An insulator loop resides between the synthetically interacting elements of the human/rat conserved breast cancer susceptibility locus MCS5A/Mcs5a

Abstract

Many low-penetrance breast cancer susceptibility loci are found to be located in non-protein-coding regions, suggesting their involvement in gene expression regulation. We identified the human/rat-conserved breast cancer susceptibility locus MCS5A/Mcs5a. This locus has been shown to act in a non-mammary cell-autonomous fashion through the immune system. The resistant Mcs5a allele from the Wistar-Kyoto (WKy) rat strain consists of two non-protein-coding genetic elements that must be located on the same chromosome to elicit the phenotype. In this study, we show the presence of a conserved higher order chromatin structure in MCS5A/Mcs5a located in between the synthetically interacting genetic elements. The looped elements are shown to be bound by CTCF and cohesin. We identify the downregulation of Fbxo10 expression in T cells as a strong candidate mechanism through which the interacting genetic elements of the resistant Mcs5a allele modulate mammary carcinoma susceptibility. Finally, we show that the human MCS5A polymorphisms associated with breast cancer risk are located at both sides of the looped structure and functionally interact to downregulate transcriptional activity, similar to rat Mcs5a. We propose a mechanistic model for MCS5a/Mcs5a in which a CTCF-mediated insulator loop encompassing the TOMM5/Tomm5 gene, resides in between and brings into closer physical proximity the synthetically and functionally interacting resistant genetic variants.

Figure 1.

Analysis of the higher order chromatin structure of rat and human Mcs5a/MCS5A. (A) Maps of the rat and human Mcs5a/MCS5A locus. The relative position of Mcs5a1/MCS5A1, Mcs5a2/MCS5A2, all BglII restriction site, the transcripts Fbxo10/FBXO10, Tomm5/TOMM5 and Frmpd1/FRMPD1, and the human breast cancer risk-associated variants is indicated. The primers matching the fixed fragments used to generate panels (B–E) are indicated by black vertical triangles. (B–D) 3C profiles of the Mcs5a locus in T cells of susceptible congenic control (susc.) and Mcs5a resistant congenic (Mcs5a) rats. The position of the fixed fragments is indicated with a black bar and F, and the primer within the fixed fragment is indicated with a vertical triangle. In (B) the fixed fragment contains the CpG island associated with the predicted promoter of Fbxo10 in Mcs5a1. In (C) the fixed fragment is located five BglII restriction fragments (~10 kb) upstream of the previous fixed fragment that contains the CpG island in Mcs5a1. In (D) the fixed fragment was located in the first looping fragment in Mcs5a2. (E) Comparison of the rat and human Mcs5a/MCS5A 3 C profile. Dashed lines represent the 3C profile with the fixed fragment containing the predicted Fbxo10/FBXO10 promoter in Mcs5a1/MCS5A1 and does not display looping to Mcs5a2/MCS5A2. In the human, this fixed fragment also contains the four correlated MCS5A1 polymorphisms associated with breast cancer risk. Solid lines represents the 3C profile with the fixed fragment located five (rat) or two (human) BglII restriction fragments (~10 kb) upstream of the BglII fragment containing the predicted Fbxo10/FBXO10 promoter in Mcs5a1/MCS5A1. The chromatin fragments in Mcs5a2/MCS5A2 looping to the fixed fragment in Mcs5a1/MCS5A1 are indicated with PEAK 1 and PEAK 2. Graphed are the average ± SEM relative interaction frequencies of a fixed BglII fragment with other BglII fragments in Mcs5a/MCS5A. The genomic distance (in kilobases) represents the distance of the midpoint of a BglII fragment to the Mcs5a1/MCS5A1-Mcs5a2/MCS5A2 border.

Figure 2.

