© 2015. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2015) 8, 1121-1127 doi:10.1242/dmm.019885

RESEARCH ARTICLE Scaffold attachment factor B2 (SAFB2)-null mice reveal non- redundant functions of SAFB2 compared with its paralog, SAFB1 Shiming Jiang1,2,*, Tiffany A. Katz‡, Jason P. Garee1,2,§, Francesco J. DeMayo1, Adrian V. Lee1,2,‡ and Steffi Oesterreich1,2,‡,¶

ABSTRACT domains. In the human genome, SAFB1 and SAFB2 map Scaffold attachment factors SAFB1 and SAFB2 are multifunctional adjacent to each other to chromosome 19p13.3, where they are proteins that share >70% sequence similarity. SAFB1-knockout oriented in a bidirectional divergent configuration and separated − − ∼ (SAFB1 / ) mice display a high degree of lethality, severe growth by an 500 bp region that can act as a bidirectional promoter retardation, and infertility in male mice. To assess the in vivo role of (Townson et al., 2003). Given the high degree of sequence SAFB2, and to identify unique functions of the two paralogs, we similarity, we hypothesized that the proteins have both shared but generated SAFB2−/− mice. In stark contrast to SAFB1−/−, SAFB2−/− also unique functions. offspring were born at expected Mendelian ratios and did not show There have been many reports on SAFB1, but less is known about any obvious defects in growth or fertility. Generation of paralog- SAFB2. SAFB1 is a multifunctional protein that binds both DNA specific antibodies allowed extensive expression analysis of SAFB1 and RNA and is involved in the attachment of chromatin to the and SAFB2 in mouse tissues, showing high expression of both nuclear matrix, and in the regulation of transcription, the stress SAFB1 and SAFB2 in the immune system, and in hormonally response and splicing (Garee and Oesterreich, 2010; Garee et al., controlled tissues, with especially high expression of SAFB2 in the 2011; Altmeyer et al., 2013). More recently it has been shown that male reproductive tract. Further analysis showed a significantly SAFB1 might play a role in embryonic stem (ES)-cell self-renewal increased testis weight in SAFB2−/− mice, which was associated with as a target of FoxD3 (Plank et al., 2014). Intriguingly, there have an increased number of Sertoli cells. Our data suggest that this is at been a number of reports that collectively imply an important role of least in part caused by alterations in androgen-receptor function and SAFB1 in DNA damage pathways. Lachepelle et al. demonstrated expression upon deletion of SAFB2. Thus, despite a high degree of that SAFB1 binds directly to Werner syndrome helicase (Wrn), sequence similarity, SAFB1−/− and SAFB2−/− mice do not totally which is important for DNA repair and replication (Lachapelle et al., phenocopy each other. SAFB2−/− mice are viable, and do not show 2011). These studies also showed that Wrn protein was required for SAFB1−/− any major defects, and our data suggest a role for SAFB2 in the immortalization and tumorigenesis in models. SAFB1 differentiation and activity of Sertoli cells that deserves further study. also interacts with, and downregulates, p53, resulting in suppression of p53-mediated gene expression (Peidis et al., 2011). Further KEY WORDS: Reproduction, Scaffold attachment factor B2 (SAFB2), demonstrating a role in DNA damage signaling, γH2AX spreading Sertoli cells is enabled by SAFB1 binding to acetylated histones (Altmeyer et al., 2013). Reactive oxygen species (ROS) generation is indirectly INTRODUCTION regulated by SAFB1, with knockdown inducing the expression of Scaffold attachment factor B2 (SAFB2) is a member of a protein xanthine oxoreductase, a key enzyme in ROS production (Lin et al., family that also contains the paralog SAFB1 and a closely related 2008). protein scaffold attachment factor-like transcriptional modulator Both SAFB1 and SAFB2 have been shown to interact with and (SLTM). SAFB2 was originally identified as KIAA0138 and repress transcriptional activity of nuclear receptors, including subsequently named SAFB2 based on its high homology to estrogen receptor alpha (ERα) (Townson et al., 2003; Hashimoto SAFB1 (Oesterreich, 2003; Townson et al., 2003; Jiang et al., et al., 2012). Similar to SAFB1, SAFB2 can block cell growth, 2006; Hammerich-Hille et al., 2009, 2010; Hong et al., 2012). because SAFB2 overexpression leads to a decrease in the proportion The two proteins display the highest conservation in the of cells in S phase and an increase in cells blocked in G1 (Townson functional SAF-Box and RNA recognition motif (RRM) et al., 2003). SAFB2 also plays a role in alternative RNA splicing, inhibiting the splicing of a tra2β variable exon (Sergeant et al., 1Department of Molecular and Cellular Biology, Baylor College of Medicine, One 2007), and binding to and regulating activity of the SR protein Baylor Plaza, Houston, TX 77030, USA. 2Lester and Sue Smith Breast Center, Baylor kinases SRPK1 and SRPK1a (Tsianou et al., 2009). More recently, College of Medicine, One Baylor Plaza, Houston, TX 77030, USA. *Present address: Department of Epigenetics and Molecular Carcinogenesis, UT it was shown that SAFB2 is a substrate for the E3 ubiquitin ligase ‡ MD Anderson Cancer Center, Houston, TX. Present address: Women’s Cancer activity of BRCA1 (also known as BARD), resulting in regulation Research Center, Department of Pharmacology and Chemical Biology, University of SAFB2 protein expression (Song et al., 2011). of Pittsburgh Cancer Institute and Magee-Womens Research Institute, University of § To identify unique physiological functions of SAFB2, we Pittsburgh, Pittsburgh, PA 15213, USA. Present address: Hondros College, − − Westerville, OH 43081, USA. generated SAFB2 / mice through targeted deletion in ES cells. Interestingly, SAFB2−/− offspring were born at expected Mendelian ¶Author for correspondence ([email protected]) ratios and did not show any obvious defects in growth or fertility. This is an Open Access article distributed under the terms of the Creative Commons Attribution Despite many shared functions, the presence of severe phenotypes License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, SAFB1−/− distribution and reproduction in any medium provided that the original work is properly attributed. of mice, including a high degree of lethality, severe growth retardation, and infertility in male mice, indicate that SAFB2

