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A novel antisense long noncoding RNA within the IGF1R gene locus is imprinted in hematopoietic malignancies.

Sun J, Li W, Sun Y, Yu D, Wen X, Wang H, Cui J, Wang G, Hoffman AR, Hu JF - Nucleic Acids Res. (2014)

Bottom Line: Using both reverse transcription-associated trap and chromatin conformation capture assays, we demonstrate that this lncRNA interacts with chromatin DNA and is involved in the formation of an intrachromosomal enhancer/promoter loop.In addition, IRAIN was downregulated both in leukemia cell lines and in blood obtained from high-risk AML patients.These data identify IRAIN as a new imprinted lncRNA that is involved in long-range DNA interactions.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell and Cancer Center, First Affiliated Hospital, Jilin University, Changchun, Jilin 130061, PR China Stanford University Medical School, VA Palo Alto Health Care System, Palo Alto, CA 94304, USA.

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Intrachromosomal loop between IGF1R promoter and the intragenic enhancer. (A) Schematic diagram of the Hind III sites in the IGF1R/IRAIN gene locus used for the 3C assay. (B) Detection of chromatin interactions in KG-1 leukemia cells as detected by 3C assay. M: 100 bp markers, input: chromatin complex aliquots collected before 3C assay. (C) Sequencing confirmation of the 3C PCR products. Left: the Hind III site in the 3C DNA is flanked by DNA sequences from the IGF1R promoter and enhancer; right: local chromatin interaction. (D) IRAIN lncRNA knockdown abolishes the intrachromosomal DNA interaction. Cells were transfected with two shRNAs. After IRAIN knockdown, cells were fixed and used to detect DNA interaction with the 3C assay. Note the requirement for IRAIN in the maintenance of the IGF1R promoter/enhancer DNA interaction.
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Figure 5: Intrachromosomal loop between IGF1R promoter and the intragenic enhancer. (A) Schematic diagram of the Hind III sites in the IGF1R/IRAIN gene locus used for the 3C assay. (B) Detection of chromatin interactions in KG-1 leukemia cells as detected by 3C assay. M: 100 bp markers, input: chromatin complex aliquots collected before 3C assay. (C) Sequencing confirmation of the 3C PCR products. Left: the Hind III site in the 3C DNA is flanked by DNA sequences from the IGF1R promoter and enhancer; right: local chromatin interaction. (D) IRAIN lncRNA knockdown abolishes the intrachromosomal DNA interaction. Cells were transfected with two shRNAs. After IRAIN knockdown, cells were fixed and used to detect DNA interaction with the 3C assay. Note the requirement for IRAIN in the maintenance of the IGF1R promoter/enhancer DNA interaction.

Mentions: KG-1 cells were fixed with 2% formaldehyde, digested with restriction enzyme Hind III and then ligated with T4 DNA ligase to examine the remote interaction between these two DNA regions that are 150 kb apart (Figure 5A). Using primers from these remote regions, we found that the IGF1R promoter DNA interacted with the putative intronic enhancer DNA (Figure 5B, lane 4). In addition, a local DNA interaction was also detected between sites d′ and h′ (lane 2). Both interactions were confirmed by DNA sequencing (Figure 5C). ChIP assay also showed that both the IGF1R promoter (h2′) and the enhancer (p′) region sites were associated with the active enhancer marker, H3K4 methylation (Supplementary Figure S5). These data suggest that IRAIN lncRNA may be actively involved in the interaction of two remote regions of the IGF1R gene.


A novel antisense long noncoding RNA within the IGF1R gene locus is imprinted in hematopoietic malignancies.

Sun J, Li W, Sun Y, Yu D, Wen X, Wang H, Cui J, Wang G, Hoffman AR, Hu JF - Nucleic Acids Res. (2014)

Intrachromosomal loop between IGF1R promoter and the intragenic enhancer. (A) Schematic diagram of the Hind III sites in the IGF1R/IRAIN gene locus used for the 3C assay. (B) Detection of chromatin interactions in KG-1 leukemia cells as detected by 3C assay. M: 100 bp markers, input: chromatin complex aliquots collected before 3C assay. (C) Sequencing confirmation of the 3C PCR products. Left: the Hind III site in the 3C DNA is flanked by DNA sequences from the IGF1R promoter and enhancer; right: local chromatin interaction. (D) IRAIN lncRNA knockdown abolishes the intrachromosomal DNA interaction. Cells were transfected with two shRNAs. After IRAIN knockdown, cells were fixed and used to detect DNA interaction with the 3C assay. Note the requirement for IRAIN in the maintenance of the IGF1R promoter/enhancer DNA interaction.
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Figure 5: Intrachromosomal loop between IGF1R promoter and the intragenic enhancer. (A) Schematic diagram of the Hind III sites in the IGF1R/IRAIN gene locus used for the 3C assay. (B) Detection of chromatin interactions in KG-1 leukemia cells as detected by 3C assay. M: 100 bp markers, input: chromatin complex aliquots collected before 3C assay. (C) Sequencing confirmation of the 3C PCR products. Left: the Hind III site in the 3C DNA is flanked by DNA sequences from the IGF1R promoter and enhancer; right: local chromatin interaction. (D) IRAIN lncRNA knockdown abolishes the intrachromosomal DNA interaction. Cells were transfected with two shRNAs. After IRAIN knockdown, cells were fixed and used to detect DNA interaction with the 3C assay. Note the requirement for IRAIN in the maintenance of the IGF1R promoter/enhancer DNA interaction.
Mentions: KG-1 cells were fixed with 2% formaldehyde, digested with restriction enzyme Hind III and then ligated with T4 DNA ligase to examine the remote interaction between these two DNA regions that are 150 kb apart (Figure 5A). Using primers from these remote regions, we found that the IGF1R promoter DNA interacted with the putative intronic enhancer DNA (Figure 5B, lane 4). In addition, a local DNA interaction was also detected between sites d′ and h′ (lane 2). Both interactions were confirmed by DNA sequencing (Figure 5C). ChIP assay also showed that both the IGF1R promoter (h2′) and the enhancer (p′) region sites were associated with the active enhancer marker, H3K4 methylation (Supplementary Figure S5). These data suggest that IRAIN lncRNA may be actively involved in the interaction of two remote regions of the IGF1R gene.

Bottom Line: Using both reverse transcription-associated trap and chromatin conformation capture assays, we demonstrate that this lncRNA interacts with chromatin DNA and is involved in the formation of an intrachromosomal enhancer/promoter loop.In addition, IRAIN was downregulated both in leukemia cell lines and in blood obtained from high-risk AML patients.These data identify IRAIN as a new imprinted lncRNA that is involved in long-range DNA interactions.

View Article: PubMed Central - PubMed

Affiliation: Stem Cell and Cancer Center, First Affiliated Hospital, Jilin University, Changchun, Jilin 130061, PR China Stanford University Medical School, VA Palo Alto Health Care System, Palo Alto, CA 94304, USA.

Show MeSH
Related in: MedlinePlus