A Shared Role for RBF1 and dCAP-D3 in the Regulation of Transcription with Consequences for Innate Immunity

et al. (2012) A Shared Role for RBF1 and dCAP-D3 in the Regulation of Transcription with Consequences for Innate Immunity. PLoS Genet 8(4): e1002618. doi:10.1371/journal.pgen.1002618 A Shared Role for RBF1 and dCAP-D3 in the Regulation of Transcription with Consequences for Innate Immunity Michelle S. Longworth James A. Walker Endre Anderssen Nam-Sung Moon Andrew Gladden Margarete M. S. Heck Sridhar Ramaswamy Nicholas J. Dyson Giovanni Bosco, Univeristy of Arizona, United States of America Previously, we discovered a conserved interaction between RB proteins and the Condensin II protein CAP-D3 that is important for ensuring uniform chromatin condensation during mitotic prophase. The Drosophila melanogaster homologs RBF1 and dCAP-D3 co-localize on non-dividing polytene chromatin, suggesting the existence of a shared, non-mitotic role for these two proteins. Here, we show that the absence of RBF1 and dCAP-D3 alters the expression of many of the same genes in larvae and adult flies. Strikingly, most of the genes affected by the loss of RBF1 and dCAP-D3 are not classic cell cycle genes but are developmentally regulated genes with tissue-specific functions and these genes tend to be located in gene clusters. Our data reveal that RBF1 and dCAP-D3 are needed in fat body cells to activate transcription of clusters of antimicrobial peptide (AMP) genes. AMPs are important for innate immunity, and loss of either dCAP-D3 or RBF1 regulation results in a decrease in the ability to clear bacteria. Interestingly, in the adult fat body, RBF1 and dCAP-D3 bind to regions flanking an AMP gene cluster both prior to and following bacterial infection. These results describe a novel, non-mitotic role for the RBF1 and dCAP-D3 proteins in activation of the Drosophila immune system and suggest dCAP-D3 has an important role at specific subsets of RBF1-dependent genes. - Funding: MSL was supported by a Leukemia and Lymphoma Society Career Development Fellowship Award, by a Charles King Trust Fellowship Award, and by Cleveland Clinic Foundation Seed funds. JAW was supported by a grant from the DOD (W81XWH-09-1-04871). NJD is the Saltonstall Scholar of the Massachusetts General Hospital Cancer Center. This study was supported by NIH grants GM53203 and CA64402 (to NJD) and CIHR grant MOP-93666 (to N-SM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The RB family proteins (pRB, p130 and p107 in humans; RBF1 and RBF2 in Drosophila) co-ordinate changes in gene expression. Understanding the types of programs that these proteins regulate is important because of the unequivocal link between the inactivation of RB proteins and human cancer. Mutation of the retinoblastoma tumor susceptibility gene (RB1) is the rate-limiting step in the genesis of retinoblastoma and over 90% of human tumors exhibit reduced pRB function [1,2]. RB family members are best-known for their roles in the regulation of E2F-dependent transcription. E2F-controlled genes are needed for cell proliferation and RB proteins suppress the expression of these targets during G0 and G1 of the cell cycle [3]. In addition, RB proteins are also important for the regulation of genes that are not involved in cell cycle progression. For example, osteoblast differentiation is modulated by pRB through its interaction with Runx2 [4]; in muscle cells, pRB promotes the expression of muscle-specific differentiation markers, enabling these cells to irreversibly exit the cell cycle [57]; in Drosophila, RBF1 cooperates with the Hippo pathway to maintain photoreceptor differentiation, independent of dE2F1 activity [8]. Such E2F-independent functions may help to explain why the inactivation of RB proteins can have very different consequences in different cellular contexts. However, many of the E2Findependent activities of RB proteins are not well-understood. At present, it is unclear if pRB has different activities in different cell types, or whether there is a yet-to-be discovered, general process that allows RB proteins to activate or repress the expression of variable sets of genes in different cell types. Recent studies have suggested that pRB family members may impact the organization of higher-order chromatin structures, in addition to their local effects on the promoters of individual genes [9]. Mutation of pRB causes defects in pericentric heterochromatin [10] and RBF1 is necessary for uniform chromatin condensation in proliferating tissues of Drosophila larvae [11]. Part of the explanation for these defects is that RBF1 and pRB promote the localization of the Condensin II complex protein, CAP-D3 to DNA both in Drosophila and human cells [11]. Depletion of pRB from human cells strongly reduces the level of CAP-D3 associated with centromeres during mitosis and causes centromere dysfunction [12]. Condensin complexes are necessary for the stable and uniform condensation of chromatin in early mitosis [1316]. They are The retinoblastoma protein (pRB) is a tumor suppressor protein known for its ability to repress transcription of E2Fdependent genes and induce cell cycle arrest. We have previously shown that RB proteins in Drosophila and human cells interact with the Condensin II subunit, CAPD3, in an E2F-independent manner. Condensins promote condensation of chomosomes in mitosis. Our previous studies suggested that the Drosophila pRB and CAP-D3 homologs, RBF1 and dCAP-D3, co-localize on DNA and may share a function in cells that never undergo mitosis. In this study, we show that one non-mitotic function shared between RBF1 and dCAP-D3 is the regulation of many non-cell-cycle-related, clustered, and cell-type-specific transcripts including a conserved family of genes that are important for the immune response in the fly. In fact, results show that normal levels of dCAP-D3 and RBF1 expression are necessary for the ability of the fly to clear infection with human bacterial pathogens. This work demonstrates that dCAP-D3 proteins can regulate a unique subset of RBF1-dependent transcripts in vivo and identifies a novel role for both RBF1 and dCAP-D3 protein in activation of innate immune genes, which may be conserved in human cells. conserved from bacteria to humans with at least two types of Condensin complexes (Condensin I and II) present in higher eukaryotes. Both Condensin I and II complexes contain heterodimers of SMC4 and SMC2 proteins that form an ATPase which acts to constrain positive supercoils [17,18]. Each type of Condensin also contains three specific non-SMC proteins that, upon phosphorylation, stabilize the complex and promote ATPase activity [14,19,20]. The kleisin CAPH and two HEAT repeat containing subunits, CAP-G and CAP-D2 are components of Condensin I, while the kleisin CAP-H2 and two HEAT repeat containing subunits, CAP-G2 and CAP-D3, are constituents of Condensin II. G (...truncated)