Received 19 January 2015; Accepted 9 June 2015 cannot fully compensate for loss of SAFB1 (Ivanova et al., 2005). Disease Models & Mechanisms

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of SAFB2 mRNA expression was confirmed by northern blot TRANSLATIONAL IMPACT analysis (Fig. 1C), and by RT-PCR assays using primers spanning the Safb2 C-terminus (exons 18-21) (Fig. 1D). RT-PCR with Clinical issue Scaffold attachment binding factors (SAFBs) are a family of primers covering exons 4-7 revealed remaining expression of the multifunctional proteins that include SAFB2, SAFB1 and the scaffold N-terminus. SAFB1 mRNA expression was not affected, as shown attachment factor-like transcriptional modulator (SLTM). SAFBs have by RT-PCR using primers spanning N-terminal (exons 1-4) and been shown to play a role in RNA splicing, DNA damage signaling and C-terminal (exons 9-11) regions of the gene (Fig. 1D). transcription, and are also involved in some forms of cancer. In addition, – – To determine whether the remaining expression of N-terminal SAFB1 / mice display severe growth retardation, as well as deficits in – – RNA product would result in expression of a truncated protein, we reproductive function. Male SAFB1 / mice are sterile and a significant amount of lethality is observed in both prenatal and neonatal pups. generated polyclonal antibodies against the SAFB2 N-terminus Whereas many mechanisms of reproductive dysfunction exist, (aa105-199, exons 3-5) (Materials and Methods, and supplementary reproductive incompetence in humans still remains unexplained. The material Fig. S1). We were unable to detect full-length or truncated − − SAFB family might play an important role in reproductive function and SAFB2 in the SAFB2 / mice, even after prolonged exposure of the infertility. film, suggesting that either there is no truncated N-terminal protein expression or the potential truncated protein is highly unstable Results – – (Fig. 1E). As expected, SAFB1 protein expression was detected in In this study, the authors generated SAFB2 / mice to determine whether both SAFB2+/+ and SAFB2−/− mice, confirming specificity of the the two paralogs, SAFB1 and SAFB2, have redundant or distinct SAFB2 β functions. They find that indeed the two proteins have likely distinct knockout. Finally, immunofluorescence for -galactosidase – – +/+ −/− functions. SAFB2 / mice do not display the severe growth retardation or was performed on testes from SAFB2 and SAFB2 mice, – – − − significant neonatal lethality of the SAFB1 / animals. SAFB2 is likely to indicating the presence of β-galactosidase in SAFB2 / Sertoli cells, play a role in male reproduction because it was found to be highly and also in the germ cells although at a lower level (Fig. 1F). We expressed throughout the male reproductive track and to be involved in SAFB2 –/– conclude that our model represents a -null mutation resulting the regulation of the androgen receptor (AR). Additionally, SAFB2 in lack of SAFB2 protein expression. mice showed a significantly increased testis weight and a higher number of Sertoli cells in the testes, compared with wild type. Finally, the study −/− includes a comprehensive expression analysis of SAFB1 and SAFB2 in SAFB2 mice do not display any gross phenotypes − − mouse tissues, showing that they have shared but also unique target We have previously shown that SAFB1 / mice display both tissues. prenatal and neonatal lethality, growth retardation (caused by defects in the IGF signaling system), infertility in male mice and Implications and future directions reduced testis weight (Ivanova et al., 2005). To determine whether SAFB1 and SAFB2 play important roles in a number of normal and SAFB2−/− mice displayed