(A) H&E staining of mouse uterine sections following Ronin^+/− crosses. Empty swollen decidua (marked by an asterisk, bar = 1 cm, left) were similar in size to those containing wild-type embryos (middle), but displayed residual resorbed embryonic tissue (right) at day E6.5. (B) Phase contrast images of blastocysts and ICM outgrowth. Ronin^−/− blastocysts isolated from Ronin^+/− crosses were morphologically indistinguishable from their Ronin^+/+ counterparts (left) but the ICM never proliferated (right) when cultured on gelatin coated culture plates, as would be expected for Ronin^+/+ cells (bar = 30 μm) (C, left) Immunofluorescent images of Cre-GFP transfected ES cells. The Ronin^{+/f lox} cells (green fluorescent) were viable while Ronin^{f lox/−} cells died rapidly (arrow indicates typical morphology of apoptotic cells). Genotyping of 88 colonies failed to identify any Ronin^−/− cell (bar = 20 μm). (C, right) Immunofluorescent images of MEFs isolated from a Ronin^{f lox/f lox} animal. After transfection with GFP adenovirus, all MEFs showed green fluorescence indicating successful transfection (left panel, insert shows phase contrast image of transduced MEFs). PCR-based genotyping of Cre-GFP-transfected cells confirmed successful Cre-mediated excision of both Ronin alleles, resulting in viable Ronin^−/− MEF cells (data not shown). (D–H) siRNA mediated knockdown of Ronin in ES cells. R1 cells were transfected with siRNA and the differentiation level assessed 4 days later (AP staining). (D) Phase contrast images showing no apparent difference in differentiation between control and Ronin siRNA while Oct4 siRNA is inducing differentiation. (E) Quantification of experiment shown in (D). (F) Quantification of colony number (independent of differentiation level); Colony number is reduced after treatment with Ronin siRNA compared to the control, but to a lesser extent than with Oct4 siRNA. (G) Proliferation rate of ES cells after treatment with siRNA. Cells treated with Ronin siRNA are showing significantly reduced proliferation rated when compared to control. (H) Quantification of Ronin expression by real time PCR of ES cells 18 hours after treatment with siRNA showing that Ronin expression level is reduced to 20%.

(A) Semiquantitative RT-PCR analysis of pluripotency factors and marker genes for all three germ layers. Ectopic expression of Ronin delayed upregulation of differentiation markers. (B) siRNA against Oct4 and Ronin was transfected into ES cells and the expression of their mRNA determined at the indicated time points by real-time quantitative PCR. Data are reported as means and standard deviations of triplicate experiments. Knockdown of Oct4 resulted in its loss, but Ronin expression was not affected. (C) Alkaline phosphatase staining of ES cells after siRNA knockdown of Oct4. EF1α-Ronin ES cells showed a substantially higher degree of self-renewal than their wildtype counterparts, demonstrating that Ronin functions independently of Oct4. (D) Quantification of result in panel C. Bars represent means and standard deviations of triplicate experiments. (E) ZBHTC4.1 [EF1α] ES cells were induced to differentiate by addition of doxycycline, while ZBHTC4.1 [EF1α-Ronin] ES cells ectopically expressing Ronin did respond to doxycyclin only minimally and did not differentiate. (F) Quantification of panel (E).

(A, left) Box plots showing results of microarray analysis of control ES cells and ES cells 24 hours after transfection with pEF1α-hRonin-Flag. Each box represent median and 75th and 25th percentile values. Comparison of the absolute quantities of hybridization (right) indicate that Ronin acts as a repressor of a broad range of genes. (B) Confocal images of 5-fluorouridine (5-FU) staining of newly transcribed RNA after induction of Ronin expression in a Ronin-inducible cell line, bar = 10 μm. (C) Quantification of ³H-Uridine incorporation into newly transcribed RNA of control A172loxP cells and A172LP-Ronin-Flag cells after induction with 1 μg/ml doxycycline. Bars show means (and standard deviations) of triplicate experiments. (D) Western blot analysis of H3K9me2 methylation. Induction of Ronin expression by doxycycline led to increased H3K9me2 methylation over time. (E) Chromatin immunoprecipitation and PCR of genomic regions containing the 3x sequence upstream of Gata4 and Gata6. Undifferentiated (control) ES cells showed enrichment for both Ronin and H3K9me2, which was lost after induction of differentiation. In addition Ronin binds the Oct4 promoter region which contains the 3x sequence but not the Nanog promoter which does not contain the 3x sequence.

