Katrin Tschöp

Conserved Antagonism between JMJD2A/KDM4A and HP1γ during Cell Cycle Progression

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.

A kinase shRNA screen links LATS2 and the pRB tumor suppressor

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 pRBs 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.

Loss of RBF1 changes glutamine catabolism

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.

Cyclin B1 transcription is enhanced by the p300 coactivator and regulated during the cell cycle by a CHR-dependent repression mechanism

Cyclin B is a central regulator of transition from the G2 phase of the cell cycle to mitosis. In mammalian cells two B‐type cyclins have been characterised, cyclin B1 and B2. Both are expressed with a maximum in G2 and their synthesis is mainly regulated on the transcriptional level. We show that a single cell cycle genes homology region, lacking a functional cell cycle‐dependent element in tandem with it, contributes most of the cell cycle‐dependent transcription from the cyclin B1 promoter. The coactivator p300 binds to the cyclin B1 promoter and synergises with the transcription factor NF‐Y in activating transcription of cyclin B1.

Identification of Tcf-4 as a transcriptional target of p53 signalling.

T-cell factor (Tcf)-4 is a main transcription factor to pass on Wnt/β-catenin signalling. The tumour suppressor protein p53 contributes as a transcription factor to cell-cycle arrest and apoptosis induction. Mutations of components in p53 and Wnt/β-catenin signalling networks play a part in tumour formation. Here, we identify the Tcf-4 gene as a downstream effector of p53. Induction of wild-type p53 in a tet-off regulated human colon cell system leads to the reduction of Tcf-4 mRNA and protein levels. Also, mRNA of the Tcf-4 target gene uPAR is downregulated after p53 induction. Expression of a luciferase reporter controlled by the Tcf-4 promoter is repressed by wild-type p53, but not by a p53 mutant deficient in DNA binding. Such a regulation is seen in cell lines of different origin. These findings directly link Wnt/β-catenin signalling and p53 tumour suppressor function and may provide a mechanism by which loss of p53 function contributes to progression in the adenoma/carcinoma sequence in colon tumours. Furthermore, since Tcf-4 is expressed in many tissues and downregulation of Tcf-4 by p53 is seen in several different cell types, this regulation likely plays a role in proliferation control of all tissues that can express p53 and Tcf-4.

Three CCAAT-boxes and a single cell cycle genes homology region (CHR) are the major regulating sites for transcription from the human cyclin B2 promoter.

Cyclins are essential regulators of the cell division cycle. Cyclin B associates with the cyclin-dependent kinase 1 (cdc2) to form a complex which is required for cells to undergo mitosis. In mammalian cells three B-type cyclins have been characterised, cyclin B1, B2 and B3. The cell cycle-dependent synthesis of cyclin B1 and B2 has been investigated in detail displaying maximum expression in G2 which is mainly regulated on the transcriptional level. We have previously shown that this regulation of the mouse cyclin B2 promoter is controlled by a cell cycle-dependent element (CDE) and the cell cycle genes homology region (CHR). Also in a number of other genes CDE/CHR elements repress transcription in G0 and G1 and lead to relief of repression later during the cell cycle. Here, we compare human and mouse cyclin B2 promoters. Both promoters share only nine regions with nucleotide identities. Three of these sites are CCAAT-boxes spaced 33 bp apart which can bind the NF-Y transcriptional activator. NF-Y binding to the human cyclin B2 promoter could be shown by chromatin immunoprecipitation (ChIP) assays. Activation by NF-Y is responsible for more than 93% of the total promoter activity as measured by cotransfecting a plasmid coding for a dominant-negative form of NF-YA. Cell cycle-dependent repression is regulated solely through a CHR. Surprisingly, in contrast to the mouse promoter the CHR in the human cyclin B2 promoter does not rely on a CDE site in tandem with it. Together with the recently described mouse cdc25C promoter, human cyclin B2 is the second identified gene which solely requires a CHR for its cell cycle regulation.

Proteomic analysis of pRb loss highlights a signature of decreased mitochondrial oxidative phosphorylation