similar defects, we first assessed if there pathophysiological processes. This study shows that loss of SAFB2 has fewer deleterious effects compared to loss of SAFB1, but analysis of the was some degree of lethality by analyzing genotypes of litters from – – +/− +/+ +/− SAFB2 / phenotypes suggests a role for SAFB2 in the male SAFB2 intercrosses. All three genotypes of SAFB2 ( , and – – − − reproductive system. This SAFB2 / mouse model provides a unique / ) were produced at the expected Mendelian distribution (1:2:1 system to study SAFB2 function in the normal male reproductive system, ratio), suggesting that SAFB2−/− did not affect viability (Table 1). as well as in pathophysiological conditions such as cancer. There were also no significant differences in body weight (Fig. 2A) or levels of circulating IGF-1 (Fig. 2B) between SAFB2+/+ and SAFB2−/− mice. Extensive expression analysis revealed high SAFB2 expression in To test whether SAFB2−/− caused defects in fertility, we bred the male reproductive system, and subsequent focused analysis SAFB2−/− males with SAFB2+/+ females, and SAFB2−/− females showed that SAFB2−/− mice have significantly bigger testes, and a with SAFB2+/+ males. The number of successful pregnancies, significant increase in Sertoli cell number, and we show data number of litters and number of pups per litter were not significantly suggesting that this is at least in part a result of altered androgen different (Table 2), suggesting that there are no major defects in the receptor (AR) activity. Our results reveal physiologically diverse development of the reproductive system. Gross histological analysis functions of SAFB1 and SAFB2, including a unique role of SAFB2 of SAFB2−/− mice also did not identify any major structural defects in Sertoli cells. in brain, spine, skin, tonsil, lung, heart, liver, spleen, stomach, intestine and kidney (data not shown). Thus, in stark contrast to RESULTS SAFB1−/− mice, SAFB2−/− does not cause any major phenotypes. Generation of SAFB2−/− mice − − SAFB2 / mice were generated by homologous recombination in SAFB2 protein is highly expressed in the male reproductive 129/Sv ES cells, replacing exons 4-10 with an IRES-lacZ-SV40pA tract, and SAFB2−/− results in increased testis weight and (β-galactosidase) marker and a PGK-Neomycin-bGHpA selection number of Sertoli cells marker (Fig. 1A). The targeting construct was linearized and To narrow down which tissue could have potential, less obvious, electroporated into ES cells, and the neomycin-resistant ES cell phenotypes in the SAFB2−/− mice, we surveyed a large panel of colonies were screened by Southern blot analysis (Fig. 1B, left mouse tissues for SAFB2 protein, and compared it to that of panel). Three recombinant clones were injected into C57BL/6 SAFB1. Immunoblot (Fig. 3) and immunohistochemistry (IHC) blastocysts, and two (B11 and G12) were transmitted to germ line. (Fig. 4) analyses were performed, using tissue from knockout The chimeric mice were bred with wild-type C57BL/6 mice to animals as controls (Fig. 4A). Immunoblot data showed high produce F1 mixed background (129/Sv and C57BL/6) expression of both SAFB1 and SAFB2 in the immune system heterozygous mutant mice (SAFB2+/−). Heterozygous F1 mice (spleen, thymus) and in hormonally regulated organs (uterus, from each line were intercrossed to produce two independent lines ovary). Of interest, in some tissues of the male reproductive tract, of homozygous mutant mice (SAFB2−/−). Genotyping was including the prostate, coagulating gland, epididymus and seminal confirmed by Southern blot analysis (Fig. 1B, right panel). Loss vesicle, SAFB2 expression was significantly higher than that of Disease Models & Mechanisms

