Expression of Runx1 Isoforms in Sub-Populations of Human Blood and Bone Marrow Cells.

Runx1 is a pivotal regulator of hematopoiesis. Deficiency of Runx1 leads to complete block of definitive hematopoiesis in mouse embryos. Runx1 protein forms a heterodimer with CBFβ and binds to the specific DNA sequence TGT/cGGT to regulate the expression of a number of hematopoietic genes. Runx 1 has been recognized to have three different isoforms (a, b, and c) due to differential splicing and promoter utilization. The distal (Runx1c) promoter region was shown to have binding sites mediating repression of Runx1 transcription. Data on the pattern of Runx1 isoforms expression in different hematopoietic lineages are scant. The functional impact of different Runx1 isoforms on commitment and differentiation of blood cells is also essentially unknown. To further explore this, we studied Runx1 isoform expression in different subpopulations of human hematopoietic cells. Total RNA was extracted from peripheral blood and unstimulated bone marrow. Also, Lin-CD34+CD38+, and Lin-CD34+CD38− cell populations were sorted from G-CSF-stimulated bone marrow and total RNA extracted. Expression of Runx1 isoforms was assessed by semiquantitative RT-PCR analysis, using GAPDH as a control. All 3 isoforms were present in all studied cell populations. Runx1a was uniformly a minor form with lowest expression. Runx1c isoform was a dominant one in peripheral blood as opposed to Runx1b in bone marrow. Most interestingly, the ratio of Runx1b to other isoforms was significantly increased in Lin-CD34+CD38- subpopulation compared to Lin-CD34+CD38+ cells. The prevalence of Runx1c isoform in peripheral blood as compaired to bone marrow is most likely related to a different cellular composition of these tissues and suggests that Runx 1c isoform may be predominantly expressed in some of the mature cell lineages. Since Lin-CD34+CD38− cells represent most primitive progenitor cell population with multi-lineage repopulating ability, the observed overexpression of Runx1b may be related to transcriptional accessibility of these cells. Upregulation of Runx 1b with G-CSF in this subpopulation can not be ruled out as well. These preliminary data suggest that the expression of Runx1 alternative splicing isoforms is both lineage and differentiation stage-specific in human hematopoietic cells. We will also test whether Runx1 isoform expression has a consistent pattern in different types of leukemia and if it is resembles such of mature blood counterparts or primitive progenitors. These studies, as well as the studies of differential expression of Runx1 isoforms in different mature blood lineages and progenitor cell sub-populations are underway and results will be presented. These data may further shed a light on functional differences in Runx1 isoforms and mechanisms of hematopoiesis.