Next Gen Dissection of Gfi1 Dependent Transcriptome in Myeloid Progenitors Reveals Global Control of Multiple Transcriptional Programs Including Coding and Non Coding RNAs

Abstract 111

Growth Factor Independence 1 (Gfi1) is a regulator of HSC maintenance, and lack of Gfi1 leads to myeloid progenitor accumulation and neutropenia. The Gfi1 transcription factor contains both a SNAG repressor domain, and a zinc-finger DNA-binding domain. A proline to alanine substitution at position 2 in the SNAG domain (P2A) blocks RCoR/Lysine Specific Demethylase 1 (LSD1) interaction and abrogates Gfi1-mediated repression. Mouse models have been developed which either delete Gfi1 exons 2–3 (Gfi1^Δex2-3) or knock in the P2A mutation (Gfi^P2A). Both models are neutropenic, and demonstrate an accumulation of arrested myeloid progenitors. Transgenic shRNA knockdown of Lsd1 in the hematopoietic system results in the accumulation of myeloid progenitors and inhibits terminal granulocytic differentiation; however, the role of Lsd1/Gfi1 in this process has not been established. To characterize the global role of Gfi1 dependent transcriptional regulation in progenitor maintenance and lineage determination, we utilized a Next Generation Sequencing (NGS) approach to profile Gfi1^Δex2-3and Gfi^P2A lineage negative bone marrow cells. Using gene-level analyses, a representative group of 1,019 genes were identified that exhibited more than 1.5 fold deregulation (by FPKM) and differential gene expression relative to WT sample profiles in at least one model. Interestingly, some of the Gfi1-dependent genes encode putative lncRNAs, which were validated independently. Using an shRNA mediated approach, we identified one lncRNA whose expression (like Gfi1) affects the granulo- monocytic balance of differentiating myeloid progenitors. Bioinformatic interrogation revealed that genes regulated by Gfi1 are mainly involved in normal and aberrant progenitor regulation, with processes such as immunology, signal transduction, and myelopoiesis modestly represented. Given the phenotype of Lsd1 knockdown in hematopoietic cells, we hypothesized that loss of Gfi1 (and/or SNAG) activity would disrupt Lsd1 activity at Gfi1-target genes, resulting in the accumulation of histone 3 lysine 4 dimethyl (H3K4me2) marks. For the subset of genes deregulated in both Gfi1^Δex2-3and Gfi^P2A models, chromatin immunoprecipitation revealed direct Gfi1 DNA binding and increased H3K4^me2 marks in a majority of the significantly deregulated genes. We previously showed that accumulation of Gfi1 null myeloid progenitors was dose dependent upon the Gfi1 target gene HoxA9. In our current experiments, HoxA9 was deregulated in both Gfi1 null and Lsd1 knockdown, bound by Gfi1 in ChIP, and showed increased H3K4me2 peaks upon Gfi1 loss; providing a mechanistic explanation for the common phenotypes of Gfi1 null and Lsd1 knockdown in hematopoietic progenitors. Our results collectively reveal multiple transcriptional circuits controlled by the Gfi1/Lsd1 complex underlying normal progenitor maintenance and differentiation.