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− − Fig. 1. Generation of SAFB2 / mice. (A) SAFB2 mouse allele, and targeting construct for deletion of the SAFB2 genomic fragment from exons 4 to 10. SB, Southern blot probe. (B) Southern blot analysis of genomic DNA from embryonic stem (ES) cells (left panel) and mice (right − − panel). SAFB2+/+ and SAFB2 / bands have sizes of 12 kb and 5 kb, respectively. (C) Northern blot analysis of − − − RNA from SAFB2+/+, SAFB2+/ and SAFB2 / mice. (D) − RT-PCR using RNA from SAFB2+/+, SAFB2+/ and − − SAFB2 / mice and primers targeting both the N and C terminal regions of SAFB2 and SAFB1. NTC, non- template control. (E) Immunoblot of lysates from either − − SAFB2+/+ or SAFB2 / mice with antibodies targeting SAFB2 and SAFB1. Lower panel shows an entire SAFB2 immunoblot to provide proof of expression of the N- terminal fragment. (F) LacZ (β-galactosidase) staining of − − SAFB2+/+ or SAFB2 / Sertoli cells. Blue, DAPI; red, Alexa Fluor 546 (lacZ).

SAFB1. In general, protein expression was concordant between COS7, 239T). We observed significant repression of AR activity by immunoblot and IHC analyses, and IHC data revealed a potential SAFB2 overexpression in LNCaP (Fig. 5D) and COS7 cells, but not weak cytoplasmic location of SAFB2 in some tissues, such as heart in 293T (data not shown). In adult mice (12 months), there was less and kidney. These results indicate that SAFB2 might play a role in AR expression in SAFB2−/− testes compared to SAFB2+/+ testes the male reproductive system. (Fig. 5E, and supplementary material Fig. S2), possibly as a result of Despite lack of gross phenotypes on fertility, further analysis negative feedback as previously described (Cai et al., 2011). There showed that SAFB2−/− mice displayed a significant increase in testis were no differences in expression of ERβ between SAFB2−/− and weight compared to SAFB2+/+ mice (Fig. 5A). This was in contrast to SAFB2+/+ testes (data not shown). Thus, SAFB2−/− mice have SAFB1−/− male mice, which we had previously shown to have increased weight of testes, associated with an increase in Sertoli cell decreased testes size (Ivanova et al., 2005). A potential cause for number, likely at least in part owing to altered AR signaling. increased testes size in SAFB2−/− mice were changes in Sertoli cells, known as the ‘nursing’ cells of the testes. Staining for DISCUSSION expression of Wilms tumor 1 (WT1), a marker of Sertoli cells, SAFB2 was originally identified based on high homology with its showed a significantly increased number of Sertoli cells in paralog, SAFB1 (Townson et al., 2003). Similar to SAFB1, SAFB2 SAFB2−/− testes compared to SAFB2+/+ testes (Fig. 5B). A representative section showing increased WT1 staining in the SAFB2−/− mice is shown in Fig. 5C. Because SAFB1 was previously shown to regulate AR activity (Mukhopadhyay et al., 2014) and AR signaling can effect numbers of Sertoli cells (Johnston et al., 2004; Tan et al., 2005), we next tested the effect of SAFB2 on androgen-stimulated AR activity in a number of cell lines (LNCaP,

Table 1. SAFB2 genetic deletion does not cause lethality Genotype Number of offspring +/+ +/−−/− Ratio +/+:+/−:−/− P-value

