Identification of an evolutionarily conserved cis-regulatory element controlling the Peg3 imprinted domain

Abstract

The mammalian Peg3 domain harbors more than 20 evolutionarily conserved regions (ECRs) that are spread over the 250-kb genomic interval. The majority of these ECRs are marked with two histone modifications, H3K4me1 and H3K27ac, suggesting potential roles as distant regulatory elements for the transcription of the nearby imprinted genes. In the current study, the chromatin conformation capture (3C) method was utilized to detect potential interactions of these ECRs with the imprinted genes. According to the results, one region, ECR18, located 200-kb upstream of Peg3 interacts with the two promoter regions of Peg3 and Zim2. The observed interaction is most prominent in brain, but was also detected in testis. Histone modification and DNA methylation on ECR18 show no allele bias, implying that this region is likely functional on both alleles. In vitro assays also reveal ECR18 as a potential enhancer or repressor for the promoter of Peg3. Overall, these results indicate that the promoters of several imprinted genes in the Peg3 domain interact with one evolutionarily conserved region, ECR18, and further suggest that ECR18 may play key roles in the transcription and imprinting control of the Peg3 domain as a distant regulatory element.

Figure 1. Evolutionarily Conserved Regions (ECRs) in the Peg3 domain.

The upper panel represents a snapshot from UCSC Genome Browser showing the chromosomal position of the Peg3 domain, mammalian conservation levels (Mammal Cons), the positions of 18 ECRs, and histone modification profiles of H3K4me1 and H3K27ac observed in brain, liver, and testis. The different levels of the enrichment are shown with varying degrees of grey colors. The high levels of the enrichment at ECR11 and 18 are evident with darker-color vertical lines, the positions of which are indicated with *. The lower panel represents the genomic structure of the Peg3 domain: paternally and maternally expressed genes are marked with blue and red colors, respectively.

Figure 2. Summary of histone modification patterns on ECRs.

The table summarizes the histone modification patterns of H3K4me1 and H3K27ac in the Peg3 domain. The histone modification status of each locus (the name on the Y-axis) was presented for each tissue (the name on the X-axis). Black rectangles indicate the regions with both H3K4me1 and H3K27ac modifications; the darker grey ones indicate the regions with H3K4me1 only; and finally the lighter grey ones indicate the regions with H3K27ac only.

Figure 3. 3C strategy for the Peg3 domain.

The upper diagram details the genomic region covered by the two BAC clones (178C5 and 117K9) that have been used for preparing two control libraries, the relative positions of 18 ECRs, the relative positions of oligonucleotides that have been used for 3C experiments. Each oligonucleotide has been named with a numeric value to indicate its relative position to the transcription start site of Peg3. The gel images on the bottom panel show the results derived from the two sets of control experiments testing the efficiency and compatibility of each primer set. For both control experiments, the +1 oligonucleotide was used as a base primer, which intends to detect long-range interaction between the Peg3 promoter and other regions.

Figure 4. Interaction of the promoter of Peg3/Usp29 with ECRs.

Potential long-range interaction of the Peg3 promoter was tested using the +1 oligonucleotide as a base primer along with a set of oligonucleotides that are derived from multiple regions of the Peg3 domain. The initial survey was performed with a fixed number of cycles (36 cycles) using the three libraries derived from neonatal brain, testis and liver, and these results are shown on bottom. Quantitative PCR analyses were also performed. The results are summarized with graphs on top. For this series of analyses, the Ct (threshold cycle) value for each primer set was first calculated from the control library with ligation, and subsequently used as an internal control for the normalization of the Ct values derived from the three tissue libraries.

Figure 5. Interaction of the promoters of Zim2 and Zfp264/Zim3 with ECRs.

Potential long-range interactions between the promoters of Zim2 and Zfp264/Zim3 versus other genomic regions harboring ECRs were tested using two oligonucleotides as a base primer (-62 for Zim2 and +249 for Zfp264/Zim3). The results are shown on the middle (Zim2) and bottom (Zfp264/Zim3) panels.

Figure 6. Epigenetic modifications and evolutionary conservation of ECR18.

(A) Immunoprecipitated DNA with anti-H3K27ac antibodies was first amplified with PCR, and the subsequent PCR products were digested with an enzyme to differentiate parental alleles for a given locus. The promoter regions of imprinted genes show mono-allelic or one allele-biased patterns whereas ECR18 shows a bi-allelic pattern similar to those seen the input DNA. (B) DNA mthylation analysis on ECR18. The bisulfite-converted DNA from male and female neonates were used for PCR amplification. The amplified PCR products were digested with TaqI, and the digestion by this enzyme indicates methylation on the original DNA. (C) The panel represents a snapshot of UCSC Genome Browser showing the peaks of the two histone modifications, H3K27ac and H3K4me1, detected in the human PEG3 domain.

Figure 7. Transcriptional activity of ECR18.

The diagram on top shows schematic representations of the reporter constructs that have been used for the current study. The graphs on bottom summarize the results derived from a series of reporter assay with these constructs. These assays were conducted in triplicates in Neuro2a and NIH3T3 cells and normalized with the β-Gal activity of an independent reporter construct. This series of reporter assays were repeated three independent times.

Figure 8. ECR18 as a shared enhancer for the Peg3 domain.

(A) The diagram represents the paternal allele of the Peg3 domain with each gene being indicated with a horizontal arrow. The transcriptional level of each gene is also indicated with a vertical arrow: the thicker or thinner arrow indicates higher or lower expression levels for a given gene. Potential interaction between ECR18 and the promoter of each gene is indicated by a dotted line. (B) The diagram represents the paternal allele of Peg3 domain in the mutant animals that have a deletion in the Peg3-DMR, an ICR for the Peg3 domain. (C) The diagram represents the maternal allele of the Peg3 domain in the wild-type animals. The maternal allele in the mutant animals is not shown since the mutational effects are very minimal.