The effect of Kat۲a depletion on Ctcf and Myc binding in MLL-AF۹ model of AML

Background and Aim: Acute Myeloid Leukemia (AML) is an aggressive hematological malignancy. Kat۲a 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 KAT۲A regulatory targets by chromatin immunoprecipitation followed by next generation-sequencing (ChIP-seq) in Kat۲a wild type (WT) vs. knockout (KO) MLL-AF۹ secondary leukemias. Here we performed ChIP-qPCR analysis in selected Kat۲a acetylation target promoter peaks to investigate to what extent reprogramming of acetylation marks reflects Ctcf and Myc binding in WT vs KO MLL-AF۹ 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 H۳K۹ac and H۳K۲۷ac ChIPseq peaks in Kat۲a WT and KO primary MLL-AF۹ leukemias, q-PCR primers were designed under the respective H۳K۹ac peak for analysis of pooled mouse MLL-AF۹ secondary leukemia samples of each genotype. After performing ChIP-qPCR, results were quantified relative to rabbit IgG, using the intergenic region in mouse chromosome ۱ (mChr۱) as a reference.Results: Despite a significant association of H۳K۹ac-depleted promoters in Kat۲a 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-AF۹ leukemias. Critically, we observed that the binding of Myc at promoter regions dependent on Kat۲a for H۳K۹ac was globally reduced in Kat۲a KO leukemias as compared to WT (۹۵% CI WT-KO enrichment ۰.۰۱۲۷۳ to ۱.۵۸۹; ۲-way ANOVA p<۰.۰۵).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, Kat۲a may regulate the binding of sequence-specific TF at the promoter regions it acetylates.

کلیدواژه ها:

Acute Myeloid Leukemia (AML) ، Kat۲a ، histone acetyltransferase ، Myc ، Ctcf