Regulation of the Interleukin-6 gene expression during monocytic differentiation of HL-60 cells by chromatin remodeling and methylation.

ABSTRACT

The pro-inflammatory cytokine Interleukin (IL)-6 is involved in the proliferation and differentiation of leukocytes and non-immune cells, but its overproduction is associated with inflammatory and autoimmune disorders. The main producers of IL-6 are mature monocytes, whereas progenitor cells and the promyeloid cell line HL-60 do not synthesize IL-6. In contrast, HL-60 cells differentiated into monocytic cells were able to express IL-6 after lipopolysaccharide (LPS) stimulation. This study investigated the chromatin structure of the IL-6 promoter and the effect of methylation on IL-6 gene regulation during monopoiesis. The results show that the proximal IL-6 promoter regions I to III (+13/-329) were inaccessible in undifferentiated HL-60 cells but became significantly accessible in differentiated HL-60 cells stimulated with LPS. Region IL-6 VI (-1099/-1142) remained closed, but the upstream region IL-6 VII (-2564/-2877) relaxed after differentiation and LPS treatment. The opening of IL-6 IV (-309/-521) and IL-6V (-500/-722), containing DNA and histone methylation sites, was differentiation-dependent only. Demethylation experiments using 5-aza-2'-deoxycytidine (AZA) followed by LPS stimulation revealed a significant enhanced IL-6 mRNA expression and protein release by HL-60 cells. AZA treatment resulted in significant increased IL-6 promoter accessibilities, identifying methylation as an important repressor of IL-6 gene regulation in promyeloid cells. The histone deacetylase (HDAC) inhibitor trichostatin A (TSA) had no effect on IL-6 promoter accessibility. Our data indicate that during monopoiesis the proximal IL-6 promoter is reorganized into an accessible conformation allowing transcription of IL-6 after LPS stimulation. DNA methylation appears to be the essential epigenetic mechanism in IL-6 gene expression of mature monocytes and their progenitors by controlling the chromatin structure.