Abstract
Developmental stage-specific expression of the β-type globin genes is regulated by many cis- and trans-acting components. The adult β-globin gene contains an E-box located 60 bp downstream of the transcription start site that has been shown to bind transcription factor upstream stimulatory factor (USF) and to contribute to efficient in vitro transcription. We expressed an artificial zinc finger DNA-binding domain (ZF-DBD) targeting this site (+60 ZF-DBD) in murine erythroleukemia cells. Expression of the +60 ZF-DBD reduced the recruitment and elongation of RNA polymerase II (Pol II) at the adult β-globin gene and at the same time increased the binding of Pol II at locus control region (LCR) element HS2, suggesting that Pol II is transferred from the LCR to the globin gene promoters. Expression of the +60 ZF-DBD also reduced the frequency of interactions between the LCR and the adult β-globin promoter. ChIP-exonuclease-sequencing revealed that the +60ZF-DBD was targeted to the adult β-globin downstream promoter and that the binding of the ZF-DBD caused alterations in the association of USF2 containing protein complexes. The data demonstrate that targeting a ZF-DBD to the adult β-globin downstream promoter region interferes with the LCR-mediated recruitment and activity of Pol II.
Highlights
Transcription of the adult b-type globin gene is tightly regulated by distal and proximal cis-regulatory DNA elements [1,2]
The results show that expression of the +60 zinc finger DNA-binding domain (ZF-DBD) increased the interaction of NF-E2 with locus control region (LCR) HS2, similar to what we observed for polymerase II (Pol II)
We examined the functional role of the adult bmaj-globin downstream promoter region using an artificial ZF-DBD targeting an E-box
Summary
Transcription of the adult b-type globin gene is tightly regulated by distal and proximal cis-regulatory DNA elements [1,2]. Active chromatin marks and transcription complexes first associate with the LCR during differentiation of erythroid cells [10,11,12,13]. This suggests that the LCR serves as the primary site of transcription complex recruitment and possibly assembly, and that the assembled transcription complexes are transferred to the globin genes [14]. This model is supported by in vitro transfer experiments, ; in vivo evidence is lacking [15]
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