Targeted Deletion of Multiple CTCF-Binding Elements in the Human C-MYC Gene Reveals a Requirement for CTCF in C-MYC Expression

BACKGROUND:Insulators and domain boundaries both shield genes from adjacent enhancers and inhibit intrusion of heterochromatin into transgenes. Previous studies examined the functional mechanism of the MYC insulator element MINE and its CTCF binding sites in the context of transgenes that were randomly inserted into the genome by transfection. However, the contribution of CTCF binding sites to both gene regulation and maintenance of chromatin has not been tested at the endogenous MYC gene. METHODOLOGY/PRINCIPAL FINDINGS:To determine the impact of CTCF binding on MYC expression, a series of mutant human chromosomal alleles was prepared in homologous recombination-efficient DT40 cells and individually transferred by microcell fusion into murine cells. Functional tests reported here reveal that deletion of CTCF binding elements within the MINE does not impact the capacity of this locus to correctly organize an 'accessible' open chromatin domain, suggesting that these sites are not essential for the formation of a competent, transcriptionally active locus. Moreover, deletion of the CTCF site at the MYC P2 promoter reduces transcription but does not affect promoter acetylation or serum-inducible transcription. Importantly, removal of either CTCF site leads to DNA methylation of flanking sequences, thereby contributing to progressive loss of transcriptional activity. CONCLUSIONS:These findings collectively demonstrate that CTCF-binding at the human MYC locus does not repress transcriptional activity but is required for protection from DNA methylation

Allele-specific transcriptional elongation regulates monoallelic expression of the IGF2BP1 gene

<p>Abstract</p> <p>Background</p> <p>Random monoallelic expression contributes to phenotypic variation of cells and organisms. However, the epigenetic mechanisms by which individual alleles are randomly selected for expression are not known. Taking cues from chromatin signatures at imprinted gene loci such as the insulin-like growth factor 2 gene 2 (<it>IGF2</it>), we evaluated the contribution of CTCF, a zinc finger protein required for parent-of-origin-specific expression of the <it>IGF2 </it>gene, as well as a role for allele-specific association with DNA methylation, histone modification and RNA polymerase II.</p> <p>Results</p> <p>Using array-based chromatin immunoprecipitation, we identified 293 genomic loci that are associated with both CTCF and histone H3 trimethylated at lysine 9 (H3K9me3). A comparison of their genomic positions with those of previously published monoallelically expressed genes revealed no significant overlap between allele-specifically expressed genes and colocalized CTCF/H3K9me3. To analyze the contributions of CTCF and H3K9me3 to gene regulation in more detail, we focused on the monoallelically expressed <it>IGF2BP1 </it>gene. <it>In vitro </it>binding assays using the CTCF target motif at the <it>IGF2BP1 </it>gene, as well as allele-specific analysis of cytosine methylation and CTCF binding, revealed that CTCF does not regulate mono- or biallelic <it>IGF2BP1 </it>expression. Surprisingly, we found that RNA polymerase II is detected on both the maternal and paternal alleles in B lymphoblasts that express <it>IGF2BP1 </it>primarily from one allele. Thus, allele-specific control of RNA polymerase II elongation regulates the allelic bias of <it>IGF2BP1 </it>gene expression.</p> <p>Conclusions</p> <p>Colocalization of CTCF and H3K9me3 does not represent a reliable chromatin signature indicative of monoallelic expression. Moreover, association of individual alleles with both active (H3K4me3) and silent (H3K27me3) chromatin modifications (allelic bivalent chromatin) or with RNA polymerase II also fails to identify monoallelically expressed gene loci. The selection of individual alleles for expression occurs in part during transcription elongation.</p

Chromosome Conformation Capture Carbon Copy (5C): a massively parallel solution for mapping interactions between genomic elements

Physical interactions between genetic elements located throughout the genome play important roles in gene regulation and can be identified with the Chromosome Conformation Capture (3C) methodology. 3C converts physical chromatin interactions into specific ligation products, which are quantified individually by PCR. Here we present a high-throughput 3C approach, 3C-Carbon Copy (5C), that employs microarrays or quantitative DNA sequencing using 454-technology as detection methods. We applied 5C to analyze a 400-kb region containing the human beta-globin locus and a 100-kb conserved gene desert region. We validated 5C by detection of several previously identified looping interactions in the beta-globin locus. We also identified a new looping interaction in K562 cells between the beta-globin Locus Control Region and the gamma-beta-globin intergenic region. Interestingly, this region has been implicated in the control of developmental globin gene switching. 5C should be widely applicable for large-scale mapping of cis- and trans- interaction networks of genomic elements and for the study of higher-order chromosome structure

Acetylation status of wildtype and mutated MYC loci on human chromosome 8.