CTCF and cohesin bind to the looping fragments in Mcs5a/MCS5A and CTCF is necessary for the higher order chromatin structure. (A and B) The Mcs5a1/MCS5A1 and Mcs5a2/MCS5A2 loci are depicted as black lines. The light gray bars within the black lines are the CpG islands associated with the promoters of the Fbxo10/FBXO10, Tomm5/TOMM5 and Frmpd1/FRMPD1 genes, respectively. The locations of the three interacting chromatin looping fragments identified in the 3C assay are indicated by light gray blocks. In the human, the location of known CTCF sites from a genome-wide CTCF ChIP-seq study is indicated (22). Amplicons within and outside the looping Mcs5a/MCS5A fragments, as well as an amplicon in the H19 locus (as a positive control) were analyzed by PCR on CTCF (C), cohesin (R; Rad21) and IgG (I; negative control) antibody immunoprecipitated chromatin samples, and an input (IN, positive control) sample, prepared from JURKAT cells (human) or primary rat splenic T cells (rat). Each gel image is accompanied by a 100-bp DNA ladder of which the lower three bands (100, 200, 300 bp) are shown. CTCF and cohesin binding was found in all looping fragments and the H19 positive control locus, but not in the Mcs5a2 fragment not located in a looping fragment. (C and D) Transcript level of CTCF, Tomm5, Gapdh, Mcart1 and Dcaf10 (normalized to ActB) measured 64–68 h after nucleofection of rat primary T cells with control siRNAs (siCONTROL; light gray bars) and siRNAs against CTCF (siCTCF; dark gray bars). The transcript level of CTCF and Tomm5 was significantly reduced (indicated with an asterisk) in the siCTCF-treated T cells. The transcript levels of the housekeeping gene Gapdh and genes Mcart1 and Dcaf10 located adjacent to the Mcs5a locus were not affected. (E) ChIP analysis of CTCF binding to the looping fragment in Mcs5a1 and the negative control fragment in Mcs5a2 in primary rat T-cells 64–68 h after nucleofection with control siRNAs and siRNAs against CTCF. CTCF binding to the Mcs5a1 looping fragment was reduced in the siCTCF-treated T cells. (F) 3C analysis of the Mcs5a locus in primary rat T cells 64–68 h after nucleofection with control siRNAs (light gray line) and siRNAs against CTCF (dark gray line). The looping fragment in Mcs5a1 was taken as the fixed fragment (indicated with a black bar and F). Looping of the fixed fragment to the looping fragments in Mcs5a2 was significantly reduced (indicated with an asterisk) in the siCTCF-treated T cells.

Figure 3.

Lower Fbxo10 transcript level in T cells is associated with the mammary carcinoma resistance phenotype mediated by Mcs5a. (A) The thymic Fbxo10, and not splenic Frmpd1 transcript level is associated with the resistant Mcs5a allele. Graphed are average ± SEM thymic Fbxo10 (dark gray) and splenic Frmpd1 (light gray) transcript levels (normalized to Gapdh) scaled to the thymic Fbxo10 or splenic Frmpd1 transcript level of the susceptible congenic control line. Significantly (P < 0.05) different Fbxo10 and Frmpd1 transcript levels as compared with the susceptible congenic control line are indicated with one and two asterisks, respectively. Sample sizes were: susc., n = 9; Mcs5a, n = 6; Mcs5a1, n = 9; Mcs5a2, n = 8. (B) Lower Fbxo10 transcript level associated with the resistant Mcs5a allele is observed in unsorted and sorted thymocytes, except for double negative (CD4⁻CD8⁻) thymocytes. Graphed are average ± SEM Fbxo10 transcript levels (normalized to Gapdh) in unsorted and sorted thymocytes, scaled to the average Fbxo10 transcript level for the unsorted thymocytes of the susceptible congenic control line. Sample sizes for the susceptible congenic control line and Mcs5a congenic resistant line were, respectively: unsorted, n = 16 and n = 9; CD4⁻CD8⁻, n = 4 and n = 4; CD4⁺CD8⁺, n = 17 and n = 19; CD4⁺CD8⁻, n = 15 and n = 18; CD8⁺CD4⁻, n = 18 and n = 17. (C) Lower Fbxo10 transcript level associated with the resistant Mcs5a allele is observed in splenic T cells, but not in other splenocytes. Graphed are average ± SEM Fbxo10 transcript levels (normalized to Gapdh) in unsorted and sorted splenocytes, scaled to the Fbxo10 transcript level for the unsorted splenocytes of the susceptible congenic control line. Sample sizes for the susceptible congenic control line and Mcs5a resistant congenic line were, respectively: unsorted, n = 16 and n = 12; B cells, n = 9 and n = 8; T cells, n = 16 and n = 13; non-B-/non-T cells, n = 3 and n = 5. (D) Lower Fbxo10 transcript level associated with the resistant Mcs5a allele is observed in cultured unstimulated and conA-stimulated T cells. Graphed are average ± SEM Fbxo10 transcript levels (normalized to ActB) in cultured T cells, unstimulated (−) and stimulated (+) with conA, scaled to the average Fbxo10 transcript level of the unstimulated sample of the susceptible congenic control line. Sample sizes for the susceptible congenic control line and Mcs5a resistant congenic line were, respectively: unstimulated, n = 11 and n = 10; conA-stimulated, n = 11 and n = 12. (E) Tomm5 transcript levels in the immune system are not associated with the resistant Mcs5a allele. Graphed are average ± SEM Tomm5 transcript levels (normalized to ActB) in spleen, thymus and primary T cells, scaled to the average Tomm5 transcript level of the susceptible congenic control line. Sample sizes for the susceptible congenic control line and Mcs5a resistant congenic line were, respectively: spleen, n = 7 and n = 7; thymus, n = 8 and n = 8; primary T cells, n = 12 and n = 9. (F) Tomm5 transcript levels in cultured T cells are strongly increased after conA-stimulation. Graphed are average ± SEM Tomm5 transcript levels (normalized to ActB) in cultured T cells, unstimulated and stimulated with conA, scaled to the average Tomm5 transcript level of the unstimulated sample of the susceptible congenic control line. Sample sizes for the susceptible congenic control line and Mcs5a resistant congenic line were, respectively: unstimulated, n = 11 and n = 10; conA-stimulated, n = 11 and n = 12. In B–F, significantly different transcript level (P < 0.05) between the susceptible congenic control line (susc.; open bars) and the Mcs5a resistant congenic line (Mcs5a; filled bars) are indicated with an asterisk. In D and F, significantly different Fbxo10 or Tomm5 transcript level (P < 0.05) between samples without and with conA stimulation is indicated with two asterisks.

