Characterization of alpha-globin locus chromatin accessibility in erythroid cells following insertion of PP7 stem-loops at the Hba-a1 gene

PP7 stem-loop DNA was inserted into the Hba-a1 gene in order to monitor nascent transcription dynamics of alpha-globin during erythroid differentiation. To confirm that this genetic modification does not influence the chromatin accessibility of the locus in erythroid cells we performed ATAC-seq in wild-type and Hba-a1-PP7 (PP7) cells, as well as Hba-a1-PP7 clones with a stably integrated PP7 coat protein (PCP) expression construct. No differences were observed between these cell lines in terms of chromatin accessibility at the alpha-globin locus, indicating the PP7 modification likely did not disrupt normal gene activation in these cells. Hba-a1-PP7 cells were created by insertion of 24 PP7 repeats in the first exon of the Hba-a1 gene by recombinase-mediated cassette exchange (RMCE). PP7 repeats were synthesized (GenScript) and inserted into the first exon of Hba-a1 in a plasmid containing DNA sequence surrounding Hba-a1 recovered from a mouse BAC clone (RP22-289A22, BACPAC Resources Centre). This construct was exchanged into cells containing an acceptor site (loxP, lox511 sites) at the Hba-a1 gene. A clone in which successful exchange had occurred (PP7) was transfected with a PGK:PCP-EGFP construct (a gift from Jonathan Chubb). Three clones with stable integration of the construct (PCP B7, B11, C12) were taken forward for analysis. ATAC-seq was performed in duplicate on WT and PP7 cells and on the three PCP clones.