Critical roles of IKAROS and HDAC1 in regulation of heterochromatin and tumor suppression in T-cell acute lymphoblastic leukemia

Abstract

The IKZF1 gene encodes IKAROS - a DNA binding protein that acts as a tumor suppressor in T-cell acute lymphoblastic leukemia (T-ALL). IKAROS can act as a transcriptional repressor via recruitment of histone deacetylase 1 (HDAC1) and chromatin remodeling, however the mechanisms through which IKAROS exerts its tumor suppressor function via heterochromatin in T-ALL are largely unknown. We studied human and mouse T-ALL using a loss-of-function and IKZF1 re-expression approach, along with primary human T-ALL, and normal human and mouse thymocytes to establish the role of IKAROS and HDAC1 in global regulation of facultative heterochromatin and transcriptional repression in T-ALL. Results identified novel IKAROS and HDAC1 functions in T-ALL: Both IKAROS and HDAC1 are essential for EZH2 histone methyltransferase activity and formation of facultative heterochromatin; recruitment of HDAC1 by IKAROS is critical for establishment of H3K27me3 histone modification and repression of active enhancers; and IKAROS-HDAC1 complexes promote formation and expansion of H3K27me3 Large Organized Chromatin lysine (K) domains (LOCKs) and Broad Genic Repression Domains (BGRDs) in T-ALL. Our results establish the central role of IKAROS and HDAC1 in activation of EZH2, global regulation of the facultative heterochromatin landscape, and silencing of active enhancers that regulate oncogene expression.

Fig. 1. IKAROS binding induces de novo formation of facultative heterochromatin (H3K27me3).

A Heatmaps of H3K27me3 ChIP-seq signals at day 1 after IKAROS expression vs. day 0. Signals are centered on H3K27me3 peaks at day 1. B Heatmaps of IKAROS and H3K27me3 ChIP-seq signals with IKAROS direct binding regions at day 0 vs. day 1. Signals are centered on IKAROS and H3K27me3 peaks at day 1. C Examples of de novo-formed H3K27me3 enrichment that are induced by IKAROS binding. D De novo H3K27me3 regions classified by function of the DNA element. E, F Gene ontology and pathway enrichment analysis of genes associated with de novo H3K27me3 regions.

Fig. 2. IKAROS recruits and activates EZH2.

A Heatmaps of EZH2 and H3K27me3 ChIP-seq signals in IKAROS-null T-ALL. B, C Heatmaps of EZH2, IKAROS and H3K27me3 ChIP-seq signals in IKAROS-EZH2 occupied (B), and EZH2-only occupied (C) regions. D EZH2-only and EZH2-IKAROS occupied sites classified by function of the DNA element. E, F Gene ontology and pathway enrichment analysis of genes associated with EZH2-IKAROS complexes.

Fig. 3. IKAROS is essential for HDAC1 recruitment to the gene promoters.

A Heatmaps of HDAC1 ChIP-seq signals in IK-null T-ALL and day 1 following IKAROS re-expression. B HDAC1-only and HDAC1-IKAROS occupied sites classified by function of the DNA element. C Examples of IKAROS recruitment of HDAC1 to the promoters of IKAROS target genes. D Analysis of the differentially expressed genes directly regulated by IKAROS-HDAC1 complexes. E, F Gene ontology and pathway enrichment analysis of genes associated with IKAROS-HDAC1 complexes.

Fig. 4. Critical roles of IKAROS and Histone Deacetylase 1(HDAC1) in the global de novo formation of H3K27me3.

A, B IKAROS, HDAC1, and Enhancer of Zeste homologue 2(EZH2) occupancy associated with H3K27me3 formation Day 1 (A) and Day 2 (B) following Ikzf1 re-expression. C EZH2 target genes are regulated by the IKAROS and HDAC1-induced epigenetic switch. EZH2 DNA occupancy at target genes does not induce H3K27me3 in Ikzf1-null T-ALL (top). A large number (75%) of EZH2-target genes undergo epigenetic switch with de novo H3K27me3 formation following Ikzf1-re-expression (middle). Most of the EZH2 target genes that undergo epigenetic switch are regulated by IKAROS and HDAC1 binding (bottom).

Fig. 5. IKAROS and HDAC1 are repressors of active enhancers.

A A large number of active enhancers are occupied by IKAROS and HDAC1 after IKAROS re-expression. Pie charts showing the portions of active enhancers occupied by IKAROS, HDAC1, or both IKAROS and HDAC1 (B) Boxplot shows expression of genes regulated by the active enhancers not bound by IKAROS and/or HDAC1 (left) and occupied by IKAROS and/or HDAC1. C IKAROS and HDAC1 regulate formation of poised and intermediate enhancers. Pie charts showing the portions of primed, poised, and silenced enhancers occupied by IKAROS, HDAC1, or both IKAROS and HDAC1 in day 1 and 2 following Ikzf1 re-expression. IKAROS-HDAC1 co-occupancy is highly associated with formation of poised and intermediate enhancers in day 1, while HDAC1-only occupancy is dominant in those enhancers in day 2. D Boxplot shows expression of genes regulated by the active, primed, intermediate, and poised enhancers.

Fig. 6. IKAROS regulates formation and expansion of H3K27m3 Long organized Chromatin Lysine(K) domains(LOCKs).

A Number (left) and genomic coverage (right) of H3K27me3 LOCKs in day 1 and 2 following Ikzf1 re-expression. B Example of the genomic expansion of the H3K27me3 LOCKs from day 1 to 2 after Ikzf1 re-expression. C Number of genes regulated by the H3K27me3 LOCKs in day 1 and 2 following Ikzf1 re-expression. D Gene expression of the genes regulated by LOCKs vs. random genes.

Fig. 7. IKAROS regulates genomic occupancy and distribution of EZH2 and HDAC1 and H3K27me3 formation in human T-ALL.

A Number of H3K27me3 (left) and HDAC1 (right) peaks in wildtype MOLT-4 and in IKZF1-knockout (MOLT-IK-KO) cells. B H3K27me3 and HDAC1 occupied sites, in wildtype MOLT-4 and in IKZF1-knockout (MOLT-IK-KO) cells, classified by function of the DNA element. C, D Gene ontology and pathway enrichment analysis of genes associated with HDAC1 occupancy in wildtype MOLT-4 cells.

Fig. 8. Novel IKAROS functions in the regulation of heterochromatin formation.

IKAROS expression regulates: de novo formation of H3K27me3 by HDAC1 recruitment and activation of EZH2; repression of active enhancers; and formation and expansion of LOCKs and BGRDs.