AML3/CBFα1 Is Required for Androgen-specific Activation of the Enhancer of the Mouse Sex-limited Protein (Slp) Gene

Abstract

A complex 120-base pair enhancer, derived from the mouse sex-limited protein (Slp) gene, is activated solely by the androgen receptor (AR) in specific tissues, although it contains a hormone response element recognized by several steroid receptors. The generation of this transcriptional specificity has been ascribed to the interactions of the receptor with tissue-specific nonreceptor factors bound to accessory sites within the enhancer. Protein-DNA interaction assays revealed two factors binding the 5 part of the enhancer that differ widely in abundance between cells showing AR-specific activation of the Slp element compared with those that also permit activation by glucocorticoid receptor (GR). The factor designated B formed a complex centered on the sequence TGTGGT, a core motif recognized by members of the AML/CBF␣ transcription factor family. This complex was competed by a high affinity binding site specific for AML/CBF␣ and was specifically supershifted by an antibody to AML3/CBF␣1, placing factor B within the AML3/CBF␣1 subclass. Interestingly, this factor was shown to bind to a second site in the 3 part of the enhancer, positioned between the two critical AR binding sites. Transfection studies revealed that AML1-ETO, a dominant-negative AML/CBF␣ construct, abrogated AR induction of the enhancer, but not of simple hormone response elements. Furthermore, overexpression of AML3/CBF␣1 could rescue the AML1-ETO repression. Finally, glutathione S-transferase-AML/CBF␣ fusion proteins demonstrated direct interaction between AML/ CBF␣ and steroid receptors. Although this interaction was equivalent between AML1/CBF␣2 and AR or GR, AML3/CBF␣1 showed stronger interaction with AR than with GR. These data demonstrate that AML3/CBF␣1 is functionally required for hormonal induction of the Slp enhancer and that direct, preferential protein-protein interactions may contribute to AR-specific activation. These results demonstrate an intriguing role of AML3/ CBF␣1 in steroid-as well as tissue-specific activation of target genes.Steroid receptors mediate a wide variety of hormonal effects by their direct modulation of gene expression. Transcriptional activation stems primarily from receptor recognition of specific DNA hormone response elements (HREs) 1 (1-3). However, it remains a paradox how the signaling pathways of receptors are distinguished at the DNA level in vivo, since in vitro several receptors recognize the same consensus HRE (4 -8). DNA elements that vary from the consensus and provide preferential recognition by a single receptor exist but have not yet proven broadly applicable (6, 9). A more versatile mechanism to generate specificity invokes the participation of nonreceptor factors bound to DNA sequences neighboring HREs of hormonally responsive genes (5, 10, 11). Differential interactions between such factors and steroid receptors may organize an enhancerspecific activation complex that confers transcriptional specificity dependent on information intrinsic to both the gene and the receptor.To study transcription...

Polyomavirus Enhancer-binding Protein 2/Core Binding Factor/Acute Myeloid Leukemia Factors Contribute to the Cell Type-specific Activity of the CD11a Integrin Gene Promoter

“…Additional double-stranded oligonucleotides used as competitors and/or probes included NFkB-CONS (5Ј-AGTTGAGGGGACTTTCCCAGGC-3Ј), which contains the NFB consensus binding site, AP2CONS (5Ј-GATCGAACTGACCGC-CCGCGGCCCGT-3Ј), which contains the human metallothionein IIa promoter AP-2-binding site (22), and AML1CONS (5Ј-GGATATTT-GCGGTTAGCA-3Ј) (23). DNase I protection was performed as described (24), except that cells were lysed in 10 mM Hepes, pH 7.6, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.75 mM spermidine, 0.1 mM spermine, 2 g/ml aprotinin, 2 g/ml leupeptin, and 10 mM Na 2 MoO 4 .…”

“…2A). Moreover, the CD11a-110 element was identical to one of the consensus CBF-binding sequences (23), and therefore, we tested whether CBF/AML proteins contribute to formation of the AML-110 complex. As shown in Fig.…”

Runx2 Integrates Estrogen Activity in Osteoblasts

“…Our results are similar to those of earlier studies showing complex formation between full-length Runx2 fusion protein and either AR or GR. However, they also differ in part because those authors found that overexpression of Runx2 in monkey kidney cells failed to suppress the effect of androgen or glucocorticoid on gene expression driven by steroid hormone RE fused to a fragment of the thymidine kinase gene promoter (51). Nevertheless, in osteoblasts, Runx2 suppressed gene expression driven by consensus ERE, ARE, or MMTV-Luc, a well characterized androgen and glucocorticoid sensitive reporter plasmid.…”

A role for Runx2 in normal mammary gland and breast cancer bone metastasis

“…It is well established that the primary mechanism of activation of the b-casein gene is via STAT5 and the Glucocorticoid Receptor (GR) [Groner, 2002] and it is possible that the Runx2/Oct-1 complex acts as a ''landing pad'' for these transcription factors. Indeed, both Runx2 and Oct-1 have been shown to interact with GR [Ning and Robins, 1999;Prefontaine et al, 1999], suggesting that the Runx2/Oct-1 complex may recruit GR. Furthermore, since Runx2 has been shown to interact with STAT1 [Kim et al, 2003], it might also be able to interact with STAT5, thus providing a link between prolactin signaling and Runx2 activity.…”