The v-ErbA oncoprotein quenches the activity of an erythroid-specific enhancer.

v-ErbA is a mutated variant of thyroid hormone receptor (TRalpha/NR1A1) borne by the Avian Erythroblastosis virus causing erythroleukemia. TRalpha is known to activate transcription of specific genes in the presence of its cognate ligand, T3 hormone, while in its absence it represses it. v-ErbA is unable to bind ligand, and hence is thought to contribute to leukemogenesis by actively repressing erythroid-specific genes such as the carbonic anhydrase II gene (CA II). In the prevailing model, v-ErbA occludes liganded TR from binding to its cognate elements and constitutively interacts with the corepressors NCoR/SMRT. We previously identified a v-ErbA responsive element (VRE) within a DNase I hypersensitive region (HS2) located in the second intron of the CA II gene. We now show that HS2 fulfils all the requirements for a genuine enhancer that functions independent of its orientation and position with a profound erythroid-specific activity in normal erythroid progenitors (T2ECs) and in leukemic erythroid cell lines. We find that the HS2 enhancer activity is governed by two adjacent GATA-factor binding sites. v-ErbA as well as unliganded TR prevent HS2 activity by nullifying the positive function of factors bound to GATA-sites. However, v-ErbA, in contrast to TR, does not convey active repression to silence the transcriptional activity intrinsic to a heterologous tk promoter. We propose that depending on the sequence and context of the binding site, v-ErbA contributes to leukemogenesis by occluding liganded TR as well as unliganded TR thereby preventing activation or repression, respectively.

Leukemic transformation by the v-ErbA oncoprotein entails constitutive binding to and repression of an erythroid enhancer in vivo.

v-ErbA, a mutated thyroid hormone receptor alpha (TRalpha), is thought to contribute to avian erythroblastosis virus (AEV)-induced leukemic transformation by constitutively repressing transcription of target genes. However, the binding of v-ErbA or any unliganded nuclear receptor to a chromatin-embedded response element as well as the role of the N-CoR-SMRT-HDAC co-repressor complex in mediating repression remain hypothetical. Here we identify a v-ErbA-response element, VRE, in an intronic DNase I hypersensitive site (HS2) of the chicken erythroid carbonic anhydrase II (CAII) gene. In vivo footprinting shows that v-ErbA is constitutively bound to this HS2-VRE in transformed, undifferentiated erythroblasts along with other transcription factors like GATA-1. Transfection assays show that the repressed HS2 region can be turned into a potent enhancer in v-ErbA-expressing cells by mutation of the VRE. Differentiation of transformed cells alleviates v-ErbA binding concomitant with activation of CAII transcription. Co-expression of a gag-TRalpha fusion protein in AEV-transformed cells and addition of ligand derepresses CAII transcription. Treatment of transformed cells with the histone deacetylase inhibitor, trichostatin A, derepresses the endogenous, chromatin-embedded CAII gene, while a transfected HS2-enhancer construct remains repressed. Taken together, our data suggest that v-ErbA prevents CAII activation by 'neutralizing' in cis the activity of erythroid transcription factors.