Total: 239 57 127 55 1.0:2.2:0.96 n.s. −/− Fig. 2. Body weight and IGF-I levels in SAFB2 mice. (A) Body weight Females: 111 27 59 25 1.0:2.2:0.93 n.s. +/+ −/− measurements in male (n=5) and female (n=6) SAFB2 and SAFB2 mice, Males: 128 30 68 30 1.0:2.3:1.0 n.s. +/+ presented as mean±s.e.m. (B) Serum IGF-I levels (mean±s.e.m.) in SAFB2 −/− P-value was calculated using Chi-square test. (n=10) and SAFB2 (n=10) mice. Disease Models & Mechanisms

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Table 2. Lack of breeding phenotypes in SAFB2−/− mice Number of mice used in breeding Genotype (males×females) Males Females Number (%) of pregnant females Number of pups per litter (mean±s.d.) P-value +/+×+/+ 3 3 3 (100) 6.2±2.6 −/−×+/+ 5 5 5 (100) 7.4±2.5 n.s. +/+×−/− 5 5 5 (100) 5.3±2.3 n.s. P-value was calculated using t-test. is a multifunctional protein with roles in transcriptional repression, et al., 2007). SAFB1 is also highly expressed in the testes, and has RNA splicing and SR protein kinase signaling, and with effects on been shown to be involved in the regulation of AR function, cell growth (Tsianou et al., 2009; Garee and Oesterreich, 2010; indicating that SAFB1 might also play a role in the testes Garee et al., 2011; Song et al., 2011; Altmeyer et al., 2013). Here, (Mukhopadhyay et al., 2014). Upon detailed analysis, we found we describe a novel mouse model deficient in SAFB2. In contrast to an increased weight of the testes in SAFB2−/− male mice. We SAFB1−/− mice, which have major phenotypes, including prenatal hypothesize that this is at least in part the result of SAFB2 playing and neonatal lethality, growth retardation and fertility problems, a role in Sertoli cells, because genetic deletion of SAFB2 led to an there were no gross phenotypes in SAFB2−/− mice. Comprehensive increase in the number of Sertoli cells. expression analysis of both paralogs shows high expression of Androgen signaling controls the regulation of Sertoli cell SAFB2 in the male reproductive tract, and our results indicate a role number and activity (Johnston et al., 2004; Atanassova et al., for SAFB2 in the testes. 2005; Tan et al., 2005; Walker and Cheng, 2005), and, because we SAFB1−/− mice display severe defects, including a high rate of show here that SAFB2 can regulate AR activity and levels, it is lethality and dwarfism, and, additionally, the males are infertile feasible that this cofactor role of SAFB2 mediates the effect on (Ivanova et al., 2005). These dramatic phenotypes were not Sertoli cells. Our recent preliminary studies have shown that AR recapitulated with genetic deletion of SAFB2, suggesting that expression was increased and proliferation was decreased in pre- either SAFB1 has unique critical functions not shared with SAFB2, pubertal testes from SAFB2−/− compared to SAFB1+/+ mice (data or that SAFB1 can fully compensate for loss of SAFB2, whereas the not shown), suggesting early maturation in SAFB2−/− mice, and reverse is not true. This might indicate that, although SAFB1 and this will be the focus of future studies. We cannot exclude the SAFB2 are paralogs, having arisen from a common ancestor, they involvement of other pathways, such as altered follicle-stimulating might not have retained all their shared functions, and new functions hormone (FSH) and estrogen signaling, because both pathways have been gained. Previous work has demonstrated that SAFB1 and have been implicated in Sertoli cell functions (Smith et al., 1982; SAFB2 can heterodimerize, but only SAFB1 is able to Johnston et al., 2004; Atanassova et al., 2005; Tan et al., 2005; homodimerize (Townson et al., 2003). Elegant biochemical work Walker and Cheng, 2005; Lucas et al., 2011). Of note, we did not by Sergeant et al. (2007) has clearly shown that SAFB1 is a detect altered ERβ levels in testes when comparing the two component of a small nuclear complex, whereas SAFB2 is part of genotypes. Finally, SAFB2 might also have specific splicing higher molecular mass nuclear protein complexes. There are also functions in Sertoli cells, as previously suggested (Sergeant et al., differences in subnuclear localization between SAFB1 and SAFB2 2007), that contribute to the observed phenotypes. More detailed that are cell-type-dependent, and additional studies are necessary to mechanism-oriented questions are the topic of future studies. gain a more detailed understanding of the shared and unique In summary, we have described the mouse phenotype with functions of the paralogs. genetic loss of the multifunctional transcriptional repressor In our study, we provide the first comprehensive expression SAFB2. Genetic deletion of SAFB2 shows a mild phenotype analysis of SAFB1 and SAFB2, showing high expression of both with no gross structural defects, growth retardation or reproductive proteins in the immune system and in hormonally regulated defects. Our data suggest a role for SAFB2 in the male organs. SAFB1−/− mice have defects in the immune system (S.O. reproductive tract, especially in Sertoli cells, which deserves et al., our unpublished data) but, again, this phenotype is not further investigation. The results of this study provide the basis mirrored in SAFB2−/− mice. Expression of SAFB2 was especially for future studies to delineate the unique and overlapping high in the male reproductive tract, in agreement with previous physiological function of the two SAFB paralogs, which have studies showing high SAFB2 expression in Sertoli cells (Sergeant important roles in normal and pathobiology.

