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1 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas 77030, USA; 2 Department of Life Science and Institute of Genetics, National Yang-Ming University, Taipei 112, Taiwan
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Abstract |
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 Top Abstract ResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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[Keywords: Rbbp1/Arid4a ; Rbbp1l1/Arid4b ; Prader-Willi/Angelman domain; genomic imprinting; epigenetic modifications]
Received May 25, 2006; revised version accepted August 28, 2006.
). Imprinting of the PWS/AS domain is regulated through a bipartite cis-acting imprinting center (IC), comprised of the PWSIC and the AS-IC within and upstream of the SNRPN promoter (Buiting et al. 1995, 1999; Ohta et al. 1999). The IC is marked epigenetically according to the parent of origin, which results in differential allelic expression of genes across the PWS/AS domain (Kantor et al. 2006).
Allele-specific DNA methylation occurs at the PWS-IC, where the paternal allele is unmethylated and actively transcribed and the maternal allele is methylated and silent (Shemer et al. 1997; Gabriel et al. 1998). There are parent-of-origin-specific chromatin modifications of the PWS-IC domain with histone H3 Lys 9 (H3K9) methylated on the maternal allele and Lys 4 (H3K4) methylated on the paternal allele (Xin et al. 2001; Fournier et al. 2002), and with histones H3 and H4 more highly acetylated on the paternal allele than on the maternal allele (Saitoh and Wada 2000; Fulmer-Smentek and Francke 2001). In contrast, the AS-IC is modified with histone H4 acetylated and H3K4 methylated only on the maternal allele (Perk et al. 2002). Although epigenetic modifications that regulate imprinting are well defined, very little is known about epigenetic regulators that control genomic imprinting.
With the goal of screening for mutations affecting trans-acting factors that regulate imprinting, we combined selection for altered expression of an Snrpn-EGFP fusion gene with gene trap mutagenesis in mouse embryonic stem (ES) cells. Two gene trap clones associated with increased expression of EGFP were found to be integrations into two related Arid (AT-rich interaction domain) family genes, Arid4a and Arid4b (Wilsker et al. 2005), previously known as retinoblastoma-binding protein-related genes, Rb-binding protein 1 (Rbbp1) (Defeo-Jones et al. 1991) and Rbbp1-like 1 (Rbbp1l1) (Cao et al. 2001), respectively. This result led to studies demonstrating that Rbbp1/Arid4a, Rbbp1l1/Arid4b, and Rb play a role in the regulation of the PWS/AS imprinted domain, representing a novel epigenetic mechanism of regulation of genomic imprinting.
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Results |
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TopAbstractResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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To investigate regulation of imprinting, an EGFP gene cassette was placed in the position normally occupied by the ORF for SNRPN (Fig. 1A). Snrpn-EGFP ES clones were identified by Southern blot analysis using a 3'-flanking segment and EGFP as probes (Fig. 1B). The expression of the Snrpn-EGFP fusion gene was evaluated by flow cytometric analysis (Fig. 1C). The major peak representing 86% of targeted cells from an EGFP-neo clone expressed EGFP protein with a mean fluorescence intensity (MFI) of 6.23, while wild-type ES cells displayed a background MFI of 1.09. After the neor cassette was removed from the targeted allele, the percentage of EGFP-positive cells increased to 99.8%, and the MFI increased from 6.23 to 13.8.
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; Gabriel et al. 1998). We have reported that gene targeting at the Snrpn locus frequently resulted in exclusive recovery of paternal recombinants and demethylation and activation of expression for the nonrecombinant, normally silenced maternal allele (Tsai et al. 2003). This phenomenon occurred in the available Snrpn-EGFP ES clone with loss of methylation on the maternal allele for the Snrpn promoter (Fig. 1D).
