Epigenetic Regulation of Cell Cycle-Promoting Genes By Ikaros and HDAC1 in Acute Lymphoblastic Leukemia

B-cell acute lymphoblastic leukemia (B-ALL) is the most common childhood leukemia. Expression profiling has identified IKZF1 (Ikaros) as a major tumor suppressor in B-ALL and established reduced Ikaros function as a poor prognostic marker for this disease. Ikaros regulates expression of its target genes via chromatin remodeling. In vivo, Ikaros can form a complex with histone deacetylases HDAC1 and/or HDAC2 as well as the NuRD chromatin remodeling complex. The mechanisms by which Ikaros exerts its tumor suppressor function and regulates gene expression in B-ALL are unknown. Here we report the use of chromatin immunoprecipitation coupled with next generation sequencing (ChIP-SEQ) to identify genes that are regulated by Ikaros in vivo and to determine the role of Ikaros in chromatin remodeling in B-ALL.

Results reveal that Ikaros binds to the promoter regions of a large number of genes that are critical for cell cycle progression. These include CDC2, CDC16, CDC25A, ANAPC1, and ANAPC7. Overexpression of Ikaros in leukemia cells resulted in transcriptional repression of Ikaros target genes. Results from luciferase reporter assays performed using the respective promoters of Ikaros target genes support a role for Ikaros as a transcriptional repressor of these genes. Downregulation of Ikaros by siRNA resulted in increased expression of Ikaros target genes that control cell cycle progression. These results suggest that Ikaros functions as a negative regulator of cell cycle progression by repressing transcription of cell cycle-promoting genes. Next, we studied how Ikaros binding affects the epigenetic signature at promoters of Ikaros target genes. Global epigenetic mapping showed that Ikaros binding at the promoter region of cell cycle-promoting genes is associated with the formation of one of two types of repressive epigenetic marks – either H3K27me3 or H3K9me3. While these epigenetic marks were mutually exclusive, they were both associated with the loss of H3K9 acetylation and transcriptional repression. Serial qChIP assays spanning promoters of the Ikaros target genes revealed that the presence of H3K27me3 is associated with Ikaros and HDAC1 binding, while the H3K9me3 modification is associated with Ikaros binding and the absence of HDAC1. ChIP-SEQ analysis of HDAC1 global genomic binding demonstrated that over 80% of H3K27me3 modifications at promoter regions are associated with HDAC1 binding at surrounding sites. The treatment of leukemia cells with the histone deacetylase inhibitor – trichostatin (TSA) resulted in a severe reduction of global levels of H3K27me3, as evidenced by Wesern blot. These data suggest that HDAC1 activity in leukemia is essential for the formation of repressive chromatin that is characterized by the presence of H3K27me3.

Our data suggest that Ikaros binding at the promoters of its target genes can result in the formation of repressive chromatin by two distinct mechanisms: 1) direct Ikaros binding resulting in increased H3K9me³ or 2) Ikaros recruitment of HDAC1 with increased H3K27me³ modifications. These data suggest distinct mechanisms for the regulation of chromatin remodeling and target gene expression by Ikaros alone, and Ikaros in complex with HDAC1. In conclusion, the presented data suggest that HDAC1 has a key role in regulating cell cycle progression and proliferation in B-ALL. Our results identify novel, Ikaros-mediated mechanisms of epigenetic regulation that contribute to tumor suppression in leukemia. Supported by National Institutes of Health R01 HL095120, and the Four Diamonds Fund Endowment.