A Pentamer Transcriptional Complex Including tal-1 and Retinoblastoma Protein Downmodulates c-kit Expression in Normal Erythroblasts

Abstract

Human proerythroblasts and early erythroblasts, generated in vitro by normal adult progenitors, contain a pentamer protein complex comprising the tal-1 transcription factor heterodimerized with the ubiquitous E2A protein and linked to Lmo2, Ldb1, and retinoblastoma protein (pRb). The pentamer can assemble on a consensus tal-1 binding site. In the pRb⁻ SAOS-2 cell line transiently transfected with a reporter plasmid containing six tal-1 binding site, pRb enhances the transcriptional activity of tal-1–E12–Lmo2 and tal-1–E12–Lmo2–Ldb1 complexes but not that of a tal-1–E12 heterodimer. We explored the functional significance of the pentamer in erythropoiesis, specifically, its transcriptional effect on the c-kit receptor, a tal-1 target gene stimulating early hematopoietic proliferation downmodulated in erythroblasts. In TF1 cells, the pentamer decreased the activity of the reporter plasmid containing the c-kit proximal promoter with two inverted E box-2 type motifs. In SAOS-2 cells the pentamer negatively regulates (i) the activity of the reporter plasmid containing the proximal human c-kit promoter and (ii) endogenous c-kit expression. In both cases pRb significantly potentiates the inhibitory effect of the tal-1–E12–Lmo2–Ldb1 tetramer. These data indicate that this pentameric complex assembled in maturing erythroblasts plays an important regulatory role in c-kit downmodulation; hypothetically, the complex may regulate the expression of other critical erythroid genes.

ACKNOWLEDGMENTS

We thank R. Baer, L. Whitaker, and W. H. Lee for providing the pE1b-LUCE6 reporter plasmid and the Rb mutants. We are grateful to T. H. Rabbitts, L. W. Jurata, and K. Pulford for reagents. We also thank M. Fontana for editorial assistance and M. Teragnoli and A. Zito for graphics.

V. Lulli was supported by an AIDS fellowship from the Italian Ministry of Health (Rome, Italy).

L. Vitelli and G. Condorelli contributed equally to this work.