PGC-1 Coactivator Activity Is Required for Murine Erythropoiesis

The LSD1 inhibitor RN-1 induces fetal hemoglobin synthesis and reduces disease pathology in sickle cell mice

“…34,35 RBCs were labeled in vivo using N-hydroxysuccinimide biotin (Pierce), [36][37][38] and the lifespan of circulating RBCs was measured as described in the supplemental Methods, available on the Blood Web site. …”

“…Synthesis of complementary DNA and quantitative real-time polymerase chain reaction (QRT-PCR) was performed as described. 35 mRNA abundance of target genes was determined based on cycle threshold (C T ) values of the QRT-PCR and then normalized to the abundance of Ornithine decarboxylase antizyme (Oaz1) mRNA as the internal control. 39 Bone marrow (BM) cells were isolated from untreated and drug-treated SCD mice for chromatin immunoprecipitation (ChIP) assays as previously described.…”

“…Tissues from these organs were fixed, embedded, sectioned, and stained with hematoxylin-eosin (H&E). 34,35 Semiquantification of histologic changes was analyzed as described in the supplemental Methods.…”

“…Protein extracts from control or RN-1-treated murine erythroleukemia (MEL) cells or SCD mice BM cells were subjected to western blotting as described, 35 using the same antibodies against LSD1, H3K4me2, H3K9me2, and H3, as well as antibodies against PGC-1a and b-actin (Santa Cruz Biotechnology).…”

“…48 We recently showed that PGC-1a activity is required for both murine erythropoiesis as well as for globin gene regulation. 35 To determine whether the induction of HbF in SCD mice by RN-1 treatment might be mediated by PGC-1a, we examined the expression of PGC-1a in SCD mice treated (or not) with RN-1. We discovered that the loss of LSD1 after RN-1 treatment in SCD mice increased H3K4me2 levels at the PGC-1a promoter (supplemental Figure 8A) and induced the expression of PGC-1a in a dose-dependent manner ( Figure 7A-B), suggesting that elevated PGC-1a expression might contribute to the induction of the g-globin gene in these mice.…”