Overexpression Of The Histone Demethylase JMJD1C In Polycythemia Vera Contributes To NF-E2 Overexpression Via Epigenetic Dysregulation and An Auto-Regulatory Loop

Abstract

Expression of the transcription factor and epigenetic modulator “nuclear factor erythroid-2” (NF-E2) is aberrantly elevated in patients with Myeloproliferative Neoplasms (MPN). We have recently shown that NF-E2 overexpression in a murine model causes a phenotype similar to MPN in humans. This includes thrombocytosis, leukocytosis, expansion of the stem- and progenitor cell compartments as well as spontaneous transformation to acute leukemia. However, both the downstream pathways by which this transcription factor exerts its effects as well as the mechanisms leading to its overexpression in MPN patients remain incompletely characterized.We show here that the histone demethylases JMJD1C and JMJD2C constitute novel NF-E2 target genes. Using chromatin immunoprecipitation (ChIP) we demonstrate NF-E2 binding to the JMJD1C and JMJD2C loci. Both JMJD1C and JMJD2C protein levels are statistically significantly elevated in patients with polycythemia vera (PV) compared to healthy controls.JMJD1C and 2C proteins mediate the demethylation of histone H3K9, converting H3K9me2 via H3K9me to unmethylated H3K9. Consistent with increased demethylase activity, we observed statistically significantly decreased levels of global H3K9me and H3K9me2 in PV patients compared to healthy controls.We subsequently investigated whether the NF-E2 gene is regulated by epigenetic mechanisms and whether these are altered in PV patients. Indeed, using ChIP we demonstrate presence of the repressive H3K9me2 mark at three sites within the NF-E2 locus in healthy control granulocytes, where NF-E2 is expressed at low levels. These repressive histone marks are significantly reduced or completely absent in PV patients, which overexpress NF-E2. At the same time, binding of the repressive heterochromatin protein 1-alpha (HP1a) to the NF-E2 gene, while present in healthy controls, is significantly reduced in PV patients. HP1a chromatin binding is dependent on the presence of the H3K9me2 modification. Decreased levels of H3K9me2 and decreased HP1a binding are therefore both consistent with elevated NF-E2 expression in PV.We hypothesized that increased levels of the H3K9me2 demethylase JMJD1C in PV patients are responsible for the loss of this histone mark in the NF-E2 gene. Consistent with this model, we demonstrate significantly increased binding of JMJD1C to the NF-E2 locus in purified primary PV granulocytes.Our data demonstrate that the NF-E2 gene is regulated by epigenetic mechanisms and that these histone modifications are perturbed in PV patients. Moreover, NF-E2 participates in an auto-regulatory loop, by directly regulating transcription of the histone demethylase JMJD1C. Increased levels of NF-E2 thus cause increased JMJD1C expression. The JMJD1C protein, in turn, demethylates H3K9me2 residues in the NF-E2 locus, thereby further augmenting NF-E2 expression. Besides sustaining this novel autoregulatory loop, increased JMJD1C activity may contribute to MPN pathology by altering gene expression at additional loci.We sought to determine whether elevated NF-E2 levels could be normalized by pharmacological intervention. Treatment with the nucleoside analogue Decitabine reduced NF-E2 expression, and restored both the H3K9me2 marks and HP1a binding to the NF-E2 locus.These data provide a molecular rational for pre-clinical investigation of the effects of histone demethylase inhibitors and Decitabine on MPN cell biology and, subsequently, for a phase I trial investigating a combination of Decitabine and histone demethylase inhibitors in MPN patients. Disclosures:No relevant conflicts of interest to declare.