Integrative analysis of HIF binding and transactivation reveals its role in maintaining histone methylation homeostasis

Abstract

Adaptation to hypoxia is mediated through a coordinated transcriptional response driven largely by hypoxia-inducible factor 1 (HIF-1). We used ChIP-chip and gene expression profiling to identify direct targets of HIF-1 transactivation on a genome-wide scale. Several hundred direct HIF-1 targets were identified and, as expected, were highly enriched for proteins that facilitate metabolic adaptation to hypoxia. Surprisingly, there was also striking enrichment for the family of 2-oxoglutarate dioxygenases, including the jumonji-domain histone demethylases. We demonstrate that these histone demethylases are direct HIF targets, and their up-regulation helps maintain epigenetic homeostasis under hypoxic conditions. These results suggest that the coordinated increase in expression of several oxygen-dependent enzymes by HIF may help compensate for decreased levels of oxygen under conditions of cellular hypoxia.

Fig. 1.

HIF-1 ChIP-chip analysis and validation. (A) Representative integrated genome browser (IGB; Affymetrix) tracks showing peaks of probe intensities (vertical bars) arrayed by chromosomal position. For each locus, tracks are scaled identically. ChIP peaks were identified by using the MAT algorithm and were classified as hypoxia-unique, hypoxia-enriched, or nonspecific. (B) ChIP-chip results were validated by ChIP-qPCR using primer pairs surrounding the putative binding sites in the indicated loci. For each locus, the fold enrichment comparing HIF-1 ChIP DNA to input is represented in the bar graph (mean ± SD). To determine specificity for HIF-1, the bottom heat map depicts the fold enrichment comparing hypoxic to normoxic cells and control hypoxic cells to cells in which HIF-1α was specifically depleted with a lentiviral shRNA (sh-HIF1α). Primer pairs locate 5 and 10 kb upstream of the VEGF gene were used as controls.

Fig. 2.

Distribution of HIF-1-bound sites and association with transactivation. (A) Distribution of HIF-1 ChIP hits relative to the TSS of associated genes. (B) Distribution of core HRE-containing HIF-1 hits (HRE+) relate to the annotated structure of associated genes. (C) GSEA analysis of mRNA expression profiles for hypoxic (0.5%O₂, 12 h) vs. normoxic cells. Relative expression was rank-ordered by signal-to-noise (S2N) ratios of triplicate hypoxic samples vs. triplicate normoxic samples. Genes associated with HRE+ HIF-1-binding sites were strongly correlated with the hypoxic phenotype. In contrast, no such enrichment was evident for the ChIP-chip fragments without identifiable core HREs (HRE−). The color bar indicates up-regulated (red, positive S2N) and down-regulated (blue, negative S2N) genes.

Fig. 3.

Overrepresentation of 2-OG-dioxygenases among HIF-1 ChIP hits. (A) HIF-1 target genes in Cluster 1, with previously described HIF-1-bound targets indicated with an asterisk. (B) HIF-1-targeted 2-OG-dioxygenases in Cluster 2. Average change (Log2) in mRNA expression levels at 4, 8, and 12 h of hypoxia in HepG2, U87 and MDA-MB231 cells are indicated by using the assigned color scale. (C) Representative IGB tracks showing HIF-1 binding at promoter regions of the 2-OG-dioxygenase family genes in HepG2 and U87 cell lines. For each locus, tracks are scaled identically between hypoxia (H) and normoxia (N) samples.

Fig. 4.

Hypoxia- and HIF-dependent up-regulation of JHDMs. (A) GSEA analysis of mRNA expression profiles for hypoxic (12 h) vs. normoxic cells with gene set containing all known JmjC histone demethylases in HepG2 and U87 cells. (B) Gene expression changes under hypoxia in HepG2 cells. (C) Expression dataset for Grade IV glioblastoma was interrogated for changes in mRNA levels of the JmjC family. The most highly up-regulated JmjC family member is shown for normal tissue (N) vs. tumor (T) samples. Known HIF-1 targets are on the left. Data represented as the median (bar), 25–75th percentiles (box), and range (whiskers). (D) Quantitative RT-PCR verification of up-regulated expression of HIF-1-bound JmjC genes under different levels of hypoxia. Results are expressed as mean ± SD after normalization to β-actin. (E) shRNA depletion of HIF-1α, HIF-2α, HIF1α and 2α, or ARNT was used to determine HIF-dependent up-regulation of JmjC proteins. Data represent fold change comparing hypoxic with normoxic samples (mean ± SD).

Fig. 5.

Histone hypermethylations from loss of JHDM induction in hypoxia. (A) A representative immunoblot analysis of total histone H3 and the specified modifications in biological replicates (1 and 2) under normoxic (N) and hypoxic conditions (O2 levels indicated). (B) Quantitation of a single representative study is shown, with results for each modification normalized to total histone H3 within each sample (mean ± SD). (C) Accumulation of JARID1B protein under hypoxia was verified by Western blot analysis with a JARID1B-specific antibody, with specificity verified by shRNA depletion of JARID1B (Left). Knockdown of ARNT (sh-ARNT) (Right) reduces accumulation of JARID1B in hypoxia. (D) Global histone H3 and H3K4me3 levels were determined in unmodified HepG2 cells, control shRNA cells (sh-GFP), and ARNT depleted cells (sh-ARNT). Western blot analysis for histone H3 and H3K4me3 of duplicate biological replicates (1 and 2) is shown for a representative experiment. (E) Quantitated results represented as mean ± SD. (F) A HA-JARID1B expression construct was transfected into HepG2 sh-ARNT cells. Normoxic and hypoxic cells were stained with anti-HA (red) and anti-H3K4me3 (green) antibodies. In both conditions, overexpression of JARID1B reduced the H3K4me3 level by comparing transfected cells (with arrows) to nontransfected cells.