Volume 18, Issue 2, Pages 191-202 (February 2010) ATRX Partners with Cohesin and MeCP2 and Contributes to Developmental Silencing of Imprinted Genes in the Brain  Kristin D. Kernohan, Yan Jiang, Deanna C. Tremblay, Anne C. Bonvissuto, James H. Eubanks, Mellissa R.W. Mann, Nathalie G. Bérubé  Developmental Cell  Volume 18, Issue 2, Pages 191-202 (February 2010) DOI: 10.1016/j.devcel.2009.12.017 Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 ATRX, MeCP2, and Cohesin Interact In Vivo in the Mouse Forebrain (A) ATRX was immunoprecipitated from P17 mouse forebrain extracts, and western blot analysis was performed for SMC1, SMC3, and MeCP2 (top left panel). The results show that these three proteins are immunoprecipitated with ATRX. Top right panel: in a similar manner, SMC1, SMC3, and ATRX were detected in MeCP2 immunoprecipitates. Bottom panels: the reverse immunoprecipitations were also performed, showing that SMC1 and SMC3 immunoprecipitates contain ATRX and MeCP2 protein, confirming the interactions between these proteins. Control reactions were done with IgG. (B) SMC1, SMC3, and ATRX were immunoprecipitated from control and MeCP2 KO (TM2) forebrain extracts, followed by western blot analysis with an anti-MeCP2 antibody. In all cases, no band was observed in the MeCP2 KO (TM2) immunoprecipitates, demonstrating the specificity of these interactions. See also Figure S1. Developmental Cell 2010 18, 191-202DOI: (10.1016/j.devcel.2009.12.017) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 ATRX, MeCP2, and Cohesin Are Preferentially Bound to the Maternal H19 ICR in Mouse Forebrain (A) Genomic organization and alignment of primers utilized for PCR amplification of ChIP reactions. Numbers indicate the relative nucleotide position from the start of the H19 ICR region. Gray boxes indicate CTCF-binding sites. (B) PCR of ChIP DNA shows enrichment of ATRX at the region flanked by primer pair H19-5 of the H19 ICR. ATRX ChIP of region H19-5 was also performed on control and ATRX KO forebrain tissue (right panel). (C) A peak of enrichment of ATRX, MeCP2, and SMC1 at region H19-5 was confirmed by quantitative ChIP analysis. Graphs depict a representative enrichment profile. Statistical analysis revealed that deviation from uniform binding was significant, even accounting for interexperimental variability (ATRX-1, p = 0.003; ATRX-2, p = 0.005; MeCP2, p = 0.008; SMC1, p = 0.030). (D) Colocalization of ATRX, MeCP2, and cohesin at region H19-5′ of the H19 ICR was verified by double or triple sequential ChIP experiments. Input represents one-tenth of the input sample. (E and F) Allelic analysis of ATRX, MeCP2, and cohesin interaction with region H19-5′ of the H19 ICR shows that these proteins were enriched on the maternal allele in the mouse forebrain. Similar allelic analysis was performed for macroH2A (paternal) and acetylated histones H3 and H4 (maternal) at region H19-5′ as controls. For allelic analysis, amplicons from ChIP-isolated 129Sv/CAST F1 forebrain DNA were digested with MfeI (129Sv maternal-specific site) or SacI (CAST paternal-specific site) enzymes. Inp, input; mH2A, macroH2A; H3Ac, acetylated histone 3; H4Ac, acetylated histone 4; U, uncut; C, cut; Mat, maternal; Pat, paternal; M, standard marker. The asterisk indicates a 500 bp marker in the standard (Invitrogen). See also Figure S2. Developmental Cell 2010 18, 191-202DOI: (10.1016/j.devcel.2009.12.017) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 Effects of ATRX Loss of Function on the Epigenetic State of the H19 ICR (A) Bisulfite mutagenesis and sequencing analysis of the H19 ICR region revealed no change in DNA methylation upon loss of ATRX in forebrain tissue. At least 12 alleles from each sample were analyzed, and individual alleles are represented as a string of 16 CpGs. The total percent methylation for each sample is indicated in parentheses. Unmethylated CpGs are represented as empty circles, and methylated CpGs are represented as filled circles. (B) Quantitative ChIP analysis of histone H3 and H4 modifications in control and ATRX KO forebrain tissue at region H19-5′ of the H19 ICR. Enrichment of activating marks H3Ac and H4Ac were increased, whereas