Abstract
RUNX1, also known as AML1, is a DNA binding transcription factor that is expressed in hematopoietic stem and progenitor cells (HSPCs). As demonstrated by several mouse models, RUNX1 is necessary for definitive hematopoiesis and proper homeostasis of HSPCs. Furthermore, mutations of RUNX1have been implicated in patients with a variety of blood-related malignancies and disorders. These findings have established RUNX1 as a master regulator of hematopoiesis. As a transcription factor, RUNX1 exerts its function in hematopoiesis by binding to regulatory regions in order to guide the expression of its direct target genes. Most confirmed RUNX1 target genes are mainly expressed in differentiated blood cells. Direct targets of RUNX1 in HSPCs, however, have largely remained unexplored. Identifying direct target genes of RUNX1 offers an insightful view of how this master regulator influences HSPC function. To elucidate RUNX1 target genes in HSPCs, we have analyzed gene expression signatures from wildtype and RUNX1-deficient HSPCs (Lineage-/cKit+/Sca1+) in a previous report (Matsuura et al., Blood, 2012). With the goal of continuing the characterization of RUNX1 target genes, in this current study, we performed genome-occupancy analysis with chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) using RUNX1 antibodies and a murine HSPC cell line. Bioinformatics analysis of the ChIP-seq data revealed 6370 significant RUNX1 binding peaks (<1% FDR). The majority of these peaks were located in areas outside of promoter regions. The top de novo generated sequence motif from these peaks corresponds with the known RUNX binding consensus motif TG(T/C)GGT, suggesting that our ChIP-seq dataset is highly reliable. The combination of differential gene expression and RUNX1 genome occupancy data has revealed a list of candidate RUNX1-regulated target genes. We hypothesize that RUNX1 directly modulates the expression of these genes in normal hematopoiesis.
One of the genes identified is Hmga2. We observed three RUNX1 binding peaks in the upstream, intron, and downstream regions relative to the Hmga2 gene locus. Furthermore, we confirmed strong up-regulation of Hmga2 in RUNX1-deficient HSPCs using reverse transcription coupled with quantitative polymerase chain reaction. HMGA2, also known as High Mobility Group AT-hook 2, is a non-histone chromatin protein. Its expression is highest during embryonic development and in undifferentiated cells. Over-expression of HMGA2 in transgenic mice or in bone marrow transplantation models have been reported to cause expansion of HSPCs. These reports indicate that HMGA2 is a significant mediator of HSPC proliferation. Interestingly, a major characteristic of mice without RUNX1 in their hematopoietic cells is the expansion of HSPCs, suggesting that HMGA2 may contribute to this phenotype. To further validate Hmga2 as a RUNX1 target gene, we cloned the Hmga2 promoter sequence and DNA fragments corresponding to the three RUNX1 binding peaks into luciferase reporter constructs and performed transfection studies using K562 and 293T cells. Interestingly, while these studies demonstrated strong responses to RUNX1 in promoter-luciferase assays, the effect of RUNX1 on Hmga2 promoter activity in these two cell lines was opposite. In addition, eliminating two RUNX binding motifs in the Hmga2 promoter did not affect RUNX1-mediated promoter-luciferase activity, indicating that there are additional regulatory mechanisms that may be important for RUNX1’s effect on the Hmga2 promoter. To examine the function of the three regions containing RUNX1 binding peaks in the Hmga2 gene locus, we also used luciferase reporter constructs including these regions in transfection studies. Increase of transcriptional activity was detected in the presence of the two regions that were upstream and downstream of the Hmga2 gene, suggesting that RUNX1 can act as a positive regulator through these regions. In contrast, the RUNX1 binding fragment in the intron region of Hmga2 reduced promoter-luciferase activity. This outcome indicates that RUNX1 acts as a suppressor through the Hmga2 intron element. In summary, these results establish Hmga2 as a novel RUNX1 target gene in HSPCs and mark the first study of the transcriptional regulation of the Hmga2 gene by RUNX1 through differential control regions.
Disclosures:
No relevant conflicts of interest to declare.
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