Appropriate diversification of cellular lineages from multi-potent progenitors is essential for normal development and homeostasis. The specification of erythroid and megakaryocytic lineages represents an especially vital developmental event whose molecular regulation remains incompletely defined. We now demonstrate the role of Rgs18, a GTPase-activating protein and transcriptional target of the repressor Gfi1b, in regulating these processes in mouse and human cells. Gfi1b stringently represses Rgs18 expression in erythroid cells, whereas, during megakaryocytic differentiation, declining Gfi1b levels facilitate a robust induction of Rgs18. Concordantly, alterations in Rgs18 expression produce disparate outcomes by augmenting megakaryocytic and potently suppressing erythroid differentiation and vice versa. These phenotypes reflect the differential impact of Rgs18 on signaling through p38 MAPK family proteins, and ERK1 and ERK2 (also known as MAPK3 and MAPK1, respectively) in the two lineages, which in turn alter the balance between the mutually antagonistic transcription factors Fli1 and Klf1. Overall, these results identify Rgs18 as a new and crucial effector of Gfi1b that regulates downstream signaling and gene expression programs to orchestrate erythro-megakaryocytic lineage choices. This dual role of Rgs18 in reciprocally regulating divergent lineages could exemplify generic mechanisms characteristic of multiple family members in different contexts.
Appropriate diversification of cellular lineages from multi-potent progenitors is essential for normal development and homeostasis. The specification of erythroid and megakaryocytic lineages represents an especially vital developmental event whose molecular regulation remains incompletely defined. We now demonstrate the role of Rgs18, a GTPase-activating protein and transcriptional target of the repressor Gfi1b, in regulating these processes in mouse and human cells. Gfi1b stringently represses Rgs18 expression in erythroid cells, whereas, during megakaryocytic differentiation, declining Gfi1b levels facilitate a robust induction of Rgs18. Concordantly, alterations in Rgs18 expression produce disparate outcomes by augmenting megakaryocytic and potently suppressing erythroid differentiation and vice versa. These phenotypes reflect the differential impact of Rgs18 on signaling through p38 MAPK family proteins, and ERK1 and ERK2 (also known as MAPK3 and MAPK1, respectively) in the two lineages, which in turn alter the balance between the mutually antagonistic transcription factors Fli1 and Klf1. Overall, these results identify Rgs18 as a new and crucial effector of Gfi1b that regulates downstream signaling and gene expression programs to orchestrate erythro-megakaryocytic lineage choices. This dual role of Rgs18 in reciprocally regulating divergent lineages could exemplify generic mechanisms characteristic of multiple family members in different contexts.
Introduction: Appropriate diversification of hematopoietic lineages from multi-potent progenitors is essential for normal development and health. The molecular programs that govern the divergence of erythroid and megakaryocytic lineages remain incompletely defined. Gene targeting experiments have shown the transcriptional repressor Gfi1b (Growth factor independence 1b) to be essential for erythro-megakaryocyte lineage development. Transcriptional repression of Gfi1b target genes is mediated by the cofactors LSD (lysine demethylase) 1 and Rcor (CoREST) 1. To understand the mechanism of Gfi1b action, its target genes were identified by chromatin immunoprecipitation (ChIP on Chip) screens. Three members of the Rgs (Regulator of G protein signaling) family were prominently represented in this target gene pool. In this study we present the role of Rgs18, a GTPase activating protein (GAP), in modulating erythro-megakaryocytic lineage divergence in hematopoietic progenitors. The results presented below demonstrate Rgs18 as a key arbitrator of this process in murine and human contexts. Approach: Following identification of Rgs18 as a potential Gfi1b and LSD1 target, its regulation by these factors was ascertained in erythro-megakaryocytic cells. Subsequently, to interrogate the role of Rgs18 in erythro-megakaryocyte differentiation, cDNA and shRNA mediated manipulations were performed in primary hematopoietic progenitors and cell lines, and the resulting phenotypes were analyzed. Finally, to trace the underlying mechanistic alterations responsible for these phenotypes the status of two branches of the MAPK (mitogen activated protein kinase) pathway and gene expression patterns of the mutually antagonistic transcription factors Fli1 (Friend leukemia integration [site] 1/Klf1 (Krupple like factor 1) were determined in Rgs18 manipulated cells. Result: Rgs18 expression was found to be low in immature megakaryoblasts in keeping with strong Gfi1b and LSD1 expression, but was reciprocally upregulated in mature megakaryocytes following declining Gfi1b and LSD1 levels in cells and on the rgs18 promoter. In contrast, expression of Gfi1b was strong in immature erythroid cells and increased further in mature cells, while Rgs18 expression which was modest in immature erythroid cells exhibited a reciprocal decline during maturation. Manipulation of Rgs18 expression in murine hematopoietic progenitors and a bipotential human cell line produced divergent outcomes, with expression augmenting megakaryocytic, and potently suppressing erythroid differentiation and vice versa. These phenotypes resulted from differential impact of Rgs18 expression on the P38 and ERK branches of MAPK signaling in the erythroid and megakaryocytic lineages. Repercussions of these signaling changes impacted relative expression of the mutually antagonistic transcription factors Fli1 and Klf1 and were compensated by ectopic Fli1 expression demonstrating activity of this transcription factor downstream of Rgs18. Conclusion: These results identify Rgs18 as a critical downstream effector of Gfi1b and an upstream regulator of MAPK signaling and Klf1/Fli1 gene expression. Sustained Gfi1b expression during erythroid differentiation represses Rgs18 and limits megakaryocytic gene expression. However during progression of megakaryocytic differentiation, declining Gfi1b levels results in robust expression of Rgs18 and lineage progression. Overall, this study provides new perspectives on lineage determination by highlighting multi-tier interactions between transcriptional and signaling networks in orchestrating hematopoietic lineage divergence. These insights could exemplify generic mechanisms exhibited by this large family of signal modulators in mediating lineage diversification in various contexts. Disclosures No relevant conflicts of interest to declare.
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