Abstract

The growth factor independent 1 (Gfi1) oncoprotein is an intrinsic regulator of hematopoiesis, and its mutations are associated with congenital neutropenia. We performed a yeast two-hybrid screen with Gfi1, in order to begin dissecting the gene network through which it acts and also toward identifying other genes potentially responsible for cases of hereditary neutropenia lacking mutations in Gfi1 or ELA2 (encoding neutrophil elastase, the most common cause of hereditary neutropenia). From a limited number of proteins found to interact with Gfi1 in the yeast two-hybrid screen, we evaluated PRDM5, a previously uncharacterized zinc finger protein belonging to the PR domain (PRDI-BF1 and RIZ homology) -containing family of tumor suppressor genes. We screened for mutations in the gene encoding PRDM5 among 230 unrelated patients with otherwise genetically unexplained congenital neutropenia and discovered two with heterozygous amino acid sequence substitutions that were not detected in twice the number of controls. Consequently, we systematically characterized PRDM5, and found that it functions as a novel epigenetic regulator of hematopoiesis: First, PRDM5 is a sequence-specific DNA-binding transcription factor as revealed by random sequence selection. Second, by using chromatin immunoprecipitation (ChIP)-based cloning along with “ChIP-on-chip” assays, we identified approximately two hundred protein-coding genes (including hematopoietic transcription factors) and thirty-four microRNA genes as putative PRDM5 targets, a subset of which we confirmed by additionally performing gel retardation and transfection reporter assays. Significantly, one of the targets of PRDM5—just as is true of Gfi1—is ELA2. Third, depletion of PRDM5 by siRNA in both HeLa and HEK293 cells changed the cell cycle program, and, as revealed by microarray and real-time RT-PCR assays, PRDM5 knockdown in HEK293 cells altered the expression of ninety of the protein-coding targets and eight of the microRNA targets. Fourth, transcriptional profiling revealed that the expression of about five thousand genes, including many involved in the regulation of cell proliferation and differentiation, changed more than five-fold following PRDM5 knockdown in HEK293 cells. Fifth, we found that PRDM5 regulates transcription by associating with the histone deacetylase HDAC1 and the histone methyltransferase G9a and recruits them to its target genes. Treatment of cells with the histone deacetylase inhibitors trichostatin A (TSA) or 4-phenylbutyric acid (PBA) deregulates PRDM5 target genes. Finally, the two variants observed in patients with congenital neutropenia alter the DNA-binding activity of PRDM5 and interfere with its function as a transcriptional repressor in reporter assays, suggesting that, just as with Gfi1, rare mutations in a global regulator of hematopoiesis may contribute to the genetic mechanisms causing neutropenia.

Disclosure: No relevant conflicts of interest to declare.

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