Deletion of Meg8-DMR Enhances Migration and Invasion of MLTC-1 Depending on the CTCF Binding Sites

Abstract

The Dlk1-Dio3 imprinted domain on mouse chromosome 12 contains three well-characterized paternally methylated differentially methylated regions (DMRs): IG-DMR, Gtl2-DMR, and Dlk1-DMR. These DMRs control the expression of many genes involved in embryonic development, inherited diseases, and human cancer in this domain. The first maternal methylation DMR discovered in this domain was the Meg8-DMR, the targets and biological function of which are still unknown. Here, using an enhancer-blocking assay, we first dissected the functional parts of the Meg8-DMR and showed that its insulator activity is dependent on the CCCTC-binding factor (CTCF) in MLTC-1. Results from RNA-seq showed that the deletion of the Meg8-DMR and its compartment CTCF binding sites, but not GGCG repeats, lead to the downregulation of numerous genes on chromosome 12, in particular the drastically reduced expression of Dlk1 and Rtl1 in the Dlk1-Dio3 domain, while differentially expressed genes are enriched in the MAPK pathway. In vitro assays revealed that the deletion of the Meg8-DMR and CTCF binding sites enhances cell migration and invasion by decreasing Dlk1 and activating the Notch1-Rhoc-MAPK/ERK pathway. These findings enhance research into gene regulation in the Dlk1-Dio3 domain by indicating that the Meg8-DMR functions as a long-range regulatory element which is dependent on CTCF binding sites and affects multiple genes in this domain.

Keywords: CTCF; Dlk1; Dlk1-Dio3 domain; MLTC-1; Meg8-DMR; invasion; migration.

Figure 1

Meg8-DMR acts as an insulator dependent on the CTCF binding sites. (A) Schematic representation of the Dlk1-Dio3 imprinted domain. Maternally expressed noncoding RNAs are shown in red, whereas, protein coding paternally expressed RNAs are in blue. Black and white circles represent methylated and unmethylated. Meg8-DMR, which is methylated in the maternally inherited chromosome, but unmethylated in the paternally inherited chromosome, is located on the second intron of Rian and contains conserved CTCF-binding sites and GGCG repeats. (B) The constructs of the enhancer-blocking assay were prepared and transfected into MLTC-1 cells. Each construct contained a mouse β-globin 3′HS2 enhancer element and a human Aγ-globin promoter directing transcription of a neo reporter (Neo), and various test fragments were inserted between them. (C) The colony number for each test is expressed relative to the number observed with the control plasmid (PNI). Mock is the blank control group. Error bars, mean ± SEM. n = 3. p values were calculated using t-test. * p < 0.05.

Figure 2

Generation of Meg8-DMR knockout cell line models via CRISPR/Cas9. (A) A schematic of CRISPR/Cas9-mediated deletion of Meg8-DMR, CTCFBS, and (GGCG)n. (B) PCR identified homozygous clones, F1/R1primers were located outside of Meg8-DMR, and F2/R2 primers were located inside of Meg8-DMR. (C) The deletion clones of Meg8-DMR, CTCFBS and (GGCG)n were identified through sequencing results (Meg8-DMR-KO has double peaks close to the targets because the different copies of the chromosomes may be repaired in various ways).

Figure 3

RNA−seq analysis of the Meg8−DMR deletion cell lines. (A) Venn diagram of upregulated genes and downregulated genes in Meg8-DMR-KO, CTCFBS-KO, and (GGCG)n-KO. (B) Distribution of upregulated genes and downregulated genes in Meg8-DMR-KO, CTCFBS-KO, and (GGCG)n-KO on chromosomes. (C) RNA-seq demonstrates log-fold changes of mRNA expression on chromosome 12 in Meg8-DMR-KO, CTCFBS-KO, and (GGCG)n-KO compared with WT. Significantly reduced expression of Dlk1 and Rtl1 is observed in Meg8-DMR-KO and CTCFBS-KO. Dlk1 and Rtl1 were marked in red. (D) RT-qPCR shows a dramatic reduction in paternal Dlk1 expression and slightly increased expression of maternal Gtl2, Rian, and Mirg lncRNAs in Meg8-DMR-KO and CTCFBS-KO compared with WT. Strand-specific RT-PCR shows a dramatic reduction in paternal Rtl1 expression in Meg8-DMR-KO and CTCFBS-KO compared with WT. The relative expression level was normalized by Gapdh. Error bars, mean ± SEM. n ≥ 3. p values were calculated using t-test. ** p < 0.01.

Figure 4

DEGs Functional Analysis of the Meg8-DMR deletion cell lines. (A–C) Top 20 of KEGG pathway enrichment of genes differentially expressed in Meg8-DMR-KO, CTCFBS-KO, and (GGCG)n-KO. (D) Top 10 GO terms of cellular component (CC) of GO enrichment analysis of differentially expressed genes in Meg8-DMR-KO, CTCFBS-KO and (GGCG)n-KO.

Figure 5

Meg8-DMR deletion enhanced migration and invasion of MLTC-1. (A) Immunofluorescence staining of Phalloidin-California Red Conjugate in WT, Meg8-DMR-KO, CTCFBS-KO, and (GGCG)n-KO. (B) Confluent monolayers of four cell lines were wounded and after being incubated for an additional 48 h, the relative migration ratio was calculated. (C) Migration and invasion of four cell lines were measured via Transwell assay. (D) Cell proliferation of four cell lines was measured via MTT assay. (E) Colonies grown from cells of four cell lines were counted. Error bars, mean ± SEM. n ≥ 3. p values were calculated using t-test. * p < 0.05, ** p < 0.01, *** p < 0.001, scale bars = 200 μm.

Figure 6

Overexpression of Dlk1 in Meg8−DMR−KO suppressed cells’ migration and invasion by blocking Notch1−Rhoc−MAPK/ERK. (A) Western blot shows the expression level of Dlk1 was reduced while Nocth1 Rhoc and p-ERK1/2 were activated in Meg8-DMR-KO. When the overexpression vector pcDNA3.1-Dlk1 was transfected into Meg8-DMR-KO, the expression of Nocth1, Rhoc, and p-ERK1/2 was reduced. (B) Immunofluorescence staining of Phalloidin-California Red Conjugate in WT, Meg8-DMR-KO, and Meg8-DMR-KO with Dlk1 overexpression. (C) Confluent monolayers of cells of WT, Meg8-DMR-KO and Meg8-DMR-KO with Dlk1 overexpression were wounded and after being incubated for an additional 48 h, the relative migration ratio was calculated. (D) Migration and invasion of WT, Meg8-DMR-KO, and Meg8-DMR-KO with Dlk1 overexpression were analyzed. (E) Cell proliferation of WT, Meg8-DMR-KO and Meg8-DMR-KO with Dlk1 overexpression was measured via MTT assay. (F) Colonies grown from cells of WT, Meg8-DMR-KO, and Meg8-DMR-KO with Dlk1 overexpression were counted. Error bars, mean ± SEM. n ≥ 3. p values were calculated using t-test. * p < 0.05, ** p < 0.01, ns (non-significant), scale bars = 200 μm.

Figure 7

Methylation of the Dlk1-Dio3 domain in the absence of Meg8-DMR. (A) Bisulfite sequencing analysis of Dlk1-DMR, IG-DMR, and Gtl2-DMR in each cell line. Each CpG dinucleotide is represented with a circle. Each row of circles represents an individual clone sequenced. Black and white circles represent methylated and unmethylated CpGs, respectively. (B) Percentage statistics of methylation status.