Fig. 3. SAFB2 and SAFB1 protein expression levels in mouse tissues. Immunoblot analysis using antibodies against SAFB2, SAFB1 and Lamin A/C (as loading control). KO, knock out. Disease Models & Mechanisms

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− − Fig. 4. Immunohistochemical staining of SAFB2 and SAFB1 in mouse tissues. (A) SAFB2 and SAFB1 IHC using mouse testes from wild-type, SAFB2 / − − and SAFB1 / mice. Tissues were counterstained with hematoxylin. (B) IHC for SAFB2 and SAFB1 in mouse tissues, as indicated (scale bars: 20 μm).

− − MATERIALS AND METHODS pregnant females. Three lines of SAFB2 / mice were generated, which Gene targeting and generation of SAFB2−/− mice were analyzed in a C57BL/6J×129/Sv mixed genetic background. The mice The targeting vector resulting in deletion of exons 4 (amino acid 236) used in this study were maintained on a 12-h daylight cycle and fed a diet of through 11 was generated by amplifying a 4.3-kb region (5′ arm), or a 3.4 kb a standard food and water ad libitum in a pathogen-free facility at Baylor region (3′ arm), from 129Sv mouse brain tissue genomic DNA using primer College of Medicine (BCM). Animal care was performed in accordance set 1 or primer set 2 (supplementary material Table S1). PCR products were with BCM institutional guidelines. then cloned into pGEM 5Zf(+) using SacII/NotI sites (3′ arm) or XhoI site (5′ arm). An internal ribosome entry site (IRES)-lacZ (β-galactosidase)-neo- RNA analysis selectable cassette was inserted between the arms. After electroporation of Total RNA was extracted using the RNeasy Mini Kit (Qiagen), reverse the linearized targeting construct into 129/Sv ES cells, Southern blot transcribed, and PCR was performed as previously described (Ivanova et al., analysis was used to screen for homologous recombination in 192 G418- 2005). SAFB2 cDNA spanning exons 4 through 7, and 18 through 21, was resistant ES clones. A 600-bp probe was cloned from 129Sv genomic amplified with primers shown in supplemental material Table S1. To DNA and hybridized to BamHI-digested genomic DNA (Fig. 1A, and determine specificity of the SAFB2 knockout, SAFB1 cDNA spanning − − supplementary material Table S1). Three independent SAFB2 / ES clones exons 1 through 4, and 9 through 11, was amplified. β-actin was used as were injected into blastocytes (C57BL/6J) and implanted into pseudo- loading control. For northern blot analysis, total RNA was separated by gel Disease Models & Mechanisms

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Fig. 5. Increased number of Sertoli cells − − in SAFB2 / mice. (A) Bars represent testis weight as percent (mean±s.d.) of total body weight (BW), measured in − − SAFB2+/+ (n=12) and SAFB2 / (n=22) mice. (B) Number of Sertoli cells (mean± − − s.d.) in SAFB2+/+ (n=4) and SAFB2 / (n=14) mice. (C) Representative picture of IHC for Wilms tumor 1 (WT1) in SAFB2+/+ − − and SAFB2 / mouse testes (scale bars: 10 μm). (D) Reporter assay in LNCaP cells (mean±s.e.m., n=4) with fold change compared to ARE alone. A two-way ANOVA with a Bonferroni post-test was used to analyze the data (*P<0.05). (E) Number of AR-positive cells/tubule (mean±s.e.m., n=6). A t-test was conducted for statistical analysis (**P<0.01).