Figure 4.

Fbxo10/FBXO10 TSS analysis by the 5′ RLM-RACE assay. (A) Schematic representation of three putative Fbxo10/FBXO10 transcripts annotated in the UCSC genome browser, indicated relative to the position of the Mcs5a1/MCS5A1 locus (in black). The location of the Fbxo10/FBXO10 specific primers in the first coding exon is indicated (P). The 5′ RLM-RACE revealed a cluster of 14 rat Fbxo10 TSSs within the Mcs5a1 CpG island (indicated in light gray). A cluster of three human FBXO10 TSSs in the orthologous MCS5A1 CpG island (indicated in light gray) was identified. The position of the first non-coding exon of Fbxo10/FBXO10 within the CpG island is indicated in dark gray. The human TSS cluster was found to be located at 150-bp distance from breast cancer risk-associated SNP rs6476643. The transcripts not identified in rat or human immune tissue are indicated with a X. (B) Sequence information of the rat and human Mcs5a1/MCS5A1 CpG island, first non-coding Fbxo10/FBXO10 exon (highlighted in gray) and TSS identified (in bold). In the human sequence, the breast cancer risk-associated SNP rs6476643 (T/G) is indicated.

Figure 5.

Transcriptional activity analysis of the human breast cancer risk-associated susceptible and resistant alleles in Luciferase reporter assays. (A) Schematic representation of the position of the genomic fragments derived from the MCS5A1 and MCS5A2 loci combined into the Luciferase constructs. The MCS5A1 and MCS5A2 loci are shown as black lines. The light gray bars within the black lines represent the CpG islands located in the locus. The three genes FBXO10, FRMPD1 and TOMM5 are shown in dark gray. The breast cancer risk-associated polymorphisms are represented as vertical gray lines. The fragments subcloned into the reporter constructs are indicated as horizontal light gray bars. The susceptible alleles of the MCS5A2 polymorphisms were combined with the susceptible allele of the MCS5A1 SNP rs6476643 (SS constructs 1–10). Similarly, the resistant alleles were combined (RR constructs 1–10). (B) Map of the FBXO10 TSS-Luciferase reporter construct. A MCS5A1 fragment containing the FBXO10 TSSs and the risk-associated SNP rs6476643 was inserted upstream of the Luciferase reporter gene (Luc⁺) in reverse genomic orientation. Two versions of the construct were created, namely having the susceptible (S; T allele) or resistant (R; G allele) of SNP rs6476643. Other features of the construct include the ampicillin resistance gene (Amp^R), origin of replication derived from filamentous phage (f1 ori), origin of replication in E. coli (ori), a synthetic poly(A) signal/transcriptional pause site for background reduction [synthetic poly(A)] and a cloning site downstream of Luc⁺. (C) Box plot of the relative Luciferase activity of the R and S constructs. n = 30 transient transfection assays. (D) Average ± SEM relative Luciferase activity of constructs SS 1–10 and RR 1–10. On the x-axis, the genomic distance of the MCS5A2 polymophisms to the MCS5A1 SNP rs6476643 is plotted. The data points at genomic distance 0 correspond to the FBXO10 TSS-Luciferase constructs S and R. The measurements indicated with an asterisk are significantly different between SS and RR (P < 0.05). The entire series of RR1–10 is significantly lower as compared with the entire series of SS 1–10 (P < 10⁻⁴⁸). n = 6 or more transient transfection assays per data point.

Figure 6.

A model of the MCS5A locus in a folded configuration. The first (non-coding, 5′-UTR) and second (coding, ATG-containing) exons of the FBXO10 transcript are displayed in orange. The TOMM5 transcript is indicated in dark blue. The first (non-coding, 5′-UTR) exon of the FRMPD1 transcript is shown in light blue. The CpG islands associated with their promoters are indicated in dark green. The correlated polymorphisms that associate with breast cancer risk are depicted as purple bars. The looping fragments are shown in light green and are shown to be bound by CTCF. In this model, the breast cancer susceptibility locus MCS5A harbors an insulator loop encompassing the TOMM5 gene and regulatory region. The looped structure involves three elements that may loop simultaneously (as depicted) or loop in specific combinations in a single nucleus. The polymorphisms associated with breast cancer risk in MCS5A1 and MCS5A2 are located at both sides of the looped structure and are in closer proximity as may be derived from a linear genome view. We hypothesize that the MCS5A1 and MCS5A2 breast cancer risk variants interact to regulate the transcript levels of FBXO10.