Screening for variant expression of EGFP with gene trap mutagenesis
ES cells with the Snrpn-EGFP fusion gene were subjected to gene-trap mutagenesis using the ROSA
geo* retroviral gene trap vector (Fig. 2A; Friedrich and Soriano 1991). After infection of 107 cells in each of two experiments, three clones were recovered from the population sorted for increased EGFP expression by FACS. After flow cytometric analysis, two gene trap clones (designated GT-A and GT-B) isolated from separate retroviral infection experiments had higher levels of EGFP expression (Fig. 2B). The one other clone (designated GT-C) had no change in the level of EGFP expression compared with the original Snrpn-EGFP clone (data not shown). The GT-A clone had an MFI of 17, and the GT-B clone had an MFI of 22, while the control Snrpn-EGFP ES cells had an MFI of 13. Sequencing of the trapped genes for the GT-A and GT-B clones resulted in the identification of two related Arid family genes, Arid4a and Arid4b (Wilsker et al. 2005), previously known as Rb-binding protein-related genes, Rbbp1 (Defeo-Jones et al. 1991) and Rbbp1l1 (Cao et al. 2001), respectively. Comparison of the fusion cDNA and genomic DNA sequences predicted that insertion of the gene trap vector occurred between exons 4 and 5 for Arid4a in the GT-A clone and within intron 1 for Arid4b in the GT-B clone (Fig. 2C). RT–PCR analysis showed reduction in the transcripts for Arid4a and Arid4b in the GT-A and GT-B clones, respectively (Fig. 2D). Fusion transcripts were detected with the first four exons of Arid4a linked to the geo sequence in the GT-A clone and with exon 1 of Arid4b linked to the geo sequence in the GT-B clone (Fig. 2D). These results indicated that insertion of the gene trap vector into Arid4a or Arid4b led to increased expression of the Snrpn-EGFP fusion gene, although the Snrpn promoter on the fusion allele was unmethylated and already actively transcribed in the original Snrpn-EGFP and GT clones (Figs. 1C,D, 2E). This "superactivation" might be due to changes of other epigenetic effects, such as chromatin modifications.
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Having identified two homologous genes, Arid4a and Arid4b, we tested for protein interactions between ARID4A and ARID4B (Fig. 3A). Coimmunoprecipitation experiments combined with Western blot analysis showed that V5-tagged mouse ARID4B was associated with Flag-tagged human ARID4A (Fig. 3B, top) and with endogenous human ARID4A (Fig. 3B, bottom) in human embryonic kidney 293 cells. To examine whether ARID4A interacts with the Snrpn promoter that was used to drive EGFP expression in the gene trap mutagenesis screen, we performed chromatin immunoprecipitation (ChIP) analysis (Fig. 3C). We found that DNA at –6704, –4750, –2626, and +44 of the Snrpn promoter was associated with ARID4A, whereas DNA at +1238, exon 3, and exon 7 of the Snrpn gene was not. Taken together, the data demonstrated the interaction of ARID4A with ARID4B and with the Snrpn promoter.
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In order to evaluate the function of Arid4a and Arid4b in mice, both gene loci were independently deleted by homologous recombination in ES cells (Fig. 4A,E). The targeted ES cell clones were identified by Southern blotting and PCR analysis (Fig. 4B,F) and were used to generate chimeric mice that transmitted the mutated Arid4a or Arid4b alleles to the mouse germline. Mice heterozygous (+/–) or homozygous (–/–) for Arid4a deficiency were viable and fertile. The deleted allele was verified by Southern blotting (Fig. 4C), and absence of the transcripts was confirmed by RT–PCR (Fig. 4D). In contrast, Arid4b –/– mice were not born alive, although Arid4b +/– mice were viable and fertile (Table 1). We examined embryos from timed matings between heterozygotes, and did not find any Arid4b –/– embryos from embryonic days 7.5–13.5 (E7.5–E13.5). At E3.5, we found that two out of 12 blastocysts from two different intercrosses were homozygous for the Arid4b mutation (Fig. 4G; Table 1). These data suggested that embryonic demise occurs between E3.5 and E7.5.
