Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme of the pentose phosphate pathway, the major metabolic pathway for generation of reducing equivalents for the cell. In an oxidation- reduction reaction, G6PD oxidizes glucose-6-phosphate and transfers the electrons to nicotinamide adenine dinucleotide phosphate (NADP+) to generate NADPH. NADPH provides reducing power for biosynthetic pathways that generate macromolecules such as synthesis of nucleic and fatty acids in the liver or the mammary glands. NADPH is also essential in maintaining antioxidant capacity of the cell. G6PD is normally considered to be a cytoplasmic enzyme where its activity is regulated by NADPH/NADP+ ratio. Increased G6PD activity has been reported for many diseases ranging from cardiomyopathy to cancers. We have recently discovered that NADPH, but not NADP+, NADH or NAD+, is an allosteric activator of class I histone deacetylases (HDACs) 1 and 2 in vitro. NADPH activates HDACs through a mixed activation kinetic, increasing the affinity of the HDAC enzyme for its histone substrate as well as the velocity of the deacetylation reaction. To provide in vivo support for our findings, we surmised that pharmacological inhibition of G6PD inside cells should lead to decreased NADPH levels and in turn to decreased HDAC activity and increased histone acetylation. Indeed, we observed that G6PD inhibition in MDA-MB-453 mammary cancer cells increases global histone acetylation by up to three fold. Another breast cancer cell lines, MDA-MB-231, did not show the same response to G6PD inhibition. Further investigation revealed that G6PD is highly expressed in MDA-MB-453 cells compared to MDA-MB-231 and that a detectable fraction of G6PD is localized in the nucleus with a further fraction strikingly bound to chromatin. In the 80 years since G6PD was discovered, only two reports indicate a potential presence of G6PD in the nucleus and none on chromatin. Moreover, our preliminary ChIP-seq analysis has revealed that G6PD binding to chromatin is not spurious but rather near genes with functions related to calcium homeostasis-an important pathway in breast cancer biology. In this application, we propose that G6PD binding to chromatin reveals an additional and significant function of G6PD that has not been characterized to any extent. We further postulate that G6PD may function at specific genomic loci to produce NADPH locally for HDAC activation and histone deacetylation. We now aim to determine the distribution of G6PD across the genome and its functional relevance to HDAC binding, histone acetylation and regulation of chromatin based processes such as gene expression. We believe our data will open up a new field of inquiry into the function of this critical metabolic enzyme and will uncover a remarkable juncture where metabolism and epigenetic regulation of the genome intersect.
(lay abstract 2-3 sentences) Glucose-6-phosphate dehydrogenase (G6PD) is an important metabolic enzyme which generates a critical molecule required for cell growth and many other processes including antioxidant pathways. G6PD is normally found in the cytoplasmic compartment of cells but we have found that surprisingly in breast cancer cells it is also found in the nucleus and bound to chromatin near genes that play important roles in cancer progression. In this application, we propose to study how G6PD may regulate the expression of genes that may contribute to breast cancer biology.