The Effect of Kat2a Depletion on Ctcf and Myc Binding in Mll-Af9 Model of Aml

Background and aim: acute myeloid leukemia (aml) is an aggressive hematological malignancy. kat2a is a histone acetyltransferase (hat) that functions primarily as a transcriptional activator and capable of catalyzing multiple acetyl modifications at promoters and at enhancers. in our previous study, we defined kat2a regulatory targets by chromatin immunoprecipitation followed by next generation-sequencing (chip-seq) in kat2a wild type (wt) vs. knockout (ko) mll-af9 secondary leukemias. here we performed chip-qpcr analysis in selected kat2a acetylation target promoter peaks to investigate to what extent reprogramming of acetylation marks reflects ctcf and myc binding in wt vs ko mll-af9 secondary leukemias. methods: the candidate myc and ctcf picks were selected based on experimental dna occupancy by the transcription factors (tf) across different mouse cell types in the encode database. according to the previous data in the quantification of h3k9ac and h3k27ac chipseq peaks in kat2a wt and ko primary mll-af9 leukemias, q-pcr primers were designed under the respective h3k9ac peak for analysis of pooled mouse mll-af9 secondary leukemia samples of each genotype. after performing chip-qpcr, results were quantified relative to rabbit igg, using the intergenic region in mouse chromosome 1 (mchr1) as a reference. results: despite a significant association of h3k9ac-depleted promoters in kat2a ko leukemia cells with experimental ctcf binding in encode experiments, the results of chip-qpcr couldn’t confirm it. we showed that the transcription factors (tf) myc did indeed bind at most of the locations analyzed in mll-af9 leukemias. critically, we observed that the binding of myc at promoter regions dependent on kat2a for h3k9ac was globally reduced in kat2a ko leukemias as compared to wt (95% ci wt-ko enrichment 0.01273 to 1.589; 2-way anova p<0.05). conclusion: according to the chip-seq, we hypothesized that ctcf may be dislodged to enhancers and promote the asymmetric distribution of histone acetylation marks, with dysregulation of locus control. according to the myc results, kat2a may regulate the binding of sequence-specific tf at the promoter regions it acetylates.