A) Schematic of the TMS1 genomic locus. The TMS1 gene consists of three exons (I, II and III). Non-coding regions are indicated by black boxes. The nucleotide positions are numbered with respect to the transcription start site (T) and are shown above the gene. The location of the CpG island is marked and spans from approximately -100 to +900 bp. The positions of the hypersensitive sites (HS1-HS4) and an upstream repeat element (L1/Alu) are shown. Primer sets used (1-9) for real-time PCR in ChIP assays are shown below the gene. B) Expression of TMS1. Protein lysates prepared from MCF7, MDA-MB231, HMT.1E1 and IMR90 cells were subject to western blot analysis with antibody against TMS1. C) Distribution of histone H3 modifications across the TMS1 locus. MCF7 and MDA-MB231 cell lines were subjected to ChIP with antibodies against the indicated histone modifications or a rabbit IgG (IgG) control. Immunoprecipitated DNA was amplified by real-time PCR with primer sets indicated in (A). Percent (%) input was determined as the amount of immunoprecipiated DNA relative to input DNA. Each ChIP was repeated at least three times, and although the immunoprecipitation efficiency varied between experiments, the profile of enrichment across the locus was consistent. Shown are the mean +/- standard deviation of triplicate determinations from a representative experiment.

Effect of hMOF downregulation on the TMS1 locus. A) MCF7 cells were transfected with 100 nM of siRNA targeting hMOF (MOF1 or MOF2) or Lamin A/C (Lamin) (irrelevant control) and harvested four days post-transfection. Cells were analyzed for hMOF, TMS1 or GAPDH (loading control) protein expression by western blot analysis (left) or for hMOF, TMS1 or 18s (internal control) RNA expression by real time PCR (right). The levels of expression of hMOF and TMS1 mRNA are expressed relative to that obtained in cells treated with Lamin siRNA, after normalization to 18s. Shown is the mean +/- standard deviation of three independent experiments. Experiments were also done using scrambled non-targeting siRNA as a control and similar results were observed. B) MCF7 cells were transfected as in A) and ChIP was performed with antibodies against rabbit IgG (IgG), H3K9/14Ac or H4K16Ac. Immunoprecipitated DNA was amplified by real-time PCR with primer sets indicated in Figure 1A. Data represent the percent of input DNA recovered. Each ChIP experiment was repeated at least twice with reproducible results. Shown are the mean +/- standard deviation for triplicate determinations from a representative experiment. C) MCF7 cells infected with an empty pLKO.1 vector (none) or a pLKO.1 expressing hMOF shRNA (hMOF) were analyzed for nucleosome positioning exactly as described in Figure 2C. Preferential MNase cut sites (1-7) are indicated with arrows. Shown is the migration of a 2765bp, 1001bp and 739bp Spe I fragments from the TMS1 locus that were included as internal markers. A representative experiment is shown. D) MCF7 cells were transfected with hMOF siRNA and processed at 0 to 7 days post-transfection for western blot analysis using the indicated antibodies.

Effect of hMOF downregulation on TMS1 DNA methylation. A) MCF7 cells were transfected with siRNA (100 nM) against hMOF (MOF1 and MOF2) or an irrelevant control Lamin A/C (Lamin) and harvested 4 days post-transfection. Genomic DNA was isolated, modified by sodium bisulfite and amplified by methylation specific PCR (MSP) with primer sets specific for either methylated (M) or unmethylated (U) DNA. DNA from IMR90 and MCF7 cells served as a control for unmethylated DNA, while that from HMT.1E1 and MDA-MB231 cells served as a control for methylated DNA. The TMS1 region (32-223) amplified by MSP is shown in Part B (MSP). B) DNA from MCF7 cells transfected with Lamin A/C (Lamin) or MOF2 (hMOF) siRNA was modified with bisulfite and amplified with a primer set that spans 53 CpG sites in the TMS1 CpG island (27). Products were subcloned and sequenced. Each row indicates the sequence of an independent clone where methylated (black circles) and unmethylated (white circles) CpG sites are indicated.

Localization of H4K16Ac and H4K20me3 across the TMS1 locus. A) MCF7 and MDA-MB231 breast cancer cells or B) IMR90 and HMT.1E1 cell lines were subjected to ChIP with antibodies against rabbit IgG (IgG) or the indicated histone modifications. Immunoprecipitated DNA was amplified by real-time PCR with primer sets indicated in Figure 1A. Percent (%) input was determined as the amount of immunoprecipiated DNA relative to input DNA. Each ChIP was repeated at least three times, and although the immunoprecipitation efficiency varied between experiments, the profile of enrichment across the locus was consistent. Shown are the mean +/- standard deviation of triplicate determinations from a representative experiment. C) Nucleosome positioning at the TMS1 locus. Nuclei from MCF7 (left) or MDA-MB231 (right) cells were incubated in MNase digestion buffer alone (0), digestion buffer plus CaCl₂ (0+), or digestion buffer plus CaCl₂ and 10-200U MNase. MNase-digested DNA (10 μg) was digested with Hind III (H) and Spe I (S), separated on a 1% agarose gel, and subject to Southern blot analysis using a probe anchored to the 3′ Spe I site. Preferential MNase cut sites are depicted with arrows. Shown is the relative migration of a 2765bp, 1001bp and 739bp Spe I-anchored fragments from the TMS1 locus that were included as internal markers. A representative experiment is shown.

Effect of hMSL1 silencing and localization of the MSL complex at the TMS1 locus. MCF7 cells transfected with 100 nM of Lamin A/C (Lamin) or hMSL1 siRNA were harvested four days post-transfection and analyzed for (A) hMSL1, TMS1 and GAPDH protein expression by western blot analysis and (B) chromatin immunoprecipitation with antibodies against rabbit IgG (IgG), H3K9/14Ac and H4K16Ac, exactly as described in Figure 3C. Similar results were obtained when a scrambled siRNA was used as a negative control. Shown are the mean +/- standard deviation of triplicate determinations from a representative experiment. C) Chromatin from MCF7 and MDA-MB231 cells were subjected to immunoprecipitation with antibodies against rabbit IgG (IgG), hMOF or hMSL1 as described in Figure 3C. Each ChIP experiment was repeated at least twice with reproducible results. Shown are the mean +/- standard deviation of triplicate determinations from a representative experiment.

Role of H4K16Ac in the regulation of the ESR1 and CDH1 loci. A) Schematic of the region encompassing the CpG islands of the ESR1 and CDH1 gene. Nucleotide positions with respect to the transcription start site (T) and the CpG island are indicated above each gene diagram. Exons are shown in grey boxes. The regions amplified by primer sets (1-5) used in real-time PCR are shown below each gene. B) Localization of H3K9/14Ac and H4K16Ac at the ESR1 and CDH1 CpG island. ChIP analyses were performed for MCF7 or MDA-MB231 cells with the indicated antibodies or a negative control (IgG), followed by real-time PCR of regions depicted in A). Each experiment was conducted at least three times and although the immunoprecipitation efficiency varied between experiments, the profile of enrichment across each locus was consistent. Shown are the mean +/- standard deviation of triplicate determinations from a representative experiment. C) MCF7 cells were transfected with siRNA (100 nM) against Lamin A/C (Lamin) (irrelevant control) or hMOF (MOF1 and MOF2) and the expression of ESR1 and CDH1 protein was determined by western blotting. The same blot was also exposed to anti-hMOF and anti-GAPDH antibodies.