Analysis of proximalALOX5promoter binding proteins by quantitative proteomics

Abstract

5-Lipoxygenase (5-LO) is the initial enzyme in the biosynthesis of leukotrienes, which are mediators involved in pathophysiological conditions such as asthma and certain cancer types. Knowledge of proteins involved in 5-LO pathway regulation, including gene regulatory proteins, is needed to evaluate all options for therapeutic intervention in these diseases. Here, we present a mass spectrometric screening ofALOX5promoter-interacting proteins, obtained by DNA pulldown and label-free quantitative mass spectrometry. Protein preparations from myeloid and B-lymphocytic cell lines were screened for promoter DNA interactors. Through statistical analysis, 66 proteins were identified as specificALOX5promotor binding proteins. Among those, the 15 most likely candidates for a prominent role inALOX5gene regulation are the knownALOX5interactors Sp1 and Sp3, the related factor Sp2, two Kruppel-like factors (KLF13 and KLF16) and six other zinc finger proteins (MAZ, PRDM10, VEZF1, ZBTB7A, ZNF281 and ZNF579). Intriguingly, we also identified two helicases (BLM and DHX36) and the proteins hnRNPD and hnRNPK, which are, together with the protein MAZ, known to interact with DNA G-quadruplex structures. As G-quadruplexes are implicated in gene regulation, spectroscopic and antibody-based methods were used to confirm their presence within the GC-rich sequence of theALOX5promoter. In summary, we have systematically characterized the interactome of theALOX5promoter, identifying several zinc finger proteins as novel potentialALOX5gene regulators. Further, we have shown that theALOX5promoter can form DNA G-quadruplex structures, which may play a functional role inALOX5gene regulation.