G-Quadruplex Forming Sequences Found In The Beta-Globin Gene Cluster Regulate Erythroid Differentiation Of K562 Cells

Guanine-rich DNA sequences can form stable four stranded structures by folding of the DNA strands, forming stacks of G-tetrads called G-quadruplex. G-quadruplex forming sequences are found in eukaryotic telomeres, in promoter regions and in noncoding regions of the genome. Although the function of these structures has not yet been identified it has been suggested that they play a negative regulatory role at the transcription level. The most studied G-quadruplex sequences (AS1411 and PU27) show activity in reducing growth and invasiveness of malignant cells, as well as increasing cell death of a large array of tumors with promising therapeutic applications. We identified two G-quadruplex forming sequences in the human beta globin cluster (Hbd and Hbg2). We confirmed quadruplex formation by these sequences using circular dichroism spectroscopy. We also observed in Southern blot assay that these oligo-sequences bind to specific β-globin gene fragments after EcoRI digestion. Finally we demonstrate that neither Hbd nor Hbg2 (5 or 10µM) were growth inhibitory to four different leukemia cell lines after 6 days culture measured by MTT.

The β-globin cluster contains 5 functional globin-like genes and long range regulator elements, the expression of each of these genes is tightly regulated during development (from embryo to fetus to adult), in cell type and in the course of differentiation/maturation of the erythroid lineage. Recent studies have demonstrated the importance of folding and looping of the DNA in the control of globin genes expression. We hypothesized that the G-quadruplex forming sequences within the β-globin gene cluster participate in regulation and splicing of the different globin genes during erythroid maturation. The erythroleukemia cell line K562 was used in this study because of its ability to differentiate into erythrocytes under Hemin stimulation. K562 cells were divided into 6 groups of treatment and cultured for 3 days in the presence or absence of Hemin (30µM) with or without either Hbd or Hbg2 oligonucleotides (5µM). Erythroid differentiation was evaluated by quantitative QRT-PCT for the expression of β-, γ- and ε-globin genes as well as α-globin and by flow-cytometry for the presence of β-, γ- and ε-globin; α-globin and CD235a (Glycophorin A) were used as controls. The data reveal a difference in gene expression for all globin genes when K562 were exposed to Hbd or Hbg2 only, interestingly, a significant up-regulation of β-globin was observed only in Hbg2 treated cells. Similarly in K562 induced to differentiate with Hemin show up-regulation of γ-, ε- and α- globin, the addition of Hbd or Hbg2 oligonucleotides to Hemin at the beginning of the culture slightly improve the expression of γ- and ε- however there was no difference for the expression of β- globin in the hemin +/- Hb oligonucleotides treated cells compared to control untreated cells. Flow-cytometry experiments confirmed the increase in the expression of each globin gene in all treatments compared to control. Altogether the data suggest a role for Hbd and Hbg2 in the regulation of β-globin gene transcription and therefore in the differentiation of K562 in erythroid lineage.

The implications of our findings are multiple. 1) the presence of G-quadruplex sequences in a gene complex not involved in growth regulation or oncogenesis; 2) to our knowledge this is the first time that G-quadruplex sequences are shown to specifically alter the expression of the gene in which they are located; and 3) the potential use of globin G-quadruplex to modify the globin gene expression therapeutically.

No relevant conflicts of interest to declare.