Malik MQ , Bertke MM , Huber PW .

GM38200 NIGMS NIH HHS , R01 GM038200 NIGMS NIH HHS

	FIGURE 1. TFIIIA interacts with PIAS2b and XCtBP. A, colony lift filter assay used to measure interactions between PIAS2b and TFIIIA in a yeast two-hybrid system. Triplicate assays are presented in each column, with the following bait/prey combinations: lane 1, lamC/T antigen (negative control); lane 2, TFIIIA (fingers 1–3)/PIAS2b; lane 3, TFIIIA (fingers 1–4)/PIAS2b; lane 4, p53/PIAS2b. B, MBP-TFIIIA or MBP-L5 (negative control) fusion protein was incubated with reticulocyte lysate containing 35S-labeled PIAS2b. The fusion protein was recovered by binding to amylose resin. Precipitated samples were analyzed by SDS-PAGE followed by autoradiography. C, reticulocyte lysate containing 35S-labeled XCtBP was incubated with either TFIIIA or PIAS2b alone or with both proteins together. Complexes were collected by immunoprecipitation using either anti-TFIIIA (lanes 1 and 2) or anti-PIAS2b (lanes 3 and 4) antibody adsorbed to protein A-Sepharose. Control assays used unmodified protein A-Sepharose. Precipitated samples were analyzed by SDS-PAGE followed by autoradiography.
	FIGURE 2. Expression of PIAS2b and XCtBP during early Xenopus development. A, RNase protection analysis of PIAS2b mRNA. Total RNA from 10 oocytes at the indicated Dumont stage (85) was hybridized with an antisense 32P-labeled RNA probe. The sample was then digested with RNase One, analyzed on a denaturing polyacrylamide gel, and detected by autoradiography. B, Western blot analysis of PIAS2b. Protein extracts (2 oocyte or 1.5 embryo equivalents) were prepared from developmentally staged oocytes and embryos (86) as indicated, including progesterone-matured oocytes (M) and unfertilized eggs (E). The position of molecular weight standards (in kilodaltons) is indicated. C, Western blot analysis of XCtBP.
	FIGURE 3. Subcellular localization of PIAS2b during early development. Staged albino oocytes and embryos were stained using PIAS2b primary antibody and secondary antibody conjugated to Alexa Fluor 568. A, PIAS2b is exclusively cytoplasmic at all stages of oogenesis. B, protein extract prepared from nuclei and enucleated oocytes (1.5 oocyte equivalents per stage) was analyzed by Western blotting using PIAS2b antiserum. Oocyte stages are indicated above each lane. Top panel, a blot of extract from enucleated oocytes. Bottom panel, extract prepared from isolated nuclei. Also shown is immunohistochemical staining of embryos at mid-blastula (C and D) and early gastrula (E and F) stages for PIAS2b. D, overlay of PIAS2b and TOPRO-3 nuclear staining of the midblastula embryo shown in C. Arrows point to individual nuclei. Images were collected at ×10 magnification unless indicated otherwise.
	FIGURE 4. Depletion of SUMOylation activity prevents repression of oocyte 5 S rRNA genes. A, SUMO activation (E1) activity in extract prepared from Gam1-injected embryos was measured by Western blot analysis using a 25-kDa SUMO substrate peptide. Lane 1, peptide alone; lane 2, peptide incubated with extract from water-injected (control) embryos; lane 3, peptide incubated with extract from Gam1-injected embryos. The reactions in lanes 2 and 3 also contained E2 (Ubc9) enzyme, SUMO1, and ATP. Positions of molecular weight markers (in kilodaltons) are indicated. B, one-cell embryos were injected with [32P]UTP and TFIIIA mRNA, which activates transcription of the oocyte-type genes after the midblastula transition, or Gam1 mRNA. RNA was isolated at early gastrula stage, and three embryo equivalents were analyzed by electrophoresis/autoradiography. Lane 1, control embryos injected with [32P]UTP only; lane 2, injection with TFIIIA mRNA (7 ng); lane 3, injection with Gam1 mRNA (5 ng). C, autoradiographs for four experiments were scanned, and 5 S rRNA was quantitated relative to tRNA using ImageJ software. The ratio is given in arbitrary units. Error bars indicate mean ± S.D. D, one-cell embryos were injected with Gam1 mRNA (lanes 1, 3, and 5) or water (lanes 2, 4, and 6). Whole cell extract was prepared, and four embryo equivalents were analyzed by Western blot developed with TFIIIA antibody.
	FIGURE 5. Occupancy of the 5 S rRNA genes during early development. ChIP assays of embryos at the indicated stage of development tested for the presence of the specified protein or histone modification. A, oocyte 5 S rRNA genes. LB, late blastula stage; EG, early gastrula stage; LG, late gastrula stage; EN, early neurula stage; LN, late neurula stage. B, somatic 5 S rRNA genes. C, oocyte 5 S rRNA genes from Gam1-injected embryos. D, somatic 5 S rRNA genes from Gam1-injected embryos. The antibodies used were as follows: lane 1, beads only (negative control); lane 2, SUMO1; lane 3, SUMO2/3; lane 4, H3K9me2; lane 5, H3K27me3; lane 6, CtBP; lane 7, PIAS2b; lane 8, TFIIIA; lane 9, input.
	FIGURE 6. Localization of TFIIIA to the nuclear periphery. TFIIIA antibody directly conjugated to Alexa Fluor 568 was used to immunohistochemically stain embryos at the indicated stage of development. A, two-cell embryo. B, midblastula stage (stage 8) embryo. C, late blastula stage (stage 9) embryo. D, midneurula stage (stage 19) embryo. E, nucleus of a midneurula embryo injected with Gam1 mRNA counterstained with DAPI. F, nucleus of a midneurula embryo injected with water and counterstained with DAPI. Images were collected at ×20 magnification unless indicated otherwise. C' and D' are magnifications of the indicated regions of the images in the corresponding panels.
	FIGURE 7. SUMOylation of TFIIIA. In vitro SUMOylation of TFIIIA was tested using purified E1 and E2 enzymes. Products of the reactions were analyzed by a Western blot analysis developed with TFIIIA antibody. Lane 1, TFIIIA with SUMO1 and E1 and E2 enzymes; lane 2, TFIIIA alone. The positions of molecular weight markers (in kilodaltons) are indicated.
	FIGURE 8. Model for the repression of the oocyte-type 5 S rRNA genes following the midblastula transition. A, repression of the oocyte-type genes is largely due to histone H1-dependent positioning of a nucleosome over the binding site for TFIIIA (nucleotides 45–96). The A:T-rich sequence flanking the 3′ end of the gene, which is the binding site for the linker histone, is required for repression. In addition, ∼1% of the oocyte-type genes are bound by TFIIIA, which can interact with PIAS2b and the CtBP corepressor complex. This complex may be stabilized by an interaction between the SAP domain of PIAS2b and the A:T-rich sequence that immediately flanks the 3′ end of the oocyte-type genes. B, the somatic-type genes are flanked by G:C-rich sequences, providing no contact site for PIAS2b. Histone octamers are weakly positioned on the somatic-type gene, and the binding site for TFIIIA is exposed. Binding of the transcription factor is sufficient to reposition the octamer that possibly leads to dissociation of the H1 linker histone (18).

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