The retinoblastoma tumor suppressor (pRb) protein associates with chromatin and regulates gene expression. Numerous studies have identified Rb-dependent RNA signatures, but the proteomic effects of Rb loss are largely unexplored. We acutely ablated Rb in adult mice and conducted a quantitative analysis of RNA and proteomic changes in the colon and lungs, where Rb(KO) was sufficient or insufficient to induce ectopic proliferation, respectively. As expected, Rb(KO) caused similar increases in classic pRb/E2F-regulated transcripts in both tissues, but, unexpectedly, their protein products increased only in the colon, consistent with its increased proliferative index. Thus, these protein changes induced by Rb loss are coupled with proliferation but uncoupled from transcription. The proteomic changes in common between Rb(KO) tissues showed a striking decrease in proteins with mitochondrial functions. Accordingly, RB1 inactivation in human cells decreased both mitochondrial mass and oxidative phosphorylation (OXPHOS) function. RB(KO) cells showed decreased mitochondrial respiratory capacity and the accumulation of hypopolarized mitochondria. Additionally, RB/Rb loss altered mitochondrial pyruvate oxidation from (13)C-glucose through the TCA cycle in mouse tissues and cultured cells. Consequently, RB(KO) cells have an enhanced sensitivity to mitochondrial stress conditions. In summary, proteomic analyses provide a new perspective on Rb/RB1 mutation, highlighting the importance of pRb for mitochondrial function and suggesting vulnerabilities for treatment.

Expression of cyclin-dependent kinase subunit 1 (Cks1) is regulated during the cell cycle by a CDE/CHR tandem element and is downregulated by p53 but not by p63 or p73.

Cks1 is a member of the Cyclin-dependent kinase subunit family. These proteins are essential components of cyclin/cyclin-dependent kinase (cdk) complexes contributing to cell cycle control in all eukaryotes. Cks1 protein is found overexpressed in a number of tumors. Expression of Cks1 mRNA starts in late G1 reaching a peak in S/G2-phases of the cell cycle. We find that this expression pattern depends on transcriptional regulation and is controlled by a combination of a cell cycle-dependent element (CDE) together with a cell cycle genes homology region (CHR) in the Cks1 promoter. Furthermore, we observe Cks1 mRNA and protein to be downregulated after induced expression of the tumor suppressor p53. This repression is due to p53 downregulating transcription from the Cks1 promoter. p53-dependent repression is seen in a dose-dependent manner and in several cell types of different origin. In contrast to p53, its homologues p63 and p73 do not significantly repress transcription from the Cks1 promoter. The Cks1 promoter does not contain a p53 binding site. For some promoters the CCAAT box-binding transcription factor NF-Y had been implicated in p53-dependent repression. NF-Y is the main activator for Cks1 transcription but does not influence p53-dependent repression from the Cks1 promoter. Generally, the observation that the potential oncogene Cks1 is downregulated by the tumor suppressor p53 corresponds well with the idea that p53 employs multiple ways in order to halt the cell cycle.

Gene expression of cyclin-dependent kinase subunit Cks2 is repressed by the tumor suppressor p53 but not by the related proteins p63 or p73

Cks2 proteins are essential components of cyclin/cyclin‐dependent kinase complexes and contribute to cell cycle control. We identify Cks2 as a transcriptional target downregulated by the tumor suppressor p53. Cks2 expression was found to be repressed by p53 both at the mRNA and the protein levels. p53 downregulates transcription from the Cks2 promoter in a dose‐dependent manner and in all cell types tested. This repression appears to be independent of p53 binding to the Cks2 promoter. In contrast to p53, neither p63 nor p73 proteins can repress Cks2 transcription. Thus p53, rather than its homologues p63 and p73, may contribute to control of the first metaphase/anaphase transition of mammalian meiosis by downregulation of Cks2 expression.

Human cyclin B3. mRNA expression during the cell cycle and identification of three novel nonclassical nuclear localization signals.

Cyclins form complexes with cyclin‐dependent kinases. By controlling activity of the enzymes, cyclins regulate progression through the cell cycle. A‐ and B‐type cyclins were discovered due to their distinct appearance in S and G2 phases and their rapid proteolytic destruction during mitosis. Transition from G2 to mitosis is basically controlled by B‐type cyclins. In mammals, two cyclin B proteins are well characterized, cyclin B1 and cyclin B2. Recently, a human cyclin B3 gene was described. In contrast to the expression pattern of other B‐type cyclins, we find cyclin B3 mRNA expressed not only in S and G2/M cells but also in G0 and G1. Human cyclin B3 is expressed in different variants. We show that one isoform remains in the cytoplasm, whereas the other variant is translocated to the nucleus. Transport to the nucleus is dependent on three autonomous nonclassical nuclear localization signals that where previously not implicated in nuclear translocation. It had been shown that cyclin B3 coimmunoprecipitates with cdk2; but this complex does not exhibit any kinase activity. Furthermore, a degradation‐resistant version of cyclin B3 can arrest cells in G1 and G2. Taken together with the finding that cyclin B3 mRNA is not only expressed in G2/M but is also detected in significant amounts in resting cells and in G1 cells. This may suggest a dominant‐negative function of human cyclin B3 in competition with activating cyclins in G0 and the G1 phase of the cell cycle.