(A) Schematic diagram of the Ronin protein showing a THAP domain at the N-terminus. NLS, nuclear translocation signal; polyQ, polyglutamine tract; polyA, polyalanine sequence; THAP, Thanatos-associated protein; aa, amino acid. (B) SELEX identification of the consensus sequence recognized by Ronin DNA-binding motif (top) and gel-shift experiments with a specific DNA sequence (bottom). After the SELEX procedure, 104 sequences were analyzed, and a motif search identified a specific consensus (top) and a “3x sequence”. Binding of Ronin-Flag to the biotinylated 3x sequence was verified by gel-shift experiments that could be inhibited by the Flag antibody and either, double-stranded or single-stranded unlabeled competitor 3x molecules. Triangles indicate gel shift complexes. (C) Northern Blot analysis of multiple mouse tissues. Ronin expression was detected only in the positive control (mouse ES cell line R1); all other tissues lacked appreciable signals. (D) X-Gal staining of tissues isolated from mpRonin-lacZ reporter mice. Very strong positive staining was detected in the oocytes and in some areas of the adult brain. (E) Immunostaining of oocytes and zygotes isolated from adult females using a Ronin antiserum. Ronin was restricted to the ooplasm, but was prevalent throughout the zygote (bar = 10 μm) (F) Ronin promoter-driven lacZ expression was detected at the 2-cell stage of embryo development and intensified during the 8-cell and compact morula stages, but surprisingly subsided in the blastocyst stage. (G) Immunostaining of Ronin in morula and blastocyst stage embryos and in in vitro inner cell mass (ICM) outgrowth. Ronin protein is present throughout the cytoplasm and nucleus in morula-stage embryos but concentrated in the cytoplasm of the inner cell mass in the blastocyst. Ronin again is present in the nucleus of ICM outgrowth. (H) Confocal images of mouse ES cells stained with Ronin antibody. Ronin and DAPI are mutually exclusive. (I) lacZ expression was detected in undifferentiated ES cells grown in the presence of MEFs, while virtually no staining was apparent after differentiation in the absence of MEFs and LIF or after EB formation for 3 days. (scale bars: D left 30 μm, D right 80 μm, E left 30 μm, right 20 μm

(A) Phase contrast images of control ES cells and EF1α-Ronin ES cells, stably overexpressing Ronin, after differentiation in monolayer cultures in the absence or presence of LIF for 4 days. Cells were split at clonal density under the same conditions. Cells that remained pluripotent after 8 days of selection were expanded in the presence of LIF on MEFs and, in a second step, Ronin expression was silenced by Cre-mediated recombination to obtain EF1α-ΔRonin ES cells (bar = 20 μm) (B) Alkaline phosphatase staining of cells treated as in panel A at two different magnifications. EF1α-Ronin cells remained phosphatase positive during differentiation, indicating their undifferentiated state. Control ES cells, as well as the reverted EF1α-ΔRonin ES cells showed reduced phosphatase staining, indicating normal differentiation (bar 10x = 80 μm, bar 20x = 40 μm). (C) Quantification of 3 × 50 colonies from the experiment described in panel B. The values are means and standard deviations from triplicate experiments. (D) Chimeric mice generated by injection of EF1α-ΔRonin ES cells into blastocysts. (E) Western blot analysis of Stat3 and Phospho-Stat3 indicating no difference between the amount and phosphorylation level of wild-type and Ronin-overexpressing ES cells after LIF removal.

(A) Immunohistochemical staining of Oct4 in teratocarcinoma sections. Tumors formed by EF1α-Ronin ES cells after injection into immunocompromised mice were much larger than those formed by R1 control cells (top) and more clusters of Oct4-positive cells were present (bottom), insert: 40x magnification of typical Oct4-positive cell cluster in EF1α-Ronin derived tumor, bar (top) = 0.5 cm, bar (bottom) = 0.1 mm (B) H&E staining of EF1α-Ronin teratocarcinoma sections. EF1α-Ronin ES cells were capable of differentiating into all three germ layers, as were their wild-type counterparts (data not shown), white arrows indicate cluster of undifferentiated cells, bar = 50 μm.

(A) Directional yeast two-hybrid approach to detect direct interaction of Ronin, Ronin-C and Ronin-N with candidate interacting proteins identified by mass spectometry. Direct interaction of Ronin-C with HCF-1 is represented by growth of cotransfected MAV103 yeast on 50 mM 3AT-containing plates (AD = activation domain, DB = DNA binding domain). (B) Downregulation of HCF-1 by siRNA shows the same phenotype in control ES cells and Ronin ectopically expressing ES cells. (C) Anti-Ronin-Flag Western blot of glycerol gradient fractions of EF1α-Ronin ES cell nuclear extracts purified with wheat germ agglutinin beads. Ronin is present in the first 5 fractions and in fractions 20 to 23 with a peak in 20. Comparison with the elution peaks of protein standards with known sizes (top) demonstrates that Ronin is involved in a very large protein complex. (D) Western blot of Myc-tagged proteins after GST immunoprecipitation. Ronin interacts via its C-terminus with itself, HCF-1 and Sin3A and via its N-terminus with HDAC3 and Thap7. R, Ronin; C, Ronin C-terminus; N, Ronin N-terminus; D, empty destination vector. (E) Proposed model for the mechanism of Ronin function. In contrast to the site specific gene modulatory activity of canonical pluripotency factors (e.g., Oct4, Sox2 and Nanog), Ronin appears to repress transcription more broadly to ensure the maintenance of ES cell pluripotency.