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The trapping of the Arid4a and Arid4b genes led us to assess their function in the regulation of imprinting for the PWS/AS region. To determine whether the Arid4a or Arid4b mutation was accompanied by any imprinting defect, we examined the epigenetic status of the PWS-IC located at the Snrpn promoter, beginning with DNA methylation. Both Arid4a –/– mice and Arid4b +/– mice displayed normal differential DNA methylation at the Snrpn promoter (data not shown). Therefore, we inter-crossed Arid4a –/– mice and Arid4b +/– mice to produce Arid4a +/– Arid4b +/– mice. The double-heterozygous mice were then used to derive mice with triallelic mutations (Arid4a –/– Arid4b +/–), since double-null mutations (Arid4a –/– Arid4b –/–) were not viable. By Southern blot analysis using the methylation-sensitive SacII enzyme, one out of seven Arid4a –/– Arid4b +/– mice showed loss of DNA methylation (Fig. 5A, mouse 5); in contrast, the other six Arid4a –/– Arid4b +/– mice displayed differential methylation patterns similar to those of wild-type mice (Fig. 5A). Use of bisulfite sequencing to analyze 16 CpGs at exon 1 of Snrpn showed a variable pattern of hypomethylation in the Arid4a –/– Arid4b +/– mice (Fig. 5B). In mutant mice 1 and 2, there were no significant changes in the methylation pattern compared with wild-type mice. In mutant mice 3 and 4, the Snrpn CpGs were less methylated than those in wild-type controls. In mutant mouse 5, both parental chromosomes were unmethylated, which confirmed the lack of methylation shown in Southern blot analysis. We have analyzed a similar number of wild-type littermates that all have normal methylation patterns at Snrpn by Southern blotting and bisulfite sequencing analysis (data not shown).
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Trimethylated H3K9 and H4K20 are marks of repressive chromatin states (Bannister and Kouzarides 2005). In the promoter region of human SNRPN, H3K9 is more methylated on the maternal allele than the paternal allele (Xin et al. 2001). We examined allelic differential modifications for H3K9 and H4K20 trimethylation using mice with a 4.8-kb deletion at Snrpn exon 1 (Bressler et al. 2001), the equivalent of a human PWS-IC deletion. ChIP analysis showed that the maternal copy of the Snrpn promoter was modified with both H3K9 and H4K20 trimethylation, which was present in mice with the paternal 4.8-kb deletion (
4.8 m+/p–), whereas there was dramatic reduction of both modifications on the paternal copy, which was present in mice inheriting the 4.8-kb deletion maternally (4.8 m–/p+) (Fig. 5D). Taken together, these results indicate that Arid4a and Arid4b play a role in controlling maternal-specific H4K20 and H3K9 trimethylation and DNA methylation at the PWS-IC with the maternal allele in the Arid4a –/– Arid4b +/– mice being changed toward a more paternal epigenotype.
We next determined whether the observed acquisition of the paternal epigenotype at the PWS-IC on the maternal chromosome was correlated with increased expression of paternally expressed genes and decreased expression of maternally expressed genes in the PWS/AS region. Quantitative RT–PCR analysis demonstrated a 1.7-fold increased abundance of Snrpn transcripts in the Arid4a –/– Arid4b +/– mice compared with wild-type mice (Fig. 5E). A similar result was found for another paternally expressed gene, necdin (Ndn), with a 1.8-fold increased abundance of mRNA (Fig. 5E). In contrast, Western blot analysis showed a slight decrease in E6-AP encoded by the maternally expressed gene Ube3a in the Arid4a –/– Arid4b +/– mice when compared with wild-type mice (Fig. 5F).