electrophoresis, transferred to a membrane, and then hybridized with a polyclonal SAFB1 (B1-1753) antibodies were used at a dilution of 1:150 and [32P]-dCTP-labeled cDNA probe (619 bp) targeting the mouse SAFB2 1:500, respectively. Sertoli cells were stained with WT1 monoclonal antibody C-terminal sequence (position 2666 bp to 3284 bp) (Fig. 1A, and 6F-H2 (DakoCytomation). An anti-AR antibody (Santa Cruz sc-816) was supplementary material Table S1). used for IHC at a dilution of 1:50. β-galactosidase immunofluorescent staining was performed as described previously (Ismail et al., 2002). Briefly, Immunoblot analysis testes were embedded in OCT medium (Sakura Finetechnical Co., Ltd, To analyze SAFB2 expression in mouse tissues and confirm loss of Tokyo, Japan), cryosectioned at 20 µm, and fixed for 15 min with 2% expression of SAFB2 in the SAFB2−/− mice, we generated an antibody formaldehyde, 0.2% glutaraldehyde, 0.02% Nonidet P-40, then washed targeting the mouse SAFB2 N-terminus (amino acids 105 to 199). To and incubated overnight at 37°C in x-gal staining solution [1.3 mM generate mouse SAFB2 N-terminal antibody, a cDNA fragment targeting MgCl2,15mMNaCl,5mMK3Fe(CN)6, 5 mM K4Fe(CN6).3H2O, 0.02% amino acids 105 to 199 (RYGQDGVVILQSSQDRDTMDTGVPDGME- Nonidet P-40, 44 mM HEPES (pH 7.9), and 0.05% (wt/vol) 4-chloro-5- AEDLSVPCLGKADTVNQILHAFDDSKEYVAAQLGQLPAQLLKHA- bromo-3-indolyl-β-D-galactopyranoside]. The sections were washed and VDEEVFKNTLEASVSDLKVTLAD) was amplified using primer pairs counterstained with Nuclear Fast Red. All images were captured using an with BamHI and EcoRI restriction sites (see supplementary material Olympus IX70 inverted scope under a 40× objective with a CCD camera. Table S1 for sequence of primer pairs), and cloned in frame with GST into BamHI/EcoRI sites in pGEX-2TK vector. The GST-mB2 N-terminal AR reporter assay peptide was expressed in B21 E. coli (supplementary material Fig. S1A) LNCaP and COS7 cells were maintained in RPMI 10% FBS, and 293T cells and purified with Glutathione Sepharose 4 Fast Flow beads (supplementary were maintained in DMEM 5% FBS, and all cells were plated for material Fig. S1B). Polyclonal serum was generated (Sigma), affinity- experiments in phenol red free RPMI+10% CSS. Lipofectamine LTX/Plus purified using the GST-mB2 N-terminal peptide, and verified with (Invitrogen) was used to transiently transfect 1 μg of hSAFB2 or empty immunoblotting (supplementary material Fig. S1C,D). vector (pcDNA3.1), 100 ng of androgen response element (ARE), and/or For the analysis of SAFB1 protein expression, our previously described 10 ng AR into LNCaP cells for 24 h. All cells were transfected with 10 ng of (Ivanova et al., 2005) polyclonal antibody (B1-1753) was used. Antibodies null-renilla. Cells were then treated with R1881 (1 nM) or vehicle for 24 h. against β-actin (Sigma A5441, mouse monoclonal) or lamin A/C antibodies A dual luciferase reporter assay (Promega) was then conducted as per the (Cell Signaling 2032, rabbit polyclonal) were used to blot for loading manufacturer’s instructions. controls. Bands were imaged and analyzed with an Alpha Innotech CCD camera and AlphaEaseFC (FluorChem8000) software, respectively. Statistical analysis Number of pups, number of embryos, serum IGF1 levels, AR-positive cells IGF-I measurements and body weight were assessed using descriptive statistics, including t-tests, − Serum was stored at 80°C until assayed for hormones. IGF1 was measured Wilcoxon rank-sum tests, ANOVA and Chi-square tests. Number of samples with enzyme-linked immunosorbent assay kits in accordance with the and statistical analyses are indicated in figures and/or figure legends. manufacturer’s (Diagnostic Systems Laboratories) protocol. Acknowledgements Histology and immunohistochemistry analysis The authors would like to acknowledge outstanding technical support from Mrs Ora With the exception of testes that were fixed in Bouin’s solution, all tissues Britton. were fixed in 4% paraformaldehyde, processed, embedded in paraffin, and sectioned (5 µm). To detect the expression pattern of SAFB2 and SAFB1 in Competing interests tissues, the newly generated N-terminal polyclonal SAFB2 (see above) and The authors declare no competing or financial interests. Disease Models & Mechanisms

