Differential Transcriptional Regulation of meis1 by Gfi1b and Its Co-Factors LSD1 and CoREST

Abstract

Gfi1b (growth factor independence 1b) is a zinc finger transcription factor essential for development of the erythroid and megakaryocytic lineages. To elucidate the mechanism underlying Gfi1b function, potential downstream transcriptional targets were identified by chromatin immunoprecipitation and expression profiling approaches. The combination of these approaches revealed the oncogene meis1, which encodes a homeobox protein, as a direct and prominent target of Gfi1b. Examination of the meis1 promoter sequence revealed multiple Gfi1/1b consensus binding motifs. Distinct regions of the promoter were occupied by Gfi1b and its cofactors LSD1 and CoREST/Rcor1, in erythroid cells but not in the closely related megakaryocyte lineage. Accordingly, Meis1 was significantly upregulated in LSD1 inhibited erythroid cells, but not in megakaryocytes. This lineage specific upregulation in Meis1 expression was accompanied by a parallel increase in di-methyl histone3 lysine4 levels in the Meis1 promoter in LSD1 inhibited, erythroid cells. Meis1 was also substantially upregulated in gfi1b−/− fetal liver cells along with its transcriptional partners Pbx1 and several Hox messages. Elevated Meis1 message levels persisted in gfi1b mutant fetal liver cells differentiated along the erythroid lineage, relative to wild type. However, cells differentiated along the megakaryocytic lineage, exhibited no difference in Meis1 levels between controls and mutants. Transfection experiments further demonstrated specific repression of meis1 promoter driven reporters by wild type Gfi1b but neither by a SNAG domain mutant nor by a DNA binding deficient one, thus confirming direct functional regulation of this promoter by the Gfi1b transcriptional complex. Overall, our results demonstrate direct yet differential regulation of meis1 transcription by Gfi1b in distinct hematopoietic lineages thus revealing it to be a common, albeit lineage specific, target of both Gfi1b and its paralog Gfi1.

Antagonistic actions of Rcor proteins regulate LSD1 activity and cellular differentiation

“…MEL cells were cultured and differentiated as described previously (8,31 Mononucleosome Preparation and Demethylation Assays. Mononucleosomes were prepared as described previously (32).…”

“…Polyclonal anti-mouse rabbit serum was produced against the C-terminal 17 amino acids of Rcor3, affinity-purified, and used for Western blot analyses and ChIP. ChIP primers used were as described previously (8,31) and listed in SI Materials and Methods.…”

Reciprocal regulation of alternative lineages by Rgs18 and its transcriptional repressor Gfi1b

“…Examination of Rgs18 promoter sequences obtained from the mouse promoter arrays of these ChIP-on-chip screens (Chowdhury et al, 2013;Saleque et al, 2007) revealed the presence of quasi-consensus (AAATCT) and consensus (AAATCA) Gfi1-and Gfi1b-binding sites (Tong et al, 1998) in the 5′ untranslated region (UTR) and proximal protein coding regions of Rgs18, respectively (Fig. S1).…”

“…ChIP experiments were performed in MEL and L8057 cells as previously described (Chowdhury et al, 2013;Saleque et al, 2007) with anti-Gfi1b (Sc8559, Santa Cruz Biotechnology) and anti-LSD1 (ab17721, Abcam) antibodies. Primers used for qPCR amplification of ChIP DNA were: Rgs18 promoter (upstream), 5′-TCATTTCCTTCAACAATTCAGTAC-A-3′ and 5′-CGAATCTTTCCTCAGATTTTTCTTA-3′; Rgs18 promoter (downstream), 5′-ATGTGTGAATCAAAAGAGAAAACTTT-3′ and 5′-CACAGATATTCATCAATCATGCTACTT-3′; and Sµ, 5′-CTTGA-GCCAAAATGAAGTAGACTGT-3′ and 5′-ACAGTCCAGTGTAGG-CAGTAGAGTT-3′.…”

Transcriptional Regulation of Platelet Formation: Harnessing the Complexity for Efficient Platelet Production In Vitro

“…GFI1B and its cofactors lysine (K)-specific demethylase 1A (LSD1, encoded by the Kdm1A gene) and rest corepressor 1 (RCOR1) have been identified in ChIP-Seq studies as key regulators of Meis1 [189]. The evidence that GFI1B plays a role in MK maturation has been recently further exemplified with the identification of a mutation creating a dominant-negative form of GFI1B leading to an autosomal dominant form of gray platelet syndrome [190].…”