Intrinsic Transcriptional Activation-Inhibition Domains of the Polyomavirus Enhancer Binding Protein 2/Core Binding Factor α Subunit Revealed in the Presence of the β Subunit

Abstract

A member of the polyomavirus enhancer binding protein 2/core binding factor (PEBP2/CBF) is composed of PEBP2␣B1/AML1 (as the ␣ subunit) and a ␤ subunit. It plays an essential role in definitive hematopoiesis and is frequently involved in the chromosomal abnormalities associated with leukemia. In the present study, we report functionally separable modular structures in PEBP2␣B1 for DNA binding and for transcriptional activation. DNA binding through the Runt domain of PEBP2␣B1 was hindered by the adjacent carboxyterminal region, and this inhibition was relieved by interaction with the ␤ subunit. Utilizing a reporter assay system in which both the ␣ and ␤ subunits are required to achieve strong transactivation, we uncovered the presence of transcriptional activation and inhibitory domains in PEBP2␣B1 that were only apparent in the presence of the ␤ subunit. The inhibitory domain keeps the full transactivation potential of full-length PEBP2␣B1 below its maximum potential. Fusion of the transactivation domain of PEBP2␣B1 to the yeast GAL4 DNA-binding domain conferred transactivation potential, but further addition of the inhibitory domain diminished the activity. These results suggest that the activity of the ␣ subunit as a transcriptional activator is regulated intramolecularly as well as by the ␤ subunit. PEBP2␣B1 and the ␤ subunit were targeted to the nuclear matrix via signals distinct from the nuclear localization signal. Moreover, the transactivation domain by itself was capable of associating with the nuclear matrix, which implies the existence of a relationship between transactivation and nuclear matrix attachment.The polyomavirus enhancer binding protein 2 (PEBP2), also called core binding factor (CBF), is a transcription factor complex composed of ␣ and ␤ subunits (reviewed in references 21 and 51). The ␣ subunit binds to DNA and harbors the transactivating activity, while the ␤ subunit enhances the DNA binding activity of the ␣ subunit. In mammals, members of the ␣ subunit family are encoded by three genes, PEBP2␣A/ CBFA1/AML3, PEBP2␣B/CBFA2/AML1, and PEBP2␣C/ CBFA3/AML2, and all belong to the Runt domain gene family, which includes the Drosophila genes runt and lozenge. The ␤ subunit is encoded by a single gene, PEBP2␤/CBFB, whereas two genes, brother and big brother have been identified in Drosophila.Among the three mammalian ␣ subunit genes, PEBP2␣B/ CBFA2/AML1 (2, 36, 51) is disrupted in chromosomal translocations associated with several types of leukemia, including the M2 subtype of the French-American-British classification of leukemia, which is characterized by the 8-to-21 chromosome translocation [t(8;21)], and childhood acute lymphoblastic leukemia with the associated t(12;21) translocation. The t(8;21) and t(12;21) translocations produce the chimeric proteins, AML1/ETO(MTG8) and TEL-AML1, respectively (13,19,37). These proteins retain the entire Runt domain in their PEBP2␣B/AML1 portions, which is essential and sufficient for dimerization with the ␤ subunit and for DNA binding. In addition, it is...

Chloride binding by the AML1/Runx1 transcription factor studied by NMR

“…Cl 3 might act as a competitive inhibitor to DNA or force the sidechain of R139 to adopt a conformation that is optimal for DNA binding. Another possibility is that Cl 3 might a¡ect the negative in£uences that N-and Cterminal sequences exert on the DNA binding of the Runt domain [11,24]. All scenarios could be consistent with Cl 3 being excluded from the DNA bound state.…”

RUNX1 associates with histone deacetylases and SUV39H1 to repress transcription

“…While the simple interpretation of this result is that SUV39H1 and perhaps HDAC1 recruitment are critical for RUNX1-mediated repression, and possibly silencing of CD4, this motif is also sufficient to target a heterologous protein to the nuclear matrix (Kanno et al, 1998;Zeng et al, 1998). The dual function for this motif clouds the interpretation of this result, but this domain does not appear to be absolutely required to direct RUNX1 to the nuclear matrix, as RUNX1 may contain a second nuclear matrix targeting domain (Kanno et al, 1998). Thus, the loss of transcriptional repression upon deletion of residues 347-387 may be due to disrupting the conformation or stability of the histone-modifying complex, rather than inappropriate subnuclear localization.…”

Molecular basis of tissue‐specific gene expression mediated by the Runt domain transcription factor PEBP2/CBF

“…The AD is subdivided into two regions, transcription activation element (TE) 1 and TE2, each of which independently exhibits a transcriptional activation activity when linked to the GAL4 DNA binding domain. A third element, TE3, shows only weak activity and appears to be active only in collaboration with C/EBP␣ (Kanno et al 1998a). …”

“…It therefore appears that the region between amino acids 184 and 291 masks the DNA-binding surface, and that binding of the ␤ subunit to the Runt domain changes the conformation of residues 1-291 in such a way as to unmask the DNAbinding surface. The region containing amino acids 184-291 was designated the negative regulatory domain for DNA binding (NRDB) by Kanno et al (1998a). It now appears that the region N-terminal to the Runt domain also participates in the masking of DNA binding.…”