An intron 1 enhancer element mediates oestrogen-induced suppression of ERBB2 expression

Abstract

Overexpression of the ERBB2 gene in human breast cancer is associated with a poor prognosis and resistance to hormonal treatment and chemotherapy. Oestrogen receptor (ER) positive tumour-derived cell lines are known to express relatively low levels of ERBB2 protein under oestrogenic conditions, but markedly higher levels following withdrawal of oestrogens or administration of tamoxifen. Expression of the closely related ERBB3 gene, which co-operates with ERBB2 in cellular transformation, is now shown to respond to oestrogenic manipulation in a similar way, both responses being mediated largely by transcriptional changes. Six previously undescribed DNase I hypersensitive sites occur within the ®rst intron of ERBB2 in cells that overexpress the gene. A 409 base pair DNA fragment containing one of these sites conferred ER dependent oestrogen inhibition on the ERBB2 promoter in two types of transient transfection assay. DNase I footprinting revealed four separate transcription factor binding sites within this fragment consistent with a role as a transcriptional enhancer. These ®ndings implicate intron 1 sequences in the control of ERBB2 expression for the ®rst time and demonstrate that one site within this region is involved in mediating the transcriptional response to oestrogens. Additionally, there is likely to be synergism between ERBB2 and ERBB3 signalling when both are overexpressed in response to oestrogen inhibition, thereby driving transformed cell behaviour.

AP-2 transcription factors in the regulation of ERBB2 gene transcription by oestrogen

“…One of the four footprinted sequences described in the ERBB2 ®rst intron oestrogen-dependent enhancer (Bates and Hurst 1997a), called FPC, is extremely similar to FP3 (Figure 3a). The EMSA analysis shown in Figure 3c demonstrates that a FPC oligonucleotide forms a complex that is indistinguishable from FP3wt, is competed by canonical AP-2 and supershifted by the anti-AP2 antibody.…”

“…ZR75.1 cells were grown in oestrogenic (CM or SM+17b-oestradiol) Figure 3 Biochemical characterization of the oestrogen-dependent negative element in the ERBB2 promoter. (a) The sequence of the footprinted region around an AP-2 site at position 7397 in the ERBB2 promoter (FP3wt) (Hollywood and Hurst, 1993), compared to the sequence present in plasmid pE2P.Pp.CAT (FP3mod) and to the sequence of the footprint C (FPC) of the oestrogen-dependent enhancer found in the ®rst intron of human ERBB2 (Bates and Hurst, 1997a). The boxed part represents the`core binding sequence', as de®ned by methylation interference.…”

“…Mechanisms of reciprocal repression have been identi®ed: ®rst, the signaling pathway activated by the ErbB2 receptor permanently down-regulates ER expression (Pietras et al, 1995). Second, we and others have shown that oestrogen-activated ER represses ERBB2 expression in vitro and in vivo (Dati et al, 1990;Read et al, 1990;Warri et al, 1991;Russel and Hung, 1992;Antoniotti et al, 1994;Bates and Hurst, 1997a). Antioestrogens such as Tamoxifen and ICI 182,780 counteract the e ect of oestrogen on ERBB2, i.e.…”

“…Oestrogen downregulates ERBB2 mostly, if not solely, by transcriptional repression, as demonstrated by run-on analysis Bates and Hurst, 1997a) and, when su cient ER is present, reduces the activity of transiently as well as stably transfected ERBB2 promoter-CAT constructs (Russel and Hung, 1992;Antoniotti et al, 1994;Taverna et al, 1994).…”

“…More recently, an oestrogen-responsive enhancer was identi®ed in the ®rst intron of the human ERBB2 gene, which is capable of mediating oestrogen-dependent repression of transcription when cloned in proximity of the ERBB2 promoter (Bates and Hurst, 1997a).…”