1126 RESEARCH ARTICLE Disease Models & Mechanisms (2015) 8, 1121-1127 doi:10.1242/dmm.019885

Author contributions Jiang, S., Meyer, R., Kang, K., Osborne, C. K., Wong, J. and Oesterreich, S. S.J. and J.P.G. conducted experiments, and S.J. and S.O. wrote the paper. T.A.K. (2006). Scaffold attachment factor SAFB1 suppresses estrogen receptor alpha- helped to analyze the data, and contributed to writing the paper. S.O. and A.V.L. mediated transcription in part via interaction with nuclear receptor corepressor. conceived and designed the overall study, and S.O., A.V.L. and S.J. designed the Mol. Endocrinol. 20, 311-320. experiments. F.J.D. was instrumental in the generation of the mouse model. Johnston, H., Baker, P. J., Abel, M., Charlton, H. M., Jackson, G., Fleming, L., Kumar, T. R. and O’Shaughnessy, P. J. (2004). Regulation of Sertoli cell number Funding and activity by follicle-stimulating hormone and androgen during postnatal The study was in part funded by NIH R01CA097213. T.A.K. is supported by T32 development in the mouse. Endocrinology 145, 318-329. Lachapelle, S., Oesterreich, S. and Lebel, M. (2011). The Werner syndrome CA186873. Research reported in this publication was supported by the National helicase protein is required for cell proliferation, immortalization, and Cancer Institute of the National Institutes of Health under award number tumorigenesis in Scaffold attachment factor B1 deficient mice. Aging 3, 277-290. T32CA186783. The content is solely the responsibility of the authors and does not Lin, J., Xu, P., LaVallee, P. and Hoidal, J. R. (2008). Identification of proteins necessarily represent the official views of the National Institutes of Health. binding to E-Box/Ku86 sites and function of the tumor suppressor SAFB1 in transcriptional regulation of the human xanthine oxidoreductase gene. J. Biol. Supplementary material Chem. 283, 29681-29689. Supplementary material available online at Lucas, T. F. G., Pimenta, M. T., Pisolato, R., Lazari, M. F. M. and Porto, C. S. http://dmm.biologists.org/lookup/suppl/doi:10.1242/dmm.019885/-/DC1 (2011). 17beta-estradiol signaling and regulation of Sertoli cell function. Spermatogenesis 1, 318-324. References Mukhopadhyay, N. K., Kim, J., You, S., Morello, M., Hager, M. H., Huang, W.-C., Altmeyer, M., Toledo, L., Gudjonsson, T., Grøfte, M., Rask, M.-B., Lukas, C., Ramachandran, A., Yang, J., Cinar, B., Rubin, M. A. et al. (2014). Scaffold Akimov, V., Blagoev, B., Bartek, J. and Lukas, J. (2013). The chromatin attachment factor B1 regulates the androgen receptor in concert with the growth scaffold protein SAFB1 renders chromatin permissive for DNA damage signaling. inhibitory kinase MST1 and the methyltransferase EZH2. Oncogene 33, Mol. Cell 52, 206-220. 3235-3245. Atanassova, N. N., Walker, M., McKinnell, C., Fisher, J. S. and Sharpe, R. M. Oesterreich, S. (2003). Scaffold attachment factors SAFB1 and SAFB2: innocent (2005). 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