The KDM4/JMJD2 family of histone demethylases is amplified in human cancers. However, little is known about their physiologic or tumorigenic roles. We have identified a conserved and unappreciated role for the JMJD2A/KDM4A H3K9/36 tridemethylase in cell cycle progression. We demonstrate that JMJD2A protein levels are regulated in a cell cycle-dependent manner and that JMJD2A overexpression increased chromatin accessibility, S phase progression, and altered replication timing of specific genomic loci. These phenotypes depended on JMJD2A enzymatic activity. Strikingly, depletion of the only C. elegans homolog, JMJD-2, slowed DNA replication and increased ATR/p53-dependent apoptosis. Importantly, overexpression of HP1γ antagonized JMJD2A-dependent progression through S phase, and depletion of HPL-2 rescued the DNA replication-related phenotypes in jmjd-2(-/-) animals. Our findings describe a highly conserved model whereby JMJD2A regulates DNA replication by antagonizing HP1γ and controlling chromatin accessibility.

E2F transcription factors are important regulators of cell proliferation and are frequently dysregulated in human malignancies. To identify novel regulators of E2F function, we used Drosophila as a model system to screen for mutations that modify phenotypes caused by reduced levels of dE2F1. This screen identified components of the Pumilio translational repressor complex (Pumilio, Nanos, and Brain tumor) as suppressors of dE2F1-RNAi phenotypes. Subsequent experiments provided evidence that Pumilio complexes repress dE2F1 levels and that this mechanism of post-transcriptional regulation is conserved in human cells. The human Pumilio homologs Pum 1 and Pum 2 repress the translation of E2F3 by binding to the E2F3 39 untranslated region (UTR) and also enhance the activity of multiple E2F3 targeting microRNAs (miRNAs). E2F3 is an oncogene with strong proliferative potential and is regularly dysregulated or overexpressed in cancer. Interestingly, Pumilio/miRNA-mediated regulation of E2F3 is circumvented in cancer cells in several different ways. Bladder carcinomas selectively downregulate miRNAs that cooperate with Pumilio to target E2F3, and multiple tumor cell lines shorten the 39 end of the E2F3 mRNA, removing the Pumilio regulatory elements. These studies suggest that Pumilio-miRNA repression of E2F3 translation provides an important level of E2F regulation that is frequently abrogated in cancer cells.

pRB-mediated inhibition of cell proliferation is a complex process that depends on the action of many proteins. However, little is known about the specific pathways that cooperate with the Retinoblastoma protein (pRB) and the variables that influence pRB's ability to arrest tumor cells. Here we describe two shRNA screens that identify kinases that are important for pRB to suppress cell proliferation and pRB-mediated induction of senescence markers. The results reveal an unexpected effect of LATS2, a component of the Hippo pathway, on pRB-induced phenotypes. Partial knockdown of LATS2 strongly suppresses some pRB-induced senescence markers. Further analysis shows that LATS2 cooperates with pRB to promote the silencing of E2F target genes, and that reduced levels of LATS2 lead to defects in the assembly of DREAM (DP, RB [retinoblastoma], E2F, and MuvB) repressor complexes at E2F-regulated promoters. Kinase assays show that LATS2 can phosphorylate DYRK1A, and that it enhances the ability of DYRK1A to phosphorylate the DREAM subunit LIN52. Intriguingly, the LATS2 locus is physically linked with RB1 on 13q, and this region frequently displays loss of heterozygosity in human cancers. Our results reveal a functional connection between the pRB and Hippo tumor suppressor pathways, and suggest that low levels of LATS2 may undermine the ability of pRB to induce a permanent cell cycle arrest in tumor cells.

Inactivation of the retinoblastoma tumor suppressor (pRB) alters the expression of a myriad of genes. To understand the altered cellular environment that these changes create, we took advantage of the Drosophila model system and used targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) to profile the metabolic changes that occur when RBF1, the fly ortholog of pRB, is removed. We show that RBF1-depleted tissues and larvae are sensitive to fasting. Depletion of RBF1 causes major changes in nucleotide synthesis and glutathione metabolism. Under fasting conditions, these changes interconnect, and the increased replication demand of RBF1-depleted larvae is associated with the depletion of glutathione pools. In vivo 13 C isotopic tracer analysis shows that RBF1-depleted larvae increase the flux of glutamine toward glutathione synthesis, presumably to minimize oxidative stress. Concordantly, H 2 O 2 preferentially promoted apoptosis in RBF1-depleted tissues, and the sensitivity of RBF1-depleted animals to fasting was specifically suppressed by either a glutamine supplement or the antioxidant N-acetyl-cysteine. Effects of pRB activation/inactivation on glutamine catabolism were also detected in human cell lines. These results show that the inactivation of RB proteins causes metabolic reprogramming and that these consequences of RBF/RB function are present in both flies and human cell lines.