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	Figure 1. Immunohistochemistry of CTCFL Expression in Developing Testis(A) Characterization of α-CTCFL antibody. Myc-tagged CTCFL expressed in 293T cells was used to assess antibody titer and specificity. First (left) panel shows different anti-sera (dilution 1:1,000); pre-immune; 1: first bleed; 2: second bleed; 3: final bleed. Second panel: verification of myc-tagged CTCFL by α-myc antibody. Third panel: determination of α-CTCFL antibody specificity. The α-CTCFL antibody (dilution 1:10,000) was pre-incubated with GST-N-terminal CTCFL (GA) and GST (G). The antibody, pre-incubated with specific antigen, was neutralized, whereas α-CTCFL pre-incubated with GST alone retained its reactivity. Fourth panel: α-CTCFL reacted with a Western blot of adult testis extract. The black arrow indicates the position of myc epitope-tagged CTCFL, whereas the grey arrow indicates the position of endogenous CTCFL.(B–G) CTCFL expression in developing and adult testis. The pre-immune and adjacent α-CTCFL panels are at the same magnification (400×). A 5× magnification of the α-CTCFL image is given in the rightmost panel.(B) CTCFL is not detected at 13.5 dpc.(C) Mitotically arrested gonocytes (marked with white arrows) exhibit CTCFL staining at 14.5 dpc.(D) A few centrally localized gonocytes and cells at the periphery of seminiferous tubules express CTCFL at 17.5 dpc.(E) CTCFL is localized in gonocytes in newborn mice.(F) At 15 d after birth, nuclei of spermatogonia expressed CTCFL, as did their counterparts in adult testis (G).(H) Immunohistochemical detection of CTCF expression in adult testis. Normal rabbit serum (NRS) (left panel) and α-CTCF are presented as for (B–G). CTCF is expressed uniquely in Sertoli cells (marked with arrows).
	Figure 2. ChIP Analysis of CTCFL Association with the H19 ICR and Gtl2 DMR(A) Diagrams illustrate the regions within H19 ICR and Gtl2 DMR analyzed by ChIP.(B) Adult testis analysis. Upper panels show agarose gels of real-time PCR products for H19 ICR and standard PCR products for Gtl2 DMR. A no–DNA control (−)is indicated. Input (In) represents 10% of total amount of samples used. Pre-immune (PI) was used as a control for the enrichment of CTCFL immunoprecipitated with α-CTCFL (α-CL) antibody. For Gtl2 DMR, normal rabbit serum (NRS) was used. Lower panel shows real-time PCR analysis of H19 ICR. Control represents genomic region lacking CTCF binding consensus sequence.(C) The 15.5-dpc testis analysis. Figure is organized as for (B). In this case, control is included in the upper panel.
	Figure 3. N-terminal CTCFL Interacts with PRMT7(A) Diagrams show the domain structure of the PRMT7 and CTCFL proteins. MTase I and II represent the methyltransferase regions within PRMT7. The position of the recovered yeast two-hybrid clone of PRMT7 is indicated.(B) Reciprocal GST pull-downs of CTCFL and PRMT7. Scheme of the tested interactions is given in diagrams. GST and GST fusion proteins (CMV-GST vector) were co-expressed in 293T cells with myc- or V5-tagged preys. N-ter., N-terminal.(C) Co-immunoprecipitation of CTCFL and PRMT7. The 293T extracts containing over-expressed CTCFL-Myc or CTCFL-Myc&PRMT7-V5 were immunoprecipitated with α-V5 antibody or normal mouse serum (NMS). CTCFL was detected with α-Myc antibody. N-ter., N-terminal.(D) No interaction between CTCF and PRMT7 is detectable by GST pull-downs.
	Figure 4. CTCFL Interacts with Histones H1, H2A, and H3(A) Farwestern analysis. Western blots containing 1-μg and 0.25-μg histones were reacted with transfected 293T cell lysates expressing myc-tagged CTCFL. The presence of CTCFL was detected by α-Myc and α-CTCFL antibodies. Individual histones are indicated. The lower panel shows the ponceau red–stained Western blot. The migration of the individual histones are indicated to the left.(B) N-terminal and full-length CTCFL interacts with histones H1, H2A, and H3. Bacterial-produced GST histone fusion proteins were reacted with lysates from 293T cells transfected with either full-length or N-terminal CTCFL expression vectors. No detectable interaction of PRMT7 with histones is observed.
