The effect of -196 C>T mutation on Aγ and Gγ-globin in k562
The effect of -196 C>T mutation on Aγ and Gγ-globin in k562
Maryam Maleki Tehrani,1Co-first author, Tahere Dabestani,2Azadeh Ahmadifard,3Dr. Mehdi Banan,4,*
1. Genetics Research Center, University of social welfare and rehabilitation sciences, Tehran, Iran 2. Genetics Research Center, University of social welfare and rehabilitation sciences, Tehran, Iran 3. Genetics Research Center, University of social welfare and rehabilitation sciences, Tehran, Iran 4. Genetics Research Center, University of social welfare and rehabilitation sciences, Tehran, Iran
Introduction: The -196 C>T mutation is a non-deletional hereditary persistence of fetal hemoglobin (ndHPFH) mutation that increases the amount of human fetal hemoglobin (HbF) by disturbing the Zinc Finger and BTB Domain Containing 7A (ZBTB7A) transcription factor binding site. Observations have shown that individuals with the -196 C>T mutation in their Aγ-globin promoter have greater fetal hemoglobin levels than persons with the mutation in their Gγ-globin promoter (Wienert, Martyn, Funnell, Quinlan, & Crossley, 2018). Given that the promoters of these genes are identical up until the -200 region, this discrepancy may be caused by the differential existence of the -196 C>T mutation in the Aγ or Gγ promoters.
In order to investigate this disparity, we introduced the -196 C>T mutation into the left homology arm (i.e., Gγ or Aγ promoters) of a cassette containing EGFP and Neomycin resistance gene (NeoR). These modified EGFP cassettes were then knocked into the γ-globin gene(s) of K562 cells. Finally, EGFP fluorescence levels were assessed in the subsequent cell lines.
Methods: First, a previously assembled plasmid (Jafari, Hesami, Safi, Ghasemi, & Banan, 2019) includes an EGFP. (IRES). NeoR. pA cassette (IRES stands for internal ribosome entry site, and pA signifies a polyadenylation signal sequence), with flanking Left and Right homology arms (LHA and RHA) corresponding to the Gγ-globin gene was used to create the plasmid containing the Aγ-globin specific left homology arm, therefore, the Gγ specific LHA was removed through enzymatic digestion and then the Aγ specific LHA which was PCR amplified from K562 genomic DNA was cloned into the plasmid.
Subsequently, these two plasmids harboring either Aγ or Gγ Left homology arms were used as templates in the overlap-PCR (Heckman & Pease, 2007; Vallejo, Pogulis, & Pease, 2008) to generate two distinct LHA fragments containing the Aγ -196 C>T and Gγ -196C>T ndHPFH mutations, then these two LHA fragment were replaced the original LHAs in the EGFP plasmids, and by Sanger sequencing and restriction enzyme mapping the accuracy of products was verified.
Following the Creation of mutated EGFP plasmids, these two plasmids alongside the CRISPR/Cas9 PX459 plasmid (RRID: Addgene_62988) (Jafari et al., 2019) harboring the gamma-globin-specific sgRNA were co-transfected into K562 cell by using Lipofectamine 2000. In parallel, the original EGFP plasmids without mutations were also co-transfected to produce control cell lines so that we could compare the effect of mutation with control cell lines.
After selection through puromycin dihydrochloride (resistance in the CRISPR plasmid) and G418 (resistance in the EGFP plasmid), DNA was extracted from these cell lines to verify the target integration. Finally, Fluorescence levels were assessed in all these four cell lines.
Results: The Sanger sequencing readouts confirmed the target integration of -196 C>T and WT promoters into either the Aγ or the Gγ genes.
The percentage of EGFP+ cells was 90% for Aγ-196 C>T HPFH mutation and 60% for WT: Aγ promoter. For Gγ-196 C>T HPFH mutation and WT: Gγ promoter, the percentages were 75% and 83% respectively. In terms of fluorescence intensity, the Gγ-196 C>T mutation caused no change compared to control cells, while the Aγ-196 C>T HPFH mutation decreased fluorescence levels compared to WT: Aγ control cells.
Conclusion: The -196 C>T mutation has been previously reported to disturb the binding site of the ZBTB7A transcription factor in adult erythroid cells and increase the amount of γ-globin gene production (Martyn et al., 2018; Mingoia et al., 2021; Weber et al., 2020), however, this was not the case in our study on fetal-like K562 cells, nevertheless, our results are consistence with research results of studies that are done on K562 cells. A study reports unchanged γ-globin expression after ZBTB7A knock-down which is in line with our result on Gγ (Chondrou et al., 2022). Another study indicated a reduction in γ-globin expression after ZBTB7A knock-out which is compatible with our result on Aγ-globin (Kang et al., 2019), therefore, it seems that the mutation has different consequences in adult and fetal cell line models, suggesting the possible different functions of the ZBTB7A factor during adult and fetal stages.
Finally, our result indicates the difference between the effect of the -196 C>T Gγ and Aγ mutations, but it cannot explain the difference between individuals with the mutation in Gγ and Aγ, proposing further investigations on adult model cell lines.