Knockdown of HNF4A Gene Increases Fetal Hemoglobin Synthesis in Hudep-2

Sickle cell anemia is a recessive inherited disease caused by a single nucleotide polymorphism in the β-globin gene the resulting substitution of glutamic acid by valine causes red blood cell sickling when deoxygenated. Some hypomethylating agents are able to induce the expression of γ-globin by inhibiting DNA methylation at the gene promoter. However, it is not completely understood how this regulation occurs and which genes are involved in this process. To understand fetal hemoglobin (HbF) regulation, CD34⁺ cells were treated with 1µM of decitabine on the 9^th day of culture to induce HbF production. The expression levels of transcription factors and chromatin modifiers were evaluated through the PCR Array platform (Qiagen™ Germany). The transcription factor, HNF4A (Hepatocyte Nuclear Factor 4 Alpha), was highly upregulated in cells treated with decitabine, compared to the control cells, and was chosen as a candidate for CRISPR/Cas9 knockout in HUDEP-2 cells (immortalized human erythroid progenitor cells). The HNF4A gene has been reported as a transcription factor, which regulates the expression of several hepatic genes, and is able to play a role in the development of the liver, kidney, and intestines. Moreover, HNF4A is expressed in the hematopoietic tissue. To the best of our knowledge, the association between HNF4A and gamma globin gene synthesis has not been previously described. HUDEP-2 cells were cultured and then treated with 50nM decitabine. After 72 hours, HbF levels were measured with anti-HbF antibody by flow cytometry in three biological replicates. The percentage of cells positive for HbF in decitabine-treated HUDEP-2 were 12.27 ± 0.7%, N=3, while in control cells the percentage was 1.0 ± 0.06%, N=3 (p<0.0001). These results corroborate the increased expression measured in CD34⁺ cells. To knock out HNF4A in HUDEP-2 cells, we used the CRISPR/Cas9 system. We generated INDELs in heterozygosity for HNF4A. Briefly, HUDEP-2 cells were nucleofected with Cas9 high fidelity ribonucleoprotein (104 pmol), crRNA:tracrRNA (120 pmol) complex and 1µM of gRNA HNF4A using a CD34⁺ human cell kit and the E-001 program in an AMAXA Nucleofector 4D-device (Lonza). Two days after nucleofection, edited HUDEP-2 cells were submitted to clonal selection and expanded for approximately 28 days. Genomic DNA from clones was analyzed by a Sanger Sequencer, and four edited HNF4A-HUDEP-2 clones were selected with INDELs in the fourth exon of HNF4A gene. These clones were expanded in culture with controls, and the HbF levels were quantified by flow cytometry. HbF levels in these four clones were 8.2 ± 2.4%, while in the wild type HUDEP-2, HbF was 0.9 ± 0.02% (p< 0.05). The edited clones expressed significantly more HbF than the controls, although this expression was not homogeneous. Western blotting of edited HNF4A-HUDEP-2 clones demonstrated decreased HNF4A at the protein level (0.35 ± 0.9 AU N=8), compared to controls (2.02± 1.1 AU N=3, p< 0.05). Results suggest that HNF4A may play a role in the gamma globin gene transcription in HUPED-2 cells. The mechanism of how HNF4A may regulate gamma globin gene expression remains to be clarified.