Abstract
Posttranslational histone modifications serve to store epigenetic information and control both nucleosome assembly and recruitment of non-histone proteins. Histone methylation occurs on arginine and lysine residues and is involved in the regulation of gene transcription. A dynamic control of these modifications is exerted by histone methyltransferases and the recently discovered histone demethylases. Here we show that the hypoxia-inducible factor HIF-1alpha binds to specific recognition sites in the genes encoding the jumonji family histone demethylases JMJD1A and JMJD2B and induces their expression. Accordingly, hypoxic cells express elevated levels of JMJD1A and JMJD2B mRNA and protein. Furthermore, we find increased expression of JMJD1A and JMJD2B in renal cancer cells that have lost the von Hippel Lindau tumor suppressor protein VHL and therefore display a deregulated expression of hypoxia-inducible factor. Studies on ectopically expressed JMJD1A and JMJD2B indicate that both proteins retain their histone lysine demethylase activity in hypoxia and thereby might impact the hypoxic gene expression program.
Highlights
Methylation of histones contributes to dynamic changes in chromatin structure [1,2,3,4,5] and thereby influences gene expression, DNA replication, and repair [6, 7]
Among the group of 160 significantly upregulated mRNAs in the hypoxia-treated population we found the transcripts of the Jumonji C (JmjC) proteins JMJD1A and JMJD2B
We speculated that hypoxia-inducible factor (HIF) could induce the expression of some JmjC proteins in normal and transformed cells and that they might contribute to cancer progression
Summary
Cell Culture—Human renal proximal tubule epithelial cells (obtained from Lonza) were maintained in REGM with supplements and growth factors (Lonza) according to the provider’s instructions. Quantitative Real-time Reverse Transcription PCR—Total RNA was isolated from cells using an RNeasy mini kit (Qiagen) and treated with DNase I. The QuikChange site-directed mutagenesis kit (Stratagene) was used to create JMJD2B(H189G/E191Q) and JMJD1A(H1120G/D1122N) Both PCR products were verified by sequencing and transferred into a Gateway-compatible pCMV-HA expression vector. Analysis of the samples was performed by real time PCR as described above. Fragments were PCR-amplified from human genomic DNA using the primers described under supplemental information. To confirm staining specificity for H3K9me and H3K9me, fluorescence-activated cell sorting (FACS) was performed with and without preincubation of antibodies with H3 peptides containing methylated lysine residues, according to the procedure described by Chadwick et al [60]. Immunofluorescence—HeLa cells transfected with pCMVHA-hJMJD1A or pCMV-HA-hJMJD2B using FuGENE (Roche) were immediately transferred into hypoxia and after 24 h fixed in 4% paraformaldehyde/phosphate-buffered saline. Student’s paired t test was applied to reveal statistical significances. p values less than 0.05 were considered significant
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