciferase RNA expression in undifferentiated female GLI1- or GLI2-3xFLAG ESCs transfected with luciferase plasmids. Cells were cultured ± 1 μg/mL doxycycline and 100 nM SAG for 48 hr. Luciferase plasmids with no promoter, Tsix promoter, or Tsix promoter with DXPas34 were used. Average relative fold change normalized to NeoR from two biological replicates with SDs. No promoter −dox samples set to 1. Significance determined by t-test where indicated. ∗p < 0.001. See also Figures S4 and S5. Developmental Cell 2017 43, 359-371.e6DOI: (10.1016/j.devcel.2017.09.027) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 GLI Binds the 5′ End of Tsix (A) Coomassie blue stained gel of recombinant proteins used in EMSA. (B–D) DNA EMSA with the indicated proteins and probes. Free probe (arrows). Shifted probe (∗). GLI motifs (green) and mutated positions (red) in the probes are shown. 2 pmol of DNA probes; 2 pmol of GFP; and 0.5, 1, 2 pmol of GLI1-zfd were used in (B) and (C). 2 pmol of GFP or GLI1-zfd; 2 pmol DXPas34 probe; and 5, 10, and 20 pmol of DXPas34 wild-type or mutated cold competitor probes were used in (D). (E) ChIP-PCR from GLI2-3xFLAG ESCs grown for 72 hr with 100 nM SAG and 1 μg/mL doxycycline. GLI2-FLAG IP, normal IgG IP, and input PCR products for each amplicon are shown. (F) GLI1-3xFLAG and GLI2-3xFLAG ChIP-seq for positive control, Ptch1. Peaks were called from intersected datasets from two biological replicates following input normalization. (G) CEAS analysis of GLI1-3xFLAG (middle) and GLI2-3xFLAG (right) ChIP-seq from undifferentiated female ESCs treated with SAG. Genomic elements are defined in the accompanying key (left). Genomic distribution of each element indicated as a %. (H) ChIP-seq analysis of GLI1 (scale, 0–3) in d0 and d3 male and female ESCs, with the d0 female CTCF track for comparison. Tracks are displayed in IGV for the Xite-Tsix-Xist region. Because of the highly repetitive nature of DXPas34 that renders ChIP difficult, GLI coverage may be underestimated in this region. (I) Probability plot for the likelihood of finding a CTCF site at indicated distances (in kb) from a GLI site. Solid line, observed; dotted line, randomized model wherein CTCF sites are randomly shuffled throughout the genome. Developmental Cell 2017 43, 359-371.e6DOI: (10.1016/j.devcel.2017.09.027) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 Genetic Analysis Shows a Functional Intersection between IHH Signaling and Tsix (A) Genotype data from Ihh+/− intercrosses at E12.5–13.5. Observed number of embryos per genotype is shown with expected number in parentheses. E12.5–13.5: ∗p = 0.003, ∗∗p = 0.005 (χ2). (B) Examples of E13.5 Ihh+/− intercross embryos from two separate litters. Adjacent ruler gradations are 1 mm each. (C) Genotype data from Ihh+/− intercrosses at E3.5–4.0. Observed number of embryos per genotype is shown with expected number in parentheses. ∗p = 0.794 (χ2). (D) E4.0 female Ihh+/+ and Ihh−/− blastocysts are stained with the indicated antibodies. Epi, epiblast (OCT4 labeled); PE, primitive endoderm (GATA4 labeled); TE, trophectoderm. Single Z plane for each embryo shown except in the last H3K27me3 panel where all Z planes are compressed into one. Presence of Xi-H3K27me3 foci only in TE and PE (arrowheads). (E) Observed E13.5 genotype data from male TsixΔCpG Ihh+/− crosses to female Tsix+/+ Ihh+/−. Observed number of embryos per genotype is shown with expected number in parentheses. ∗p = 0.950 ∗∗p = 0.968 (χ2). (F) Examples of Tsix+/ΔCpG Ihh+/− and Tsix+/ΔCpG Ihh−/− female embryos from a single litter. Adjacent ruler gradations are 1 mm each. (G) A model for HH-GLI signaling at the initiation of random XCI. Our data suggest that HH is a non-cell-autonomous signal that controls the timing of XCI in responding cells. Our model posits that cell-to-cell communication between the visceral endoderm (VE) and the epiblast maintains two Xs in the active state and controls the timing of XCI. Prior to XCI, HH produced by the VE binds PTCH, relieving PTCH repression of SMO, which in turn enables GLI binding to the 5′ end of Tsix. Tsix expression is promoted by GLI binding and blocks initiation of XCI. Downregulation of HH activity would then result in PTCH repression of SMO causing loss of GLI signaling that would lead to downregulation of Tsix, setting in motion the XCI steps of random XCI. See also Figure S6. Developmental Cell 2017 43, 359-371.e6DOI: (10.1016/j.devcel.2017.09.027) Copyright © 2017 Elsevier Inc. Terms and Conditions