	Figure 5. Developmental Expression and Methyltransferase Activity of PRMT7(A) Developmental expression of PRMT7. Immunohistochemistry with α-PRMT7 on testis at the indicated stages of development.(B) PRMT7 in vitro methyltransferase reactions with total histones were performed either with α-V5–immunopurified PRMT7-V5 or α-V5–co-immunopurified PRMT7-V5&CTCFL-Myc. Upper panel shows Coomassie staining. Lower panel shows fluorogram (7-d exposure). Methylation of histones H2A and H4 is stimulated by co-immunoprecipitating CTCFL.(C) Discrimination between H2A and H2B methylation. For these histone methyltransferase reactions, CTCFL-myc and PRMT7-V5 were immunopurified separately using α-Myc and α-V5 antibodies, respectively, in reactions containing either H2A or H2B. Upper panel shows Coomassie staining. Lower panel shows fluorogram (7-d exposure). Stimulation of histone H2A methylation is observed with the addition of immuno-purified CTCFL.
	Figure 6. Analysis of De Novo H19 ICR Methylation by Nuclear Injection of Expression Plasmids into Xenopus OocytesThe experiment tests the ability of combinatorial expression of proteins encoded by the expression plasmids to methylate co-injected H19 ICR. A H19 ICR plasmid was co-injected with variety of cDNA expression plasmids; GFP fluorescence was used to select oocytes that had been properly injected. Efficiency of bisulfite modification was determined by evaluating transformation to T of non-CpG Cs. The maximum non-transformed Cs observed was one out of 51.(A) The specific expression plasmids co-injected with H19 ICR are indicated on the right side of the figure. Left panels indicate the methylation status of the 11 CpGs within the m3 region of the H19 ICR. Grey-shaded parts indicate the position of the CTCF consensus sequence within m3. Open circle (○) indictates non-methylated CpG; filled circle (•) indicates methylated CpG. Optimal methylation is dependent on the expression of CTCFL, PRMT7, and all Dnmt3s. Oocytes were analyzed 48 h after injection.(B) Methylation analysis of co-injected control DNA plasmid. Organization of presented data is as for (A). The control fragment contains a unique sequence fragment present on Chromosome 8 and was also used as a control in ChIP analysis (Figure 2B and 2C). No significant methylation was observed with expression of CTCFL, PRMT7, and the Dnmt3a, -b, and -L, confirming sequence specificity of the ICR methylation analyzed in (A).(C) Functional analysis of the Dnmt3 isoforms. Organization of presented data is as for (A). Omission of Dnmt3L reduces methylation to 0%. Removal of either Dnmt3a or Dnmt3b significantly reduced observed methylation. Maximal ICR methylation is observed when all three Dnmt3s are co-injected with CTCFL and PRMT7. Oocytes were analyzed 72 h after injection.(D) Detection of SYM-R3H4 in sulfuric acid–extracted proteins from microinjected Xenopus oocytes. Expression plasmids injected are indicated on the figure. An increase in SYM-R3H4 is observed in the presence of both CTCFL and PRMT7. The middle panel shows reaction with an α-H3 antibody as a control for histone loading, whereas the lower panel shows ponceau red staining.
	Figure 7. Developmental Accumulation of SYM-R3H4 in Mouse Testis(A) Significant SYM-R3H4 is first observed in the nuclei of cells composing the testis at 17.5 dpc. Subsequent stages, newborn and adult, exhibit strong nuclear staining of the majority of cells within the testis.(B) ChIP analysis of SYM-R3H4 in adult testis. The right panel shows the quantitative PCR analysis of the m1, m3, and m4 regions whereas the left panel shows an ethidium bromide–stained gel of the PCR products. Also shown are the PCR products of the ChIP analysis of the b region of Gtl2 DMR.
	Figure 8. Model of H19 ICR Methylation(A) An initial complex of CTCFL and PRMT7 localizes to the H19 ICR, resulting in symmetrical dimethyl modification of arginine residues in histones H2A and H4 composing the adjacent nucleosomes. The localization to ICR is assured by the zinc finger portion of CTCFL, whereas the interactions with histones and PRMT7 are taking place via the N-terminal portion of the protein.(B) Following disengagement of the CTCFL complex, the de novo DNA methyltransferases Dnmt3a and Dnmt3b are recruited by either their PWWP motifs or through a bridging protein to the methylated histones. Dnmt3L is recruited by direct interaction with Dnmt3a and Dnmt3b. Subsequent to their recruitment, the de novo DNA methyltransferases methylate adjacent CpGs, resulting in ICR methylation.

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