Butyrate Downregulates the Expression of a Number of Repressors of Fetal Hemoglobin In Definitive Erythroid Cells

Elevated fetal hemoglobin (HbF) is ameliorative for beta-globin gene disorders. Butyrate, a short chain fatty acid, is a potent inducer of fetal hemoglobin with limited clinical applicability. We wanted to examine non-globin gene targets of butyrate that are regulated in definitive erythroid cells prior to the induction of embryonic/fetal beta-type globin genes. Mechanistic insights may improve clinical utility for short chain fatty acids by identifying novel molecular therapeutic targets.

Induced embryonic/fetal globin gene expression is detectable in murine fetal liver-derived definitive erythroid cells (FL EryD) from wildtype and human beta-globin YAC transgenic mice after 19 hours in culture with butyrate & erythropoietin (EPO), but not in EPO alone. Differential regulation of non-globin gene targets in wildtype FL EryD was studied on a Mouse Gene 1.0ST Affymetrix Array after culture in EPO only or butyrate & EPO at 6 hours (when no embryonic globin gene expression is detectable, n=3). Data from biological replicates were normalized by robust multichip average and analyzed with expression console software. As shown in Table 1, several confirmed and putative repressors of embryonic/fetal beta-type globin gene expression, including SOX6, Bcl11A, and Ikaros 1 (but not cMyb) were significantly down regulated by Butyrate at 6 hours (n=3); this was confirmed by RT-PCR. The histone deacetylase inhibitor trichostatin A (TSA), which also induces embryonic globin gene expression in murine FL EryD, has a directionally similar effect (Table 1).

Down regulation of some fetal/embryonic globin gene repressors, relative to identically handled EPO-only treated samples, was detectable by RT-PCR as early as 60 to 120 minutes after butyrate induction. These repressors included Bcl11A (60min: 0.66±0.005, p<.001, n=2; 120min: 0.4±0.24, p<.01, n=4), Sox6 (60min: 0.55± 0.18, p=0.08, n=2; 120min: 0.63± 0.06, p<.001, n=4) and Ikaros1 (60min: 0.63±0.45, p=0.36, n=2; 120min: 0.42±0.15, p<.001, n=4).

The proximate molecular mechanisms through which butyrate act, while unknown in detail, have been posited to include ‘stress’ signaling via p38 and/or direct activation of gamma-globin gene expression through inhibited histone deacetylation. We found no evidence for butyrate-mediated enhancement of p38 phosphorylation in FL EryD at 0–120 minutes in culture. However, bulk histone acetylation measured by western for histone 3 (H3), was >1.5 fold greater with butyrate induction at 60–90 minutes relative to baseline, while less than baseline in EPO-only treated FL EryD (n=2).

Cumulatively, these data suggest that the down regulation by butyrate of major molecular repressors of embryonic/fetal globin gene expression, likely mediated directly or indirectly through epigenetic modifications, is a key underlying mechanism for the induction of fetal hemoglobin in definitive erythroid cells by short chain fatty acids.

No relevant conflicts of interest to declare.