Gene-Selective Histone Hyperacetylation and Enhanced Sp1 Occupancy Underpin Transcriptional Modulation of Genes of the Glycolytic-Pentose Phosphate Pathway in Response to Histone Deacetylase Inhibitors - Therapeutic Implications
Authors: Makarona Kalliopi , Caputo Valentina , Roper David , Costa Joana , Cowan Gillian , Chaidos Aristeidis , O'Connor David , Georgiou Elisabeth , Papaioannou Maria , Layton Mark , Roberts Irene , Karadimitris Anastasios 
Published: October 2019

Abstract
Abstract 977
Transcriptionally active genes, including housekeeping genes, are characterised by co-occupancy and antagonistic actions of histone deacetylases (HDAC) and acetyltransferases (HAT). Transcription is facilitated through the prevailing action of HAT, which maintain histone acetylation. HDAC inhibitors (HDACi) induce widespread histone hyperacetylation and as a consequence are expected to increase expression of transcriptionally active genes. We have previously demonstrated this effect in inherited glycosylphosphatidylinositol (GPI-anchor) deficiency, an autosomal recessive disorder characterised by histone hypoacetylation and transcriptional repression of PIGM due to an in cis mutation which disrupts binding of the transcription factor Sp1 to its core promoter cognate motif. We surmised HDACi-mediated hyperacetylation might lead to increased transcription of other housekeeping genes, such as those of the anaerobic glycolytic and pentose phosphate pathways (GPPP) disruption of which is ameliorated by relatively modest increases in enzymatic activity. HDACi could therefore be of therapeutic value in these disorders.
To address these hypotheses, EBV B cell lines were treated with the HDACi sodium butyrate (NaBu; 3mM) and mRNA levels for GPPP genes assessed by RQ-PCR. Of 9 genes tested (glucose-6-phosphate dehydrogenase, G6PD; glucose-6-phosphate isomerase, GPI; triosephosphate isomerase, TPI; pyruvate kinase, PK; 6-phosphogluconolactonase, PGLS; phosphogluconate dehydrogenase, PGD; ribulose-5-phosphate-3-epimerase, RPE; ribose-5-phosphate isomerase A, RPIA; transketolase, TKT), only G6PD mRNA levels increased, in a time-dependent fashion, in response to NaBu (n=3; p<0.01), an effect that was also observed in B cells from a patient with G6PD Brighton a severe, Class I G6PD deficiency (n=3; p<0.01). The increase in G6PD mRNA was observed within 4hrs post NaBu exposure and was not abrogated by the protein synthesis inhibitor cycloheximide suggesting a direct effect of NaBu on G6PD transcription. G6PD protein and enzymatic activity increased commensurately with G6PD mRNA level in both normal (n=4; p<0.01) and G6PD Brighton (n=4; p<0.01) B cells. In G6PD deficient B cells, enzymatic activity was restored to normal levels within 24hrs of treatment with HDACi (n=3; p<0.01). The selective effect of HDACi on transcription of G6PD but not other GPPP genes was also observed in other cell types, including 293T cells, and primary CD36+CD71+ erythroblasts generated from normal cord blood CD34+ cells. Notably, in NaBu-treated (1mM) primary erythroblasts a 2.3-fold increase in G6PD mRNA (n=3; p<0.01) accompanied by a 2.5-fold increase of G6PD protein levels (n=3; p<0.05) and 2.6-fold increase in enzymatic activity (n=3; p<0.05) were observed.
The epigenetic correlates of G6PD mRNA induction were assessed by ChIP-Q-PCR. This revealed a dynamic, time-dependent, 3 to 4-fold increase in levels of histone 3 and 4 acetylation and a 4-fold increase in Sp1 and Polymerase II occupancy in the promoter of G6PD but not TPI or GPI. Preliminary pharmacological and shRNA experiments suggest that HDACi-mediated transcriptional upregulation of G6PD is Sp1-dependent. No differences were observed in baseline levels of histone acetylation or Sp1 occupancy of G6PD, TPI and GPI implying a yet to be defined cis acting determinant is required for the selective increase in Sp1 binding and histone hyperacetylation.
Finally, in erythroblasts generated from peripheral blood mononuclear cells of two patients with Class I G6PD deficiency (G6PD Brighton and G6PD Harilaou), we confirmed the ability of NaBu to increase mutant G6PD mRNA and protein levels leading to increased G6PD enzymatic activity and its restoration to normal within 24hrs.
In conclusion, we show that even within the same metabolic pathway, transcriptional upregulation of active genes in response to HDACi is selective rather than universal and is underpinned by enhanced Sp1 binding and histone hyperacetylation. Our findings raise the prospect of using HDACi to treat severe Class I G6PD deficiency.

Disclosures:
No relevant conflicts of interest to declare.