Mutations of Arid4a, Arid4b, or Rb suppressed an AS-IC imprinting defect
Although a murine equivalent to the human AS-IC has not yet been identified, we have recently reported a mouse model of an AS imprinting defect with the ASICan mutation (Wu et al. 2006). To further investigate the roles of Arid4a and Arid4b in the regulation of imprinting for the PWS/AS region, we examined the genetic interaction of these two genes with the AS-IC by mating female mice with the AS-ICan mutation to male mice carrying the Arid4a or Arid4b mutations (Fig. 6). Using DNA derived from the offspring for methylation analysis of the Snrpn and Ndn CpG islands by Southern blotting, mice inheriting only the AS-ICan mutation had an absence of methylation on both alleles for Snrpn and reduced methylation of DNA at the Ndn locus (Fig. 6A, mouse 5) as previously reported (Wu et al. 2006). Interestingly, transmission of both the AS-ICan and Arid4b mutations to double-heterozygous offspring demonstrated an approximately equal intensity of methylated and unmethylated fragments at Snrpn and Ndn CpG islands (Fig. 6A, mice 3 and 4), which was similar to the results for wild-type littermates (Fig. 6A, mouse 1) and for mice carrying only the Arid4b mutation (Fig. 6A, mouse 2). Data from additional litters showed that progeny inheriting only the AS-ICan mutation displayed a complete absence of methylation at Snrpn, and progeny inheriting both Arid4b and AS-ICan mutations fell into two clear methylation patterns (Fig. 6B). Five out of 12 double-heterozygous mice had the normal differential methylation pattern, while seven double-heterozygous mice showed a lack of methylation. Similar results were observed in the offspring from the mating of male mice carrying the Arid4a deletion to female mice with an ASICan mutation (Fig. 6C). Four out of 18 double-heterozygous progeny showed normal differential methylation. Since Arid4a is an Rb-binding protein (Defeo-Jones et al. 1991), we mated female mice with an AS-ICan mutation to male mice carrying an Rb-null mutation (Fig. 6D; Lee et al. 1992). Three out of 10 mice heterozygous for both the AS-ICan and Rb mutations had the corrected differential methylation pattern. These results indicated that haploinsufficiency for Arid4a, Arid4b, or Rb suppressed the imprinting defect in a fraction of mice with the AS-ICan mutation. In all cases, the suppression of the imprinting defect must occur after fertilization, since the two mutations are not together until that time.
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). We examined Ube3a expression in mice carrying the AS-ICan mutation together with Arid4a or Arid4b mutations (Fig. 6E). Using Western blotting to analyze E6-AP encoded by the Ube3a gene, double-heterozygous mice with the imprinting defect (absence of methylation at Snrpn as in mouse 5 for AS-ICan Arid4b +/– and mouse 7 for AS-ICan Arid4a +/–) showed a severe decrease in E6-AP, which was similar to the result shown for mice inheriting only the AS-ICan mutation (mouse 3). In contrast, double-heterozygous mice with suppression of the imprinting defect (normal differential methylation pattern at Snrpn as in mouse 4 for AS-ICan Arid4b +/– and mouse 8 for AS-ICan Arid4a +/–) showed a level of protein comparable to that found in wild-type mice, Arid4a +/– mice, or Arid4b +/–mice (Fig. 6E, mice 1, 6, and 2, respectively). These results indicated that Arid4a and Arid4b mutations suppressed the repression of Ube3a caused by the AS-ICan mutation. This suppression was correlated with the correction of the differential methylation pattern at Snrpn. |
Discussion |
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TopAbstractResultsDiscussionMaterials and methodsAcknowledgmentsReferences |
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), as well as two repression domains—repression I and II (Fig. 3A; Lai et al. 1999a). A part of the repression I domain overlaps the Arid domain. The ARID domain contains a helix–turn– helix structure with DNA-binding activity (Patsialou et al. 2005). Chromodomains, as well as Tudor domains, mediate binding to methylated histones H3 and H4 (Bannister and Kouzarides 2005). For example, the chromo-domain of heterochromatin protein 1 (HP1) binds to methylated H3K9 (Bannister et al. 2001; Lachner et al. 2001), and the Tudor domain of Cut5-repeat-binding protein 2 recognizes methylated H4K20 (Sanders et al. 2004). Both ARID4A and ARID4B have been reported to be associated with the mSIN3-histone deacetylase complex (Lai et al. 2001; Fleischer et al. 2003). The ARID4A sequence, but not ARID4B, contains a LVCHE sequence as a conserved LXCXE motif known to interact with RB (Fig. 3A). ARID4A has been defined as a repressor of E2F transcription recruited by RB (Lai et al. 1999a, b, 2001). In the past few years, there is growing evidence that RB is a component of chromatin-remodeling complexes that mediate repression of transcription (Zhu 2005). RB is necessary to direct H3K9 methylation and HP1 to promoters (Nielsen et al. 2001). In addition, the RB family maintains trimethylation of H4K20 at constitutive heterochromatin (Gonzalo et al. 2005). Here, we show that Arid4a and Arid4b participate in chromatin modifications affecting H4K20 and H3K9 trimethylation at the PWS-IC. Our analysis of the Arid4a and Arid4b deletion mice for epigenetic alterations at the PWS-IC and for suppression of the AS-IC imprinting defect suggests an in vivo function of Arid4a and Arid4b on the regulation for the PWS/AS domain. The effects could be through direct interaction of ARID4A and ARID4B with chromatin complexes binding to PWS-IC or AS-IC, or it might occur through intermediate steps involving various pathways that are modulated through chromatin remodeling.
