A Genome-Wide RNA Interference Screen Identifies a Differential Role of the Mediator CDK8 Module Subunits for GATA/ RUNX-Activated Transcription in Drosophila

Abstract

Transcription factors of the RUNX and GATA families play key roles in the control of cell fate choice and differentiation, notably in the hematopoietic system. During Drosophila hematopoiesis, the RUNX factor Lozenge and the GATA factor Serpent cooperate to induce crystal cell differentiation. We used Serpent/ Lozenge-activated transcription as a paradigm to identify modulators of GATA/RUNX activity by a genomewide RNA interference screen in cultured Drosophila blood cells. Among the 129 factors identified, several belong to the Mediator complex. Mediator is organized in three modules plus a regulatory "CDK8 module," composed of Med12, Med13, CycC, and Cdk8, which has long been thought to behave as a single functional entity. Interestingly, our data demonstrate that Med12 and Med13 but not CycC or Cdk8 are essential for Serpent/Lozenge-induced transactivation in cell culture. Furthermore, our in vivo analysis of crystal cell development show that, while the four CDK8 module subunits control the emergence and the proliferation of this lineage, only Med12 and Med13 regulate its differentiation. We thus propose that Med12/Med13 acts as a coactivator for Serpent/Lozenge during crystal cell differentiation independently of CycC/Cdk8. More generally, we suggest that the set of conserved factors identified herein may regulate GATA/RUNX activity in mammals.During hematopoiesis, multipotent progenitors or stem cells generate a large spectrum of specialized cell types through the progressive deployment of cell-specific gene expression programs (59). In that respect, it is of particular interest to understand how combinatorial inputs from general and lineage-specific transcription factors converge to modulate RNA polymerase II machinery and establish the gene expression programs intrinsic to cell diversification. Over the last decade, cross-species conservation has shown that Drosophila is a valuable model system to gain insights into the mechanisms controlling blood cell fate choice and differentiation at the transcriptional level (36). Indeed, several key transcription factors and cofactors regulating blood cell development in vertebrates also participate in hematopoiesis in Drosophila.Reminiscent of the situation in vertebrates, hematopoiesis in the fruit fly occurs in two temporally and spatially distinct waves: blood cell progenitors (prohemocytes) arise first from the head mesoderm in the early embryo and then from a specialized organ, the lymph gland, in the larva (36). These progenitors differentiate into two main cell types, most closely related to vertebrate myeloid lineages: plasmatocytes, which function as macrophages, and crystal cells, which participate in melanization and clotting (53). So far, we have a better understanding of the transcriptional network controlling blood cell development in the embryo. In particular, it appears that transcription factors of the GATA and RUNX families, which regulate several steps of hematopoiesis in vertebrates, act together to control Drosophila blood cell fate choice and...

Dual role of Med12 in PRC1-dependent gene repression and ncRNA-mediated transcriptional activation

“…In particular, studies in the fruit fly suggested a functional separation of Cdk8 and Med12 in regulating key developmental genes. 37,62 In agreement with these findings our study suggests that Med12 fulfills an important role in silencing developmental genes together with PRC1. Notably, apart from its repressive function, Med12 also keeps the ES self-renewal network active.…”

The Metazoan-Specific Mediator Subunit 26 (Med26) Is Essential for Viability and Is Found at both Active Genes and Pericentric Heterochromatin in Drosophila melanogaster

“…This result contrasts previous results examining the kinase module that found Med13 was stable upon loss of Med12 (16,17). To confirm our findings, we repeated the experiment with dsRNA directed against both Med12 and Med13.…”

“…This contrasts the findings in Drosophila Kc cells (16) and in imaginal discs in vivo (17) where Med13 is stable upon loss of Med12. One possible explanation is the presence of a gene product that can substitute for Med12 in Kc cells and imaginal discs that is not present in S2 cells.…”

“…The subunits of the module are essential for organismal viability but not for cellular growth and proliferation (13,14). More recent work indicates that there are distinct and separable roles for each of the subunits of the Cdk8 module in Drosophila development (15)(16)(17)(18)(19).…”

Myeloid leukemia factor

“…22 Third, in a genome wide RNAi screen, dMLF scored as a potential co-activator of LZ. 23 Further analysis revealed that mlf participates in a positive feedback loop to control the activity of LZ. 24 Indeed, dMLF expression is activated in the crystal cell lineage by LZ and required for stabilizing LZ, thereby contributing to the fine-tuning of crystal cell number.…”