The human β-globin genes are regulated by a locus control region (LCR) and are expressed at extremely high levels in erythroid cells. How transcriptional fidelity of highly expressed genes is regulated and maintained during the cell cycle is not completely understood. Here, we analyzed the association of transcription factor USF, the co-activator CBP, topoisomerase I (Topo I), basal transcription factor TFIIB, and RNA polymerase II (Pol II) with the β-globin gene locus at specific cell-cycle stages. The data demonstrate that while association of Pol II with globin locus associated chromatin decreased in mitotically arrested cells, it remained bound at lower levels at the γ-globin gene promoter. During early S-phase, association of CBP, USF, and Pol II with the globin gene locus decreased. The re-association of CBP and USF2 with the LCR preceded re-association of Pol II, suggesting that these proteins together mediate recruitment of Pol II to the β-globin gene locus during S-phase. Finally, we analyzed the association of Topo I with the globin gene locus during late S-phase. In general, Topo I association correlated with the binding of Pol II. Inhibition of Topo I activity reduced Pol II binding at the LCR and intergenic regions but not at the γ-globin gene promoter. The data demonstrate dynamic associations of transcription factors with the globin gene locus during the cell cycle and support previous results showing that specific components of transcription complexes remain associated with highly transcribed genes during mitosis. J. Cell. Biochem. 114: 1997–2006, 2013. © 2013 Wiley Periodicals, Inc.