There are seemingly opposite effects of Arid4a/4b deficiency on DNA methylation at the PWS-IC with decreased methylation in mice with a wild-type PWS/AS domain compared with increased methylation in mice with the AS-IC mutation. On the AS-IC and PWS-IC, there are opposite parent-of-origin-specific chromatin modifications. The PWS-IC domain on the paternal allele is associated with active chromatin modifications with Lys 4 (H3K4) methylated (Xin et al. 2001; Fournier et al. 2002), and with histones H3 and H4 more highly acetylated (Saitoh and Wada 2000; Fulmer-Smentek and Francke 2001). On the maternal allele, the PWS-IC is modified with repressive chromatin states, such as H4K20 and H3K9 trimethylation (Fig. 5D). In contrast, the human AS-IC on the maternal allele is associated with active chromatin modifications with histone H4 acetylated and H3K4 methylated (Perk et al. 2002). Deficiency of Arid4a and Arid4b in mice with a wild-type PWS/AS domain changed the repressive chromatin modifications to the active states at the PWS-IC on the maternal allele with a significant decrease in trimethylation of histone H4K20 in all of the Arid4a –/– Arid4b +/– mice, and with partially reduced H3K9 trimethylation and DNA methylation (Fig. 5). It is possible that the alterations in H3K9 trimethylation and DNA methylation patterns are intimately linked with altered H4K20 trimethylation. On the other hand, in the mouse model of an AS imprinting defect with the AS-ICan mutation (Wu et al. 2006), the active chromatin modifications on the AS-IC of the maternal allele may become repressive due to the insertion of the targeting vector, which caused loss of methylation at the Snrpn promoter. Arid4a, Arid4b, or Rb mutations might shift the abnormal repressive histone modifications to a more active state at the AS-IC. Therefore, the imprinting defect with the ASICan mutation could be suppressed and the Snrpn promoter on the maternal allele could turn to be normally methylated. Taken together, Arid4a, Arid4b, and Rb might have a major role in shifting repressive histone modifications toward a more active state at both the PWS-IC and the AS-IC. The apparent opposite effects of deficiency of Arid4a and Arid4b on DNA methylation at the Snrpn promoter involve two different genotypes at the PWS/AS domain; we observed decreased DNA methylation in the Arid4a –/– Arid4b +/– mice with the wild-type PWS/AS domain (Fig. 5), and gain of DNA methylation in the AS-ICan mice together with the Arid4a, Arid4b, or Rb mutations (Figs. 6, 7). In both cases, we believe that deficiency of Arid4a and Arid4b is shifting chromatin from a more repressed to a more active state, in one case acting at the wild-type PWS-IC and in the other case acting through the mutant AS-IC. Loss or gain of DNA methylation could be the secondary consequences linked with the chromatin states, since histone modifications have been considered to play a dominant role in the cytosine methylation (Quina et al. 2006). In ES cells, deficiency of Arid4a or Arid4b leads to super-activation of the Snrpn-EGFP fusion allele (Fig. 2B), in which the Snrpn promoter was unmethylated and already actively transcribed in the original Snrpn-EGFP and GT clones (Figs. 1C,D, 2E). This superactivation might be due to changes of the chromatin modifications. The chromatins, although active, may be pushed into an even more active configuration.
