Limits...
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.

Show MeSH

Related in: MedlinePlus

Chromatin DNA interaction of the IRAIN lncRNA. (A) Detection of IRAIN lncRNA–DNA interaction. A reverse transcription-associated trap (RAT) assay was used to detect the IRAIN lncRNA-specific interaction with IGF1R promoter DNAs. Alphabetic numbers: location of lncRNA binding sites. (B) IRAIN lncRNA–DNA interaction in KG-1 and K562 leukemia cells. Note the enriched binding of IRAIN lncRNA at IGF1R promoter (a4 and a5 sites) and intragenic enhancer (p site). Input: RNA–chromatin complex aliquots collected before RAT. (C) Allelic interaction of the IRAIN lncRNA with chromatin DNAs. Two polymorphic restriction enzymes Alu I and Sac II were used to distinguish the paternal and maternal alleles. Note the monoallelic binding of the IRAIN lncRNA. (D) Sequencing confirmation of allelic IRAIN lncRNA binding with the IGF1R promoter chromatin DNA. Left: K562 genomic DNA with both ‘A/T’ alleles; right: the IRAIN ChIP DNA with the single ‘A’ allele. (E) The lncRNA/DNA interaction after depletion of nascent RNA with α-amanitin. Cells were pretreated with the RNA polymerase inhibitor α-amanitin. After depletion of the nascent RNA, cells were fixed and used to detect DNA interaction with the RAT assay. The α-amanitin treatment does not interfere with lncRNA/DNA interaction.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150754&req=5

Figure 4: Chromatin DNA interaction of the IRAIN lncRNA. (A) Detection of IRAIN lncRNA–DNA interaction. A reverse transcription-associated trap (RAT) assay was used to detect the IRAIN lncRNA-specific interaction with IGF1R promoter DNAs. Alphabetic numbers: location of lncRNA binding sites. (B) IRAIN lncRNA–DNA interaction in KG-1 and K562 leukemia cells. Note the enriched binding of IRAIN lncRNA at IGF1R promoter (a4 and a5 sites) and intragenic enhancer (p site). Input: RNA–chromatin complex aliquots collected before RAT. (C) Allelic interaction of the IRAIN lncRNA with chromatin DNAs. Two polymorphic restriction enzymes Alu I and Sac II were used to distinguish the paternal and maternal alleles. Note the monoallelic binding of the IRAIN lncRNA. (D) Sequencing confirmation of allelic IRAIN lncRNA binding with the IGF1R promoter chromatin DNA. Left: K562 genomic DNA with both ‘A/T’ alleles; right: the IRAIN ChIP DNA with the single ‘A’ allele. (E) The lncRNA/DNA interaction after depletion of nascent RNA with α-amanitin. Cells were pretreated with the RNA polymerase inhibitor α-amanitin. After depletion of the nascent RNA, cells were fixed and used to detect DNA interaction with the RAT assay. The α-amanitin treatment does not interfere with lncRNA/DNA interaction.

Mentions: As several lncRNA molecules regulate gene function by directly binding to their target chromatin DNAs (28–30,49,50), we determined if IRAIN lncRNA bound to IGF1R chromatin DNA, particularly the promoter region. We developed a ‘RAT’ assay that overcomes the high noise to signal background of existing methodologies to detect RNA/DNA interactions (Figure 4A). Cells were treated with formaldehyde to fix the structure of the lncRNA-chromatin conformations. The chromatin DNA-interacting lncRNA was then reverse transcribed into cDNA using strand-specific oligonucleotides and a thermo-stable reverse transcriptase in the presence of biotin-dCTP. After digestion with restriction enzyme or sonication to shear chromatin DNAs, the biotinylated IRAIN cDNA–DNA complex was then separated from other DNA–DNA products by streptavidin pull-down using paramagnetic Dynabeads (Dynal, Invitrogen) and analyzed by PCR using interacting DNA-specific primers.


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)

Chromatin DNA interaction of the IRAIN lncRNA. (A) Detection of IRAIN lncRNA–DNA interaction. A reverse transcription-associated trap (RAT) assay was used to detect the IRAIN lncRNA-specific interaction with IGF1R promoter DNAs. Alphabetic numbers: location of lncRNA binding sites. (B) IRAIN lncRNA–DNA interaction in KG-1 and K562 leukemia cells. Note the enriched binding of IRAIN lncRNA at IGF1R promoter (a4 and a5 sites) and intragenic enhancer (p site). Input: RNA–chromatin complex aliquots collected before RAT. (C) Allelic interaction of the IRAIN lncRNA with chromatin DNAs. Two polymorphic restriction enzymes Alu I and Sac II were used to distinguish the paternal and maternal alleles. Note the monoallelic binding of the IRAIN lncRNA. (D) Sequencing confirmation of allelic IRAIN lncRNA binding with the IGF1R promoter chromatin DNA. Left: K562 genomic DNA with both ‘A/T’ alleles; right: the IRAIN ChIP DNA with the single ‘A’ allele. (E) The lncRNA/DNA interaction after depletion of nascent RNA with α-amanitin. Cells were pretreated with the RNA polymerase inhibitor α-amanitin. After depletion of the nascent RNA, cells were fixed and used to detect DNA interaction with the RAT assay. The α-amanitin treatment does not interfere with lncRNA/DNA interaction.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150754&req=5

Figure 4: Chromatin DNA interaction of the IRAIN lncRNA. (A) Detection of IRAIN lncRNA–DNA interaction. A reverse transcription-associated trap (RAT) assay was used to detect the IRAIN lncRNA-specific interaction with IGF1R promoter DNAs. Alphabetic numbers: location of lncRNA binding sites. (B) IRAIN lncRNA–DNA interaction in KG-1 and K562 leukemia cells. Note the enriched binding of IRAIN lncRNA at IGF1R promoter (a4 and a5 sites) and intragenic enhancer (p site). Input: RNA–chromatin complex aliquots collected before RAT. (C) Allelic interaction of the IRAIN lncRNA with chromatin DNAs. Two polymorphic restriction enzymes Alu I and Sac II were used to distinguish the paternal and maternal alleles. Note the monoallelic binding of the IRAIN lncRNA. (D) Sequencing confirmation of allelic IRAIN lncRNA binding with the IGF1R promoter chromatin DNA. Left: K562 genomic DNA with both ‘A/T’ alleles; right: the IRAIN ChIP DNA with the single ‘A’ allele. (E) The lncRNA/DNA interaction after depletion of nascent RNA with α-amanitin. Cells were pretreated with the RNA polymerase inhibitor α-amanitin. After depletion of the nascent RNA, cells were fixed and used to detect DNA interaction with the RAT assay. The α-amanitin treatment does not interfere with lncRNA/DNA interaction.
Mentions: As several lncRNA molecules regulate gene function by directly binding to their target chromatin DNAs (28–30,49,50), we determined if IRAIN lncRNA bound to IGF1R chromatin DNA, particularly the promoter region. We developed a ‘RAT’ assay that overcomes the high noise to signal background of existing methodologies to detect RNA/DNA interactions (Figure 4A). Cells were treated with formaldehyde to fix the structure of the lncRNA-chromatin conformations. The chromatin DNA-interacting lncRNA was then reverse transcribed into cDNA using strand-specific oligonucleotides and a thermo-stable reverse transcriptase in the presence of biotin-dCTP. After digestion with restriction enzyme or sonication to shear chromatin DNAs, the biotinylated IRAIN cDNA–DNA complex was then separated from other DNA–DNA products by streptavidin pull-down using paramagnetic Dynabeads (Dynal, Invitrogen) and analyzed by PCR using interacting DNA-specific primers.

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