Transcriptional autoregulation of the bone related CBFA1/RUNX2 gene.

The runt related transcription factor CBFA1 (AML3/PEBP2alphaA/RUNX2) regulates expression of several bone- and cartilage-related genes and is required for bone formation in vivo. The gene regulatory mechanisms that control activation and repression of CBFA1 gene transcription during osteoblast differentiation and skeletal development are essential for proper execution of the osteogenic program. We have therefore defined functional contributions of 5' regulatory sequences conserved in rat, mouse and human CBFA1 genes to transcription. Deletion analysis reveals that 0.6 kB of the bone-related rat or mouse CBFA1 promoter (P1, MASNS protein isoform) is sufficient to confer transcriptional activation, and that there are multiple promoter domains which positively and negatively regulate transcription. Progressive deletion of promoter segments between nt -351 and -92 causes a striking 30- to 100-fold combined decrease in promoter activity. Additionally, 5' UTR sequences repress reporter gene transcription 2- to 3-fold. Our data demonstrate that CBFA1 is a principal DNA binding protein interacting with the 5' region of the CBFA1 gene in osseous cells, that there are at least three CBFA1 recognition motifs in the rat CBFA1 promoter, and that there are three tandemly repeated CBFA1 sites within the 5' UTR. We find that forced expression of CBFA1 protein downregulates CBFA1 promoter activity and that a single CBFA1 site is sufficient for transcriptional autosuppression. Thus, our data indicate that the CBFA1 gene is autoregulated in part by negative feedback on its own promoter to stringently control CBFA1 gene expression and function during bone formation.

Cell cycle-dependent modifications in activities of pRb-related tumor suppressors and proliferation-specific CDP/cut homeodomain factors in murine hematopoietic progenitor cells.

The histone H4 gene promoter provides a paradigm for defining transcriptional control operative at the G1/S phase transition point in the cell cycle. Transcription of the cell cycle-dependent histone H4 gene is upregulated at the onset of S phase, and the cell cycle control element that mediates this activation has been functionally mapped to a proximal promoter domain designated Site II. Activity of Site II is regulated by an E2F-independent mechanism involving binding of the oncoprotein IRF2 and the multisubunit protein HiNF-D, which contains the homeodomain CDP/cut, CDC2, cyclin A, and the tumor suppressor pRb. To address mechanisms that define interactions of Site II regulatory factors with this cell cycle control element, we have investigated these determinants of transcriptional regulation at the G1/S phase transition in FDC-P1 hematopoietic progenitor cells. The representation and activities of histone gene regulatory factors were examined as a function of FDC-P1 growth stimulation. We find striking differences in expression of the pRb-related growth regulatory proteins (pRb/p105, pRb2/p130, and p107) following the onset of proliferation. pRb2/p130 is present at elevated levels in quiescent cells and declines following growth stimulation. By contrast, pRb and p107 are minimally represented in quiescent FDC-P1 cells but are upregulated at the G1/S phase transition point. We also observe a dramatic upregulation of the cellular levels of pRb2/p130-associated protein kinase activity when S phase is initiated. Selective interactions of pRb and p107 with CDP/cut are observed during the FDC-P1 cell cycle and suggest functional linkage to competency for DNA binding and/or transcriptional activity. These results are particularly significant in the context of hematopoietic differentiation where stringent control of the cell cycle program is requisite for expanding the stem cell population during development and tissue renewal.