<p>ChIP experiments were performed with antibodies specific for H3K9/14ac using chromatin from mouse B78 clones carrying wildtype or mutated MYC loci on human chromosome 8. The binding sites for CTCF within the wildtype MYC promoter, the positions of primers sets used to amplify regions (amplicons) J through R, and the location of deletions introduced into the human MYC promoter are shown above. Arrows indicate transcriptional start sites of the P1 and P2 promoter. The fold enrichment of acetylated histone H3 was normalized to the signals from the endogenous mouse β-globin gene promoter as an internal standard (mean±stdev, n = 3).</p

Deletion of CTCF binding sites protects the MYC promoter from DNA hypermethylation.

<p>Top, scheme of DNA methylation analysis in the MYC promoter region. <i>Aci</i>I restriction enzyme sites across the 2.5 kb human MYC 5′ region are indicated by filled squares. Positions of CTCF binding sites N and A, and of amplified regions using primer pairs CM1 to 3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006109#pone.0006109-Ishihara1" target="_blank">[21]</a> and P2 are indicated. DNA of 4 different clones for each allele (wt, ΔCTCF-N, ΔCTCF-NA) was digested with the methylation-sensitive restriction enzyme <i>Aci I</i> (A) or <i>EcoR I</i> (RI). Amplification of <i>EcoR I</i>-digested DNA serves as a positive control, and indicates the efficiency of PCR amplification. The level of amplification of <i>Aci I</i>-digested DNA indicates gain of DNA methylation in the tested region. While CM1 and CM3 regions show complete or partial methylation, CM2 and P2 regions are unmethylated in the wildtype human MYC allele. Deletion of CTCF sites N or N+A leads to DNA methylation.</p

Acetylation status of the MYC locus on human chromosome 8 residing in chicken DT40 cells and in murine B78 cells.

<p>DNA recovered from chromatin immunoprecipitations with antibodies specific for lysine9/14 acetylated histone H3 (H3K9/14ac) was subjected to duplex PCR using primers specific for the human, chicken and mouse MYC genes. The enrichment of these regions in the immunoprecipitated DNA was normalized to the signal detected with primers specific for the endogenous chicken β-globin gene or the mouse β-globin. Upper graph, comparison of histone acetylation levels at the human MYC promoter regions L, K, N and the chicken MYC promoter region in DT40 cells. Primer location and CTCF sites N and A of the human MYC promoter region are shown schematically at the top. Lower panel, comparison of histone acetylation levels at human MYC promoter regions L, K, N, and the murine MYC promoter region after transfer of human chromosome 8 into mouse B78 cells.</p

CTCF does not repress MYC expression and is required for normal levels of MYC mRNA.

<p>Real time RT-PCR analysis of the wildtype and recombinant MYC alleles residing on human chromosome 8 in B78 cells. (A) The steady-state level of human and mouse MYC RNA normalized to the endogenous mouse GAPDH mRNA. Left panel shows the relative expression of human MYC as a percentage of the expression detected in cells carrying the wildtype (wt) MYC allele. This analysis was performed multiple times for each B78 cell line containing a human MYC allele (wt, n = 7; ΔLg, n = 8; ΔCTCF-N, n = 9; ΔCTCF-NA, n = 6). Each clone was independently analyzed at least twice, and each PCR reaction was performed in duplicate. The bars indicate the mean value (±SEM) for all clones with the specific type of mutation. The right panel shows the expression of the endogenous murine MYC gene relative to the GAPDH gene. Mouse MYC gene expression is expressed as a percentage of the expression that is detected in cells carrying the human chromosome 8 with the wildtype allele. The expression in each of the mutant cells lines varies less than two-fold, indicating unaltered expression of the endogenous mouse MYC gene. (B) Expression of wildtype and mutant human MYC genes in starved (0 Hr) and serum-induced cells (1 Hr, 3 Hr, 9 Hr). RNA was harvested prior to serum induction (0 Hr), and 1 hour, 3 hours, and 9 hours post serum induction and analyzed by real-time RT-PCR. MYC mRNA levels were normalized to murine GAPDH mRNA levels. MYC expression in serum-starved cells was set to 1, and RNA levels at 1, 3 and 9 Hr are expressed as fold-induction over 0 Hr timepoint.</p