Mice with the AS-ICan mutation represent a model system for the imprinting defect form of Angelman syndrome (Wu et al. 2006). The AS-IC mutation showed an imprinting defect with lack of methylation at the maternal Snrpn promoter and decreased expression of maternally expressed Angelman genes, Ube3a. In this paper, we showed that the suppression of the AS-IC imprinting defect could be achieved by mutations of Arid4a, Arid4b, or Rb. All mice with only the AS-ICan mutation (total 15 mice in Fig. 6B–D and 
50 mice in the previous publication; Wu et al. 2006) had absence of methylation at Snrpn. The null hypothesis is that all mice (100%) with the AS-ICan mutation should show an abnormal unmethylated pattern at Snrpn, and that any deviations from the 100% rate could be considered significant. We showed the corrected differential methylation pattern in mice with the AS-ICan mutation when together with the Arid4b (five out of 12 mice in Fig. 6B), Arid4a (four out of 18 mice in Fig. 6C), or Rb (three out of 10 mice in Fig. 6D) mutations. In Figure 7, we showed the corrected differential methylation pattern in all mice with the AS-IC mutation when both parents carry the Arid4a mutation. This suppression of the AS-IC imprinting defect also associated with relieving the repression of Ube3a.
Inactivation of RB is seen in many human tumors. RB has been shown to be involved in many cellular processes, such as control of the cell cycle, cell differentiation, DNA-damage responses, DNA replication, and protection against apoptosis, all of which could contribute to the role of RB as a tumor suppressor (Classon and Harlow 2002). ARID4A and ARID4B may be directly related to key aspects of cell transformation, and represent biologically plausible candidate genes for involvement in breast cancer (Cao et al. 1999, 2001; Takahashi et al. 1999). Loss of imprinting has been reported in several types of human tumors (Feinberg et al. 2002). There is very little information about the presence or absence of imprinting defects in retinoblastoma, but one report suggested abnormal imprinting in RB tumors (Kato et al. 1996). Our evidence that Rb, Arid4a, and Arid4b can regulate genomic imprinting raises the question of whether dysfunction of the RB/ARID4A/ARID4B complex could disrupt genomic imprinting and/or epigenetic regulation and thereby promote tumor formation. This can be tested in human and mouse tumors lacking these proteins or in tissues and cultured cells from mice with conditional mutations.
Epigenetics refers to mechanisms for gene regulation by modifications of the DNA–protein complexes that are stable within a cell but do not involve a change in DNA sequence, and genomic imprinting is a subset of epigenetics. In this report, we identified Arid4a and Arid4b as new members of epigenetic regulatory complexes that control the Prader-Willi/Angelman imprinted domain. We believe that the role of epigenetics is neglected as a potential disease mechanism in conditions other than cancer, particularly in complex disease traits (Jiang et al. 2004). It will be of interest to determine whether mutations in ARID4A and ARID4B genes could contribute to epigenetic mechanisms of human disease.
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Materials and methods |
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To construct the targeting vector for the Snrpn-EGFP fusion gene, a promoterless EGFP gene was removed from plasmid pIRES-EGFP (BD Bioscience Clontech), and was introduced upstream of the neomycin expression cassette flanked by loxP sites. The targeting vector included the 5'-homologous arm and 3'-homologous arm (Tsai et al. 2002) flanking the EGFP and neomycin cassettes, and there was an HSV-TK expression cassette outside the 3'-homologous region. To target the Arid4a and Arid4b loci, we isolated overlapping 
phage clones from a 129/SvEv genomic library. We constructed a targeting vector designed to delete a part of exons 1 and 2 in Arid4a by cloning a 2.7-kb EcoRI–XhoI phage clone end fragment as the 5'-homologous arm and a 4.1-kb XhoI phage clone end fragment as the 3'-homologous arm. The targeting vector included the 5'-homologous arm and the 3'-homologous arm flanking LacZ and neomycin cassettes; there was an HSV-TK expression cassette outside the 5'-homologous region. The Arid4b targeting vector was designed to delete 5.5 kb surrounding exon 1 by cloning a
3.1-kb SalI phage clone end fragment as the 5'-homologous arm and a 3.4-kb HindIII–EcoRI phage clone end fragment as the 3'-homologous arm. The targeting vector included the 5'-and 3'-homologous arms flanking the neomycin cassette; there was an HSV-TK expression cassette outside the 3'-homologous region.
