The Cell Cycle Regulators CDK4, CDK6 and Cyclin D3 As Well As the Histone Methyltransferases MLL2 and MLL4 Constitute NF-E2 Target Genes That Participate in a Novel Regulatory Loop

Background: 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 shown that NF-E2 overexpression in a murine model causes a MPN phenotype. In addition, we have identified in-del mutations in the NF-E2 gene in MPN patients, which, despite having lost the ability to bind DNA, nonetheless increase the activity of wild type NF-E2. These mutants, which are acquired secondary to the JAK2^V617F mutation, confer a proliferative advantage beyond that provided by the JAK2^V617F alone. In all patients analyzed, cells carrying both mutations displayed clonal dominance, vastly outnumbering those carrying JAK2^V617Falone. Mutant NF-E2 causes an increase in the number of cells entering S-phase mediated by increased expression of the cell cycle regulators CDK4, CDK6 and CyclinD3 that promote the G1-S transition. However, it is not known whether these cell cycle regulators constitute direct NF-E2 target genes or whether the proliferative effect is indirect. Moreover, additional downstream pathways by which this augmented transcription factor activity exerts its effects remain unknown.

Aims: We therefore investigated whether CDK4, CDK6 and Cyclin D3 constitute direct NF-E2 target genes. Moreover, we identified additional novel NF-E2 targets in an effort to delineate the molecular mechanism by which increased NF-E2 activity contributes to MPN pathophysiology.

Methods: We used ChIP and luciferase reporter assays as well as qRT-PCR and Western Blot to identify and characterize novel NF-E2 targets.

Results: Our data show that both the cell cycle regulators CDK4, CDK6 and CyclinD3 as well as the histone methyltransferases MLL2 and MLL4 constitute direct NF-E2 target genes, as the transcription factor binds these genes in vivo.

ChIP analysis of HEL cells shows NF-E2 binding to a site in the CDK4 promoter, four sites in the CDK6 and the Cyclin D3 locus each, to the MLL4 promoter as well as to three sites in the MLL2 locus, establishing these five genes as direct NF-E2 targets. Activity of NF-E2 on the MLL4 promoter was confirmed by reporter gene assays. Expression of both the cell cycle regulators and the methyltransferases was statistically significantly elevated in bone marrow from NF-E2 overexpressing mice. Most importantly, quantitative RT-PCR showed significantly increased mRNA expression of all five target genes in primary cells of patients with polycythemia vera (PV) compared to healthy controls. Hence, the expression of the cell cycle regulators and the epigenetically active enzymes is augmented in PV patients. Corresponding to the increased methyltransferase activity, primary cells from PV patients showed a significant elevation in global H3K4m1 levels, a chromatin mark conferred by the MLL proteins.

The MLL2 protein has been previously shown to interact with NF-E2. NF-E2 serves as a scaffold and is required both for recruitment of MLL2 to the beta-globin locus as well as for chromatin remodeling at these sites. Here we show that MLL2 binds the CDK6 locus at the same sites as NF-E2, strongly suggesting that NF-E2 likewise acts to direct MLL2 to the CDK6 gene.

Our data establish a novel interaction network where NF-E2 both regulates the expression levels of MLL2 while at the same time modulating its epigenetic activity at NF-E2 target genes, which include critical cell cycle regulators.

Summary / Conclusions: Here we show that the cell cycle regulators CDK4, CDK6 and Cyclin D3 as well as the histone methyltransferases MLL2 and MLL4 constitute novel NF-E2 target genes. We propose that elevated NF-E2 levels in MPN patients contribute to disease pathophysiology by participating in a novel regulatory loop, which both enhances transcription and alteres histone methylation leading to increased proliferation. These data provide a molecular basis for pre-clinical investigation into the effects of CDK 4/6 inhibitors as well as of histone methyltransferase inhibitors on MPN cell biology.