The TAL1/SCL Transcription Factor Regulates Cell-Cycle Progression in Murine Monocyte-Macrophage Precursors.

Abstract Abstract 570 The TAL1 (or SCL) gene, first identified through its involvement by a chromosomal translocation in T-cell acute lymphoblastic leukemia, encodes a basic helix-loop-helix transcription factor critical for embryonic and adult hematopoiesis and vascular remodeling. We reported that Tal1 was expressed at all stages of monocytic differentiation from the common myeloid progenitor to the mature (and activated) macrophage and that ex vivo knockout of this gene in BM macrophage (BMM) precursors from Tal1loxP/Lacz mice profoundly reduced cellular proliferation without significantly affecting survival or differentiation. To characterize the cell-cycle status of Tal1-deficient BMM precursors, we carried out flow cytometry-based BrdU pulse-chase analysis. This revealed delayed progression from G1 to S phase and S to G2 in Tal1-/- relative to control Tal1+/- cells. Moreover, when Tal1-/- cells were cultured with BrdU for extended times (48 hr), a significantly larger fraction of Tal1-/- cells (27.5%) failed to incorporate BrdU compared to Tal1+/- cells (7.6%), indicating an additional defect in exiting G0/G1. To determine whether Tal1's function in proliferation was gene dose-dependent, cell numbers were enumerated in cultures of Tal1-transduced, Tal1+/+, Tal1+/-, and Tal1-/- BMM precursor cells. Cells over-expressing Tal1 accumulated in higher numbers than Tal1+/+ cells, which had a proliferative advantage over Tal1+/- cells that was evident only late in culture. To address the mechanism by which TAL1 regulates cell-cycle progression in this lineage, we investigated the effect of increasing and decreasing its expression on expression of the cyclin-dependent kinase inhibitor p16(Ink4a), which was suggested in transient transfection experiments to be a target of TAL1-mediated repression. Indeed, p16(Ink4a) mRNA levels were ∼4-fold higher in Tal1-/- relative to Tal1+/+ cells and were reduced, although less significantly, in Tal1-over-expressing compared to Tal1+/+ cells. Quantitative chromatin immunoprecipitation (ChIP) analysis in wild-type BMM precursor cells showed direct association of Tal1 and its E protein DNA-binding partner E47 with 3 consensus E-box elements in the p16(Ink4a) upstream region that was greater in day 4 than day 7 cells and correlated inversely with p16(Ink4a) mRNA expression. In addition, we confirmed the previously reported increase in p16(Ink4a) mRNA abundance and decrease in Tal1 mRNA in M1 monocytic leukemia cells induced to differentiate into macrophages with interleukin-6 (IL-6). Quantitative ChIP analysis with sonicated chromatin from IL-6 treated (48 hr) and untreated M1 cells showed Tal1 occupancy at the same E-box elements in untreated cells as in primary BMM precursors that was completely abolished by IL-6 treatment. In contrast E2A occupancy was detected in both treated and untreated cells, consistent with relief of Tal1-directed repression of p16(Ink4a) in M1 cells upon differentiation. In summary, these studies have uncovered a critical role for Tal1 in cell cycle regulation during monocytopoiesis and suggest that TAL1 repression of p16(Ink4a) transcription likely contributes. Further, the ChIP and expression studies provide more definitive evidence for p16(Ink4a) as a target gene of TAL1. Finally, the results in Tal1-over-expressing BMM precursors may be relevant to TAL1's actions in T-lymphoid and, in particular, myeloid leukemias. Disclosures: No relevant conflicts of interest to declare.