Trichostatin A and Structurally Related Histone Deacetylase Inhibitors Induce 5-Lipoxygenase Promoter Activity

Abstract

5-Lipoxygenase (5-LO) mRNA expression in Mono Mac 6 cells is induced by the histone deacetylase inhibitor trichostatin A (TsA). In order to study the effects of TsA and several structurally related compounds such as MD85, D237 and M232 on 5-LO promoter activity, we have analyzed the response of a 5-lipoxygenase (5-LO) promoter luciferase reporter gene construct to histone deacetylase inhibitors in transiently transfected Mono Mac 6 and HeLa cells. We show that the activity of 5-LO promoter constructs comprising the sequences -778 to and of several successive deletions of the 5-LO promoter is strongly increased upon TsA treatment. The data suggest a significant involvement of histone deacetylases in the regulation of 5-LO gene transcription. The basal activity of the 5-LO promoter strongly depends on the presence of multiple Sp1-binding sites (GC-boxes), five of which are positioned in tandem. Deletion of the five tandemized GC-boxes in the 5-LO reporter gene construct revealed that the induction of 5-LO promoter activity by TsA seems to be independent of these GC-boxes. Methylation of 5-LO reporter gene constructs by M.Hpall reduced 5-LO promoter activity but did not prevent induction of promoter activity by TsA, although the activated reporter gene activities were lower compared to the unmethylated plasmid, indicating the dominance of methylation over TsA-sensitive histone deacetylation in silencing of the 5-LO gene. The structure-activity data obtained for histone deacetylase inhibitors suggest that this assay system might serve as a cellular screening tool for the development of HDAC inhibitors.

Calcitriol Upregulates Open Chromatin and Elongation Markers at Functional Vitamin D Response Elements in the Distal Part of the 5-Lipoxygenase Gene

“…The reporter gene constructs containing the 5-LO promoter are derived from pN10, a 5-LO promoter fragment containing the sequence − 778 to +53 (relative to the transcriptional start site), described in Klan et al 39 5-LO cds constructs are derived from pN10cds where the 5-LO cds was inserted into pN10 after PCR amplification as described in Seuter et al 11 Intron-containing plasmids were cloned from pN10cdsInJM, which was generated by PCR amplification of genomic DNA from MM6 cells. For detailed description, see Seuter et al 11 pN10LTA4Hcds was generated by opening pN10 with HindIII and NcoI inserting an oligo with HindIII overhang and the AflIII PCR fragment of the LTA4Hcds.…”

5-Lipoxygenase is a direct p53 target gene in humans

“…Furthermore, we found that 5-LO can inhibit the transcriptional activity of p53 36 suggesting that 5-LO acts in a negative feedback loop to limit induction of p53 target genes. 37 1. Introduction 43 p53 was first discovered in 1979 independently by several research 44 groups as an interaction partner of the oncoprotein large T antigen of 45 the simian virus 40 (SV40) and represents a tumor suppressor [1][2][3][4].…”

New Insights to Nuclear Receptor Gene Regulation from Analysis of their Response Elements in Target Genes

“…One of these REs is located far downstream of the TSS (+42 kB) and is one of the strongest known REs of the human genome [58]. No functional RE had been reported for 5-LO before, since previous studies had been restricted to the proximal promoter region [59,60]. Therefore, this study revealed candidate REs that are located more than 30 kB distant from their target gene's TSS.…”