Epigenomic analysis of the HOX gene loci reveals mechanisms that may control canonical expression patterns in AML and normal hematopoietic cells

Abstract

HOX genes are highly expressed in many acute myeloid leukemia (AML) samples, but the patterns of expression and associated regulatory mechanisms are not clearly understood. We analyzed RNA sequencing data from 179 primary AML samples and normal hematopoietic cells to understand the range of expression patterns in normal versus leukemic cells. HOX expression in AML was restricted to specific genes in the HOXA or HOXB loci, and was highly correlated with recurrent cytogenetic abnormalities. However, the majority of samples expressed a canonical set of HOXA and HOXB genes that was nearly identical to the expression signature of normal hematopoietic stem/progenitor cells. Transcriptional profiles at the HOX loci were similar between normal cells and AML samples, and involved bidirectional transcription at the center of each gene cluster. Epigenetic analysis of a subset of AML samples also identified common regions of chromatin accessibility in AML samples and normal CD34(+) cells that displayed differences in methylation depending on HOX expression patterns. These data provide an integrated epigenetic view of the HOX gene loci in primary AML samples, and suggest that HOX expression in most AML samples represents a normal stem cell program that is controlled by epigenetic mechanisms at specific regulatory elements.

Figure 1

A) Patterns of HOX gene expression in primary AML samples. HOXA and HOXB gene expression values from RNA-seq data (log2 (FPKM+1)) are shown for 179 primary AML samples following unsupervised hierarchical clustering of the samples based solely on the expression of the genes shown, which identified four patterns defined by the presence or absence of HOXA and/or HOXB genes. The mutation status and cytogenetic category for each AML is shown in the table below the heatmap (32). B) HOXA and HOXB gene expression in normal hematopoietic cells at different developmental stages from RNA-seq data, including CD34⁺ progenitors (n=20), promyelocytes (Pro, n=3), monocytes (Mono, n=3), and neutrophils (PMN, n=3). C) Kaplan-Meier analysis of overall patient survival stratified by the HOX expression groups identified in panel A (n=179, P=0.006 across all groups; performed using Cox proportional hazard regression). D) Survival analysis of 184 AML patients with intermediate risk cytogenetic status based on HOX expression phenotype from microarray expression (data obtained from (48); see Figure S5; P=0.0006, Cox proportional hazard regression).

Figure 2

Transcriptional profiles of the HOXA and HOXB gene clusters from different AML subtypes. A) and B) show the median normalized RNA-seq read depth from the HOXA and HOXB loci, respectively, from AMLs with MLL translocations (MLLX, n=11), Normal Karyotype and NPMc mutations (n=47), Normal Karyotype without NPMc mutations (n=33), and normal CD34⁺ cell (n=20). Protein-coding genes (in blue) and noncoding transcripts (in gray) are shown below each plot. Note difference in scale of the Y axis across the sample types, indicating different expression levels despite the similarity in transcriptional patterns. C) Median expression (in FPKM) of transcript isoforms for expressed genes in the HOXA and HOXB clusters for AMLs in the indicated mutation or cytogenetic category and CD34⁺ cells.

Figure 3

Epigenetic analysis of the HOX loci in primary AML samples with characteristic HOX expression patterns. A) RNA-seq expression of the HOX genes from the samples used for epigenetic analysis, including PML-RARA, RUNX1-RUNX1T1, MLL-ELL, and normal karyotype AMLs with the HSPC-like HOX expression pattern and NPMc mutations (n=3 each). B) Methylation distributions from bisulfite sequencing of the HOX loci in primary AML samples, showing skewing towards less methylation in samples with HOXA expression (MLL-ELL and normal karyotype) and HOXB expression (normal karyotype only). C) Clustering of methylation values identified from differential methylation analysis of CpGs at the HOXA and HOXB locus between each AML set. D) Aggregate chromatin accessibility profiles at HOX gene promoters from each AML type. Each curve shows the mean normalized ATAC-seq signal across all HOXA and HOXB gene promoters from the indicated AML types (n=3 each), which demonstrates that AMLs with HOX expression (MLL-ELL and normal karyotype samples) have more open chromatin compared to those without HOX expression.

Figure 4

Integrated epigenomic profiles of the HOXA and HOXB gene clusters in primary AML samples and normal CD34⁺ cells. Panels A and B show DNA methylation and transposase-mediated chromatin accessibility (ATAC-seq) profiles from primary AML samples at the HOXA and HOXB loci, respectively. Methylation values reflect the coverage-weighted mean methylation ratio across all samples in each group. Chromatin accessibility values represent the median number of tags per 1 kbp per million mapped tags across each AML set (n=3 each). The bottom tracks show differentially methylated CpGs (DMCs) at each locus, and the ChIP-seq signal for CTCF from the K562 cell line from the ENCODE consortium (UT Austin) (52), respectively.

Figure 5

Differential methylation at common chromatin-accessible regions within the HOXA (panel A) and HOXB (panel B) gene clusters. Each panel shows chromatin accessibility and methylation at chromatin-accessible sites present in all AML samples and normal CD34⁺ cells, with common chromatin accessibility patterns and hypermethylation in samples without expression of nearby HOX genes. These loci occur near previously identified CTCF binding events in H1 hESCs and the K562 erythroleukemia cell line from the ENCODE consortium (UT Austin), (52).