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.
Highlights
Growth factor independence (Gfi)1 and Gfi1b are homologous zinc finger transcriptional repressors that perform critical and essential functions in multiple developmental processes and stages
The rationale for the screen being that the common targets of Gfi1b, LSD1 and Rcor1 should be transcriptionally derepressed in LSD1 deficient cells relative to controls, given that LSD1 functions as a transcriptional repressor in the context of Gfi1b and CoREST [27]
Meis1 was neither expressed in control, nor upregulated upon LSD1 depletion, in the closely related megakaryocytic lineage, L8057 cells. Given that both lineages express high levels of Gfi1b, LSD1 and CoREST (Figure 1c) and that gfi1b itself is upregulated upon LSD1 inhibition in both lineages, the meis1 promoter appears to be targeted for differential regulation by Gfi1b and its co-factors in these closely related lineages
Summary
Growth factor independence (Gfi) and Gfi1b are homologous zinc finger transcriptional repressors that perform critical and essential functions in multiple developmental processes and stages. Gfi is required for maintaining stem cell homeostasis in the bone marrow [1,2], generating neutrophils [3,4], and ensuring proper development and maturation of other innate and adaptive lymphoid cells [5,6,7]. Gfi exhibits major oncogenic potential and has been associated with both murine and human cancers [9,13,14,15]. Over expression of Gfi co-operatively accelerates the rate of lymphomagenesis in collaboration with the oncogenes c-myc or pim1 [16]. Similar results have been reported for Gfi1b [17]. Recent reports suggest a probable connection between both factors and chronic (CML) and acute (AML) myeloid leukemias [18,19]
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