Article Figures & Data
Figures
- Figure 1.
A, schematic of the TMS1 genomic locus. The TMS1 gene consists of three exons (I, II, and III). Black boxes, noncoding regions. 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 ∼−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 chromatin immunoprecipitation assays are shown below the gene. B, expression of TMS1. Protein lysates prepared from MCF7, MDA-MB231, HMT.1E1, and IMR90 cells were subjected 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 chromatin immunoprecipitation 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 chromatin immunoprecipitation was repeated at least thrice, and although the immunoprecipitation efficiency varied between experiments, the profile of enrichment across the locus was consistent. Columns, mean of triplicate determinations from a representative experiment; bars, SD.
- Figure 2.
Localization of H4K16Ac and H4K20me3 across the TMS1 locus. MCF7 and MDA-MB231 breast cancer cells (A) or IMR90 and HMT.1E1 cell lines (B) were subjected to chromatin immunoprecipitation with antibodies against rabbit IgG or the indicated histone modifications. Immunoprecipitated DNA was amplified by real-time PCR with primer sets indicated in Fig. 1A. Percent input was determined as the amount of immunoprecipiated DNA relative to input DNA. Each chromatin immunoprecipitation was repeated at least thrice, and although the immunoprecipitation efficiency varied between experiments, the profile of enrichment across the locus was consistent. Columns, mean of triplicate determinations from a representative experiment; bars, SD. C, nucleosome positioning at the TMS1 locus. Nuclei from MCF7 (left) or MDA-MB231 (right) cells were incubated in micrococcal nuclease (MNase) digestion buffer alone (0), digestion buffer plus CaCl2 (0+), or digestion buffer plus CaCl2 and 10 to 200 units of micrococcal nuclease. Micrococcal nuclease–digested DNA (10 μg) was digested with HindIII (H) and SpeI (S), separated on a 1% agarose gel, and subjected to Southern blot analysis using a probe anchored to the 3′ SpeI site. Arrows, preferential micrococcal nuclease cut sites. Shown is the relative migration of 2,765-, 1,001-, and 739-bp SpeI-anchored fragments from the TMS1 locus that were included as internal markers. A representative experiment is shown.
- Figure 3.
Effect of hMOF down-regulation on the TMS1 locus. A, MCF7 cells were transfected with 100 nmol/L of siRNA targeting hMOF (MOF1 or MOF2) or lamin A/C (Lamin; irrelevant control) and harvested 4 d posttransfection. 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. Columns, mean of three independent experiments; bars, SD. Experiments were also done using scrambled nontargeting siRNA as a control and similar results were observed. B, MCF7 cells were transfected as in A, and chromatin immunoprecipitation was done with antibodies against rabbit IgG, H3K9/14Ac, or H4K16Ac. Immunoprecipitated DNA was amplified by real-time PCR with primer sets indicated in Fig. 1A. Data represent the percent of input DNA recovered. Each chromatin immunoprecipitation experiment was repeated at least twice with reproducible results. Columns, mean of triplicate determinations from a representative experiment; bars, SD. 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 Fig. 2C. Arrows, preferential micrococcal nuclease cut sites (1–7). Shown is the migration of 2,765-, 1,001-, and 739-bp SpeI 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 d posttransfection for Western blot analysis with the indicated antibodies.
- Figure 4.
Effect of hMSL1 silencing and localization of the MSL complex at the TMS1 locus. MCF7 cells transfected with 100 nmol/L of lamin A/C or hMSL1 siRNA were harvested 4 d posttransfection and analyzed for hMSL1, TMS1, and GAPDH protein expression by Western blot analysis (A) and chromatin immunoprecipitation with antibodies against rabbit IgG, H3K9/14Ac, and H4K16Ac (B), as described in Fig. 1C. Similar results were obtained when a scrambled siRNA was used as a negative control. Columns, mean of triplicate determinations from a representative experiment; bars, SD. C, chromatin from MCF7 and MDA-MB231 cells were subjected to immunoprecipitation with antibodies against rabbit IgG, hMOF, or hMSL1 as described in Fig. 1C. Each chromatin immunoprecipitation experiment was repeated at least twice with reproducible results. Columns, mean of triplicate determinations from a representative experiment; bars, SD.
- Figure 5.
Effect of hMOF down-regulation on TMS1 DNA methylation. A, MCF7 cells were transfected with siRNA (100 nmol/L) against hMOF (MOF1 and MOF2) or an irrelevant control lamin A/C and harvested 4 d posttransfection. Genomic DNA was isolated, modified by sodium bisulfite, and amplified by methylation-specific PCR 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, whereas that from HMT.1E1 and MDA-MB231 cells served as a control for methylated DNA. The TMS1 region (32–223) amplified by methylation-specific PCR is shown in B. B, DNA from MCF7 cells transfected with lamin A/C 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.
- Figure 6.
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. Nucleotide positions with respect to the transcription start site (T) and the CpG island are indicated above each gene diagram. Gray boxes, exons. 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. Chromatin immunoprecipitation analyses were done 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 thrice and, although the immunoprecipitation efficiency varied between experiments, the profile of enrichment across each locus was consistent. Columns, mean of triplicate determinations from a representative experiment; bars, SD. C, MCF7 cells were transfected with siRNA (100 nmol/L) against lamin A/C (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.
Volume 68, Issue 16