repressive marks H3K9me3 and H4K20me3 were decreased in the absence of ATRX. Graphed data represent the mean fold change in enrichment across three control and ATRX KO littermate pairs. Data were normalized to amplification of the Gapdh promoter region. See also Figure S3. Developmental Cell 2010 18, 191-202DOI: (10.1016/j.devcel.2009.12.017) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 Allele-Specific Control of H19 Gene Transcription by ATRX (A) Semiquantitative RT-PCR analysis showing increased transcript levels of H19 and Igf2 in three sets of control and ATRX KO littermate-matched neonatal (P0.5) forebrains, whereas β Actin transcript levels were not altered. (B) Real-time PCR analysis shows that H19 and Igf2 upregulation occurred in the ATRX KO postnatal forebrain (P0.5 and P17), but was not affected in the embryonic period (E13.5). Expression data for Atrx with primers specific for the long isoform were included as a control. Graphed data represent the mean relative expression level, and error bars depict standard error of the mean from biological replicates. The asterisk indicates p < 0.05, and double asterisks denote p < 0.005, as determined by a two-tailed t test. All results are normalized to β Actin expression. (C) Quantitative RT-PCR analysis confirms upregulation of H19 and decreased Atrx expression in the Atrx+/− forebrain tissue (bottom right). Error bars depict standard deviation of technical replicates. Real-time RT-PCR of H19 and Atrx in an F1 Atrx+/− forebrain. (D and E) Allelic melting curve analyses with hybridization probes (C57BL/6 and 129Sv homologous, CAST mismatched) revealed paternal-specific expression of (D) Snrpn and maternal-specific expression of (E) H19 in control 129Sv/CAST F1 forebrain. (F) Analysis of a 129Sv/CAST F1 forebrain heterozygous for ATRX revealed that H19 transcripts are still largely produced from the maternal allele. Homozygous C57BL/6, which has an identical sequence to 129Sv, and Mus musculus casteneous (CAST) samples were included as controls. Mat, maternal; Pat, paternal; B6, C57BL/6; Atrx+/−, ATRX heterozygote. Developmental Cell 2010 18, 191-202DOI: (10.1016/j.devcel.2009.12.017) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 ATRX, MeCP2, and Cohesin Bind within the Gtl2/Dlk1 Imprinted Domain (A) Genomic organization and alignment of regions analyzed by ChIP (GD-1, GD-2, GD-3, and GD-4). Numbers indicate the relative nucleotide position from the Gtl2 transcription start site. The asterisk denotes the predicted CTCF-binding site. (B) Quantitative ChIP analysis of ATRX, SMC1, and MeCP2 within the Gtl2 DMR. Regions analyzed are indicated on the x axis. ATRX (n = 4), p = 8.868e-07; MeCP2 (n = 2); SMC1 (n = 3), p = 0.05218. (C) Real-time RT-PCR analysis of Dlk1 and Gtl2 mRNA expression at E13.5, P0.5, and P17. Graphed data represent the mean relative expression level, and error bars depict standard error of the mean from biological replicates. Expression of Dlk1 is increased in postnatal forebrain tissue lacking ATRX protein, whereas expression of Gtl2 remains unaffected at all developmental time points examined. The asterisk indicates p < 0.01. GD, Gtl2 DMR. Developmental Cell 2010 18, 191-202DOI: (10.1016/j.devcel.2009.12.017) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 6 Occupancy of SMC1, MeCP2, and CTCF at the H19 ICR and Gtl2 DMR (A) ChIP analysis was performed across the H19 ICR in control and ATRX KO littermate-matched forebrains. Occupancy of CTCF and SMC1 was decreased in the ATRX KO samples at region H19-5, whereas MeCP2 occupancy was unchanged at this site. (B) Enrichment of SMC1, CTCF, and MeCP2 at region H19-5 was further quantified in additional brains (n = 3). (C) ChIP analysis was performed at the Gtl2 DMR in control and Atrx null forebrain (SMC1 and CTCF, n = 3; MeCP2, n = 2). Loss of ATRX in forebrain causes decreased occupancy of SMC1, CTCF, and MeCP2 at regions GD-4, GD-4, and GD-3, respectively (depicted in Figure 5A). Error bars depict standard error of the mean. p values were determined by a two-tailed t test. Developmental Cell 2010 18, 191-202DOI: (10.1016/j.devcel.2009.12.017) Copyright © 2010 Elsevier Inc. Terms and Conditions