We performed PAX5-specific chromatin immunoprecipitation analyses across the Csfr1 locus. We investigated the role of PAX5 in regulating Csf1r sense and antisense promoter activity by transient transfections and by employing a Pax5−/− pro−B-cell line expressing an inducible PAX5 protein. PAX5 interacting factors were identified by pull-down experiments. The role of the transcription factor Sp3 in driving antisense promoter expression was examined in B cells from Sp3 knockout mice.
PAX5 differentially regulates the Csf1r promoter and the promoter of the antisense transcript. PAX5 interferes with PU.1 transactivation at the sense promoter by binding to a PAX5 consensus sequence. At the antisense promoter, PAX5 does not specifically recognize DNA, but interacts with Sp3 to upregulate antisense promoter activity. Antisense promoter activation by PAX5 is dependent on the presence of its partial homeo-domain.
We demonstrate that PAX5 regulates Csf1r in B cells by reducing the frequency of binding of the basal transcription machinery to the promoter and by activating antisense RNA expression.