DNA·RNA triple helix formation can function as a cis-acting regulatory mechanism at the human β-globin locus

Significance

RNA containing only pyrimidines, transcribed from genomic DNA, can form DNA∙RNA triple helices with other sites in the genome. We characterize an intronic sequence in the human β-globin gene that forms a triplex with an upstream regulatory site in the β-globin locus, causing displacement of transcription factors and down regulation of expression of the genes in the locus. Consequently, overexpression of a full-length β-globin transcript containing the intron results in decreased expression of members of the β-globin gene cluster. This is a cis-regulatory feedback mechanism in which overexpression of the β-globin gene could result in signals to down-regulate that expression. Balance in the expression of the many genes associated with hemoglobin biosynthesis is essential for erythroid cell function.

Abstract

We have identified regulatory mechanisms in which an RNA transcript forms a DNA duplex·RNA triple helix with a gene or one of its regulatory elements, suggesting potential auto-regulatory mechanisms in vivo. We describe an interaction at the human β-globin locus, in which an RNA segment embedded in the second intron of the β-globin gene forms a DNA·RNA triplex with the HS2 sequence within the β-globin locus control region, a major regulator of globin expression. We show in human K562 cells that the triplex is stable in vivo. Its formation causes displacement from HS2 of major transcription factors and RNA Polymerase II, and consequently in loss of factors and polymerase that bind to the human ε- and γ-globin promoters, which are activated by HS2 in K562 cells. This results in reduced expression of these genes. These effects are observed when a small length of triplex-forming RNA is introduced into cells, or when a full-length intron-containing human β-globin transcript is expressed. Related results are obtained in human umbilical cord blood-derived erythroid progenitor-2 cells, in which β-globin expression is similarly affected by triplex formation. These results suggest a model in which RNAs conforming to the strict sequence rules for DNA·RNA triplex formation may participate in feedback regulation of genes in cis.