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Figure 3 | Breast Cancer Research

Figure 3

From: Phosphorylated and sumoylation-deficient progesterone receptors drive proliferative gene signatures during breast cancer progression

Figure 3

Promoter selectivity is achieved through increased recruitment of SUMO-deficient KR PR, CBP, MLL2 and histone tail modification, H3K4me2, to enhancer loci. (A) Schematic showing the MSX2 gene PRE-containing enhancer region located 15,094 bp upstream from the transcriptional start site. (B) Relative recruitment of PR to the MSX2 enhancer region was measured by ChIP-qPCR assays in T47D cells expressing constitutive PR null, WT or KR PR after treatment with R5020 for one or four hours. PR recruitment values were normalized as a percentage of input chromatin DNA values. To control for background non-specific antibody binding, immunoprecipitated chromatin contained a mixture from all samples with an IgG antibody. Similar ChIP results were obtained in T47D cells expressing inducible PR (right side). (C) The relative recruitment of CBP to the MSX2 enhancer region was measured as described in part B. (D) Levels of H3K4 dimethylation at the MSX2 enhancer were measured in the inducible PR expressing cell lines (iWT and iKR). The presence of H3K4me2 was determined at the MSX2 enhancer, up/downstream from the PRE, using overlapping qPCR products that span the region. (E) MLL2 recruitment to the MSX2 enhancer region was determined in T47D cells expressing both constitutive PR and inducible PR, as described in part B. (F) MAT2A gene expression was measured by RT-qPCR in T47D cells expressing stable WT or SUMO-deficient KR PR. Additionally, PR and MLL2 recruitment was quantified in these cells, as measured by standard ChIP-qPCR assay. Data are represented as mean of n = 3 +/- SD and significance calculated using Student's t-test. [See also Additional files 7, 5. KR, K388R PR-B mutant]. CBP, CREB-(cAMP-response element-binding protein)-binding protein; ChIP, chromatin immunoprecipitation; H3K4me2, histone H3 lysine 4 dimethylation; IgG, immunoglobulin G; KR, K388R PR-B mutant; MLL2, mixed lineage leukemia 2; PR, progesterone receptor; PRE, progesterone receptor response element; SD, standard deviation; SUMO, small ubiquitin-like modifier; WT, wild type.

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