Conserved Transcriptional Deregulation Underlies GFI1 and ELANE Mutant Neutropenia

Abstract 13

Mutations in ELANE, GFI1, G6PC3, and HAX1 account for the genetic defects in many patients with severe congenital neutropenia (SCN). Here we utilized next generation sequencing technology to characterize genome-wide transcriptome activity and chromatin structure states associated with normal and SCN–affected hematopoietic precursor cells. We profiled cells from Gfi1−/−, Gfi1P2A, LysM-Cre Mcl1flox and wild type mice. Growth factor independent-1 (Gfi1) is a zinc finger transcription repressor required for murine and human granulopoiesis. In Gfi1−/− bone marrow cells, the genetic lack of Gfi1 protein blocks granulopoietic programming. In Gfi1P2A knockin mice the Gfi1 protein is made, but the substitution of alanine for proline (P2A) inhibits the function of the SNAG transcriptional repressor domain; resulting in neutropenia. Mcl1 is a Bcl2 family member required for the survival of multiple hematopoietic lineage cells. LysM-Cre Mcl1flox mice are neutropenic due to myeloid-lineage-specific Cre-mediated deletion of Mcl1 and subsequent apoptosis of neutrophil progenitors after the metamyelocyte stage. Other hematopoietic lineages in LysM-Cre-Mcl1flox mice are normal. Thus, all three models are neutropenic; two lack a functional lineage specifying transcription factor while one lacks a survival factor. Bone marrow cells from each of these mouse models were processed through Miltenyi AutoMacs lineage-depletion. Total RNA was extracted and subjected to paired-end deep RNA-sequencing using Illumina Truseq library preparation methods. Reads were aligned to the reference mouse mm9 genome and Refseq and ENSEMBL gene transcript models using Tophat, Bowtie, and Cufflinks analysis suite. Both gene and transcript model specific gene expression level were estimated using the fragments per kilobase of exon model per million mapped reads (FKPM) approach. Using gene-level analyses, a representative group of 2,186 genes were identified that exhibited relatively high level expression (>50^th %ile by FPKM in at least one sample type) and differential gene expression relative to WT sample profiles in at least one model. First, our data indicate that the SNAG domain is critical for most Gfi1 associated functions. The SNAG domain associates with the Lysine specific demethylase-1 (Lsd1), an enzyme which removes histone 3 lysine 4 dimethyl (H3K4me2) marks. Chromatin immunoprecipitation revealed increased H3K4me2 marks in many genes deregulated in Gfi1−/− and Gfi1P2A Lin- bone marrow cells. Moreover, the group of genes overexpressed in Gfi1−/− and Gfi1P2A Lin- bone marrow cells was functionally associated with a variety of signal transduction and transcriptional control programs responsible for the development and differentiation of myeloid and lymphoid lineages, whereas genes that were commonly downregulated were associated with erythroid development. Many of the overexpressed immune myeloid genes were the targets of the ETS family of transcription factors and included a rich overrepresentation of genes associated with vesicle/lysosome/ and granule formation. Furthermore, using statistical and clustering-based analyses, approximately 70% of the dysregulated genes exhibited similar over-expression and under-expression in all three models. The large scale similarity of these patterns and the disparate genetic and biological mechanism of neutropenia in the three models suggests an underlying signature of the bone marrow progenitor activation in response to neutropenia. To confirm these findings in a different setting, we next examined the gene expression of human CD34+ cells from several ELANE mutant SCN patients and a single GFI1N382S patient, compared to normal CD34+ cells. Notably, all patients received G-CSF. In fact, a significant number of genes differentially expressed between ELANE and normal CD34+ cells were commonly deregulated in the GFI1 patient sample. Thus, human samples also demonstrate a signature associated with bone marrow progenitor activation in response to neutropenia. Our results reveal not only the transcriptional circuits controlled by Gfi1, but also cell autonomous transcriptional circuits underlying neutropenia.