Generation of mice with Arid4a and Arid4b deletions
The targeting vector for Arid4a or Arid4b deletion was electro-porated into AB2.2 ES cells from the 129/SvEv strain provided by Allan Bradley (The Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK). Neomycin (G418 sulfate, 200 mg/mL; Life Technologies) was used to select for ES cells undergoing the desired recombination event. DNA extracted from ES cells was analyzed either by Southern blot hybridization using 5'-flanking and 3'-flanking probes, or by PCR amplification using primers as follows: primer a, 5'-GCTACAGAGTGATTTTATGCTG TC-3'; primer b, 5'-CACACTAGTCATAACTATGCTTTGTG-3'; and primer c, 5'-CCTGACTAGGGGAGGAGTAGAAG-3'. The targeted clones were injected into C57BL6/J blastocysts and reimplanted into pseudopregnant female mice. Chimeric males were bred to C57BL6/J females to evaluate germline transmission, and both mutations were maintained on a hybrid C57BL6/J and 129/SvEv genetic background.
Southern blot analysis
The targeted ES clones with the Snrpn-EGFP fusion gene were identified by Southern blot hybridization using a 2-kb PCR fragment as a 3'-flanking probe after DNA extracted from ES cells was digested with PstI. For disrupting the Arid4a gene, the targeted ES clones were identified by Southern blot analysis using a 1.7-kb PCR fragment as a 5'-flanking probe after genomic DNA was digested with EcoRI and a 660-bp PCR fragment as a 3'-flanking probe after genomic DNA was digested with NcoI. For disrupting the Arid4b gene, the targeted ES clones were identified by Southern blot analysis using a 360-bp PCR fragment as a 5'-flanking probe after DNA extracted from ES cells was digested with SstI. Southern blots prepared for differential methylation analyses at the Snrpn and Ndn loci were hybridized with a 1.3-kb BssHII–EcoRI fragment from Snrpn intron 1 or a 1.5-kb SacI–HindIII fragment from the Ndn 5'-flanking region after genomic DNA isolated from tail biopsies was digested with HindIII alone or in combination with SacII as described (Bressler et al. 2001).
RT–PCR analysis
RNA from the gene trap clone was isolated using Trizol Reagent (Invitrogen) according to the manufacturer's directions. RT– PCR amplification was performed using SuperScript one-step RT–PCR (Invitrogen). The Arid4a transcripts were analyzed using the primer as follows: primer e1, 5'-ATGAAGGCGGCA GATGAGCC-3' and primer e5, 5'-CACAGTATACCAACTT GCATCTG-3'. Hprt transcripts were amplified using the primer pair 5'-ATGACCTAGATTTGTTTTGTATACC-3' and 5'-GTAGCTCTTCAGTCTGATAAAATCTAC-3'. For quantitative RT–PCR analysis, reverse transcription was performed using random hexamers to primer the first-strand cDNA synthesis using DNase I-treated (Promega) total RNA and the SuperScript First-Strand Synthesis System (Invitrogen). Quantitative RT–PCR analysis was performed using LightCycler Fast-Start DNA Master SYBR Green I (Roche). The sequences of PCR primers for Snrpn exon 2 and Ndn were as described (Yang et al. 1998; Tsai et al. 1999).
ChIP assay
We electroporated the expression vector Flag-ARID4A into AB2.2 ES cells followed by selection with 200 mg/mL Geneticin for 10 d. A stable clone expressing Flag-ARID4A protein was isolated and used to carry out ChIP assays with anti-Flag M2 antibody (Sigma) as described by the protocol of Upstate Bio-technology (available at http://www.upstate.com). For chromatin modification analysis, chromatin extracted from murine brain was immunoprecipitated with anti-trimeH3K9 and anti-trimeH4K20 antibodies (Upstate Biotechnology). The primer sets used to amplify the Snrpn gene were as follows: primer pair 1, 5'-CTGGCCACCAGTAACTAAATAAC-3' (forward) and 5'-GTGTGACTTGAACATTGAAGGCC-3' (reverse); primer pair 2, 5'-CTGTGTAGCACTGGCTAACCTG-3' (forward) and 5'-GAGTCTAGTCTGATCTACAGAGTG-3' (reverse); primer pair 3, 5'-GTCACACAGCATCCCAATTTCTTC-3' (forward) and 5'-CAGGTAAATTCTTACCACGTGGC-3' (reverse); primer pair e1, 5'-GAGTGATTTGCAACGCAATGGAGCG-3' (forward) and 5'-CTAACACACCCAAGGAGTCCGTCTG-3' (reverse); primer pair 4, 5'-CAAGTCAGAGAAGTGATTGTG TG-3' (forward) and 5'-GTCTGGCCATTCTACACAGGT TC-3' (reverse); primer pair e3, 5'-AGGAGGCTCTTTTAGAG TAAAGGTG-3' (forward) and 5'-CACCACCTTGAAGTTG CAATACTGC-3' (reverse); primer pair e7, 5'-ACTGGCATT GCTCGTGTGCCTC-3' (forward) and 5'-GCCTCCAACTG CTCGGACAGG-3' (reverse).
Cloning of cDNA
Arid4b-geo and Arid4a-geo fusion cDNAs were amplified by the 5'-RACE system (Life Technologies). We used three -gal gene-specific primers (GSP) as follows: GSP1, 5'-GCCATCA AAAATAATTCGCGTCT-3'; GSP2, 5'-CGTTGGTGTAGAT GGGCGCATCGT-3'; and GSP3, 5'-CGTGCATCTGCCAG TTTGAGG-3'. The Arid4b transcripts were analyzed using the primer as follows: primer e1, 5'-GGTGCAGGTGAAGCTGGT GTC-3' and primer e3, 5'-CACAGTATACCAACTTGCAT CTG-3'. To generate the vector expressing Flag-hARID4A, the coding region from a full-length human ARID4A cDNA clone (ATCC no. 95680) was cloned into the pCMV-Tag2 plasmid (Stratagene) with a Flag epitope in frame at the N terminus. A mouse Arid4b EST clone (Invitrogen; clone ID: 3970761) was used to screen a mouse brain cDNA library (BD Bioscience Clontech). A clone was isolated and used in combination with two mouse EST clones (Invitrogen; clone ID: 3970761 and 3603654) to construct an expression cassette containing further 5' and 3' sequences, respectively. The full-length mouse Arid4b cDNA was tagged at the C-terminal with a V5 epitope in the expression vector pcDNA3.1/V5-His B (Invitrogen).
Immunoprecipitation and Western blotting
Human embryonic kidney 293 cells were transfected using LipofectAMINE (Invitrogen). Whole-cell extracts were prepared in lysis buffer (50 mM Tris at pH 8.0, 200 mM NaCl, 0.5% [v/v] Nonidet P-40, 100 mM NaF, 0.2 mM sodium orthovanadate, and protease inhibitors [Roche]), and subjected to immunoprecipitation and Western blotting with antibodies (anti-Flag M2 antibody [Sigma], anti-V5 antibody [Invitrogen], anti-ARID4A antibody [Upstate Biotechnology, anti-RBBP1 clone LY11], or normal mouse IgG [Upstate Biotechnology]). Western blot analysis for Ube3a expression used anti-E6AP antibody (Bethyl; BL447) as described (Wu et al. 2006).
Bisulfite sequencing analysis
Bisulfite treatment of genomic DNA was carried out using the EZ DNA methylation-gold kit (ZYMO Research). Bisulfite-modified DNA was amplified by nested PCR as described (Wu et al. 2006).
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E-MAIL abeaudet{at}bcm.tmc.edu; FAX (713) 798-7773. 
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1452206.
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