Late Cell Cycle Genes Research Articles

A-MYB substitutes for B-MYB in activating cell cycle genes and in stimulating proliferation.

A-MYB (MYBL1) is a transcription factor with a role in meiosis in spermatocytes. The related B-MYB protein is a key oncogene and a master regulator activating late cell cycle genes. To activate genes, B-MYB forms a complex with MuvB and is recruited indirectly to cell cycle genes homology region (CHR) promoter sites of target genes. Activation through the B-MYB-MuvB (MMB) complex is essential for successful mitosis. Here, we discover that A-MYB has a function in transcriptional regulation of the mitotic cell cycle and can substitute for B-MYB. Knockdown experiments in cells not related to spermatogenesis show that B-MYB loss alone merely delays cell cycle progression. Only dual knockdown of B-MYB and A-MYB causes G2/M cell cycle arrest, endoreduplication, and apoptosis. A-MYB can substitute for B-MYB in binding to MuvB. The resulting A-MYB-MuvB complex activates genes through CHR sites. We find that A-MYB activates the same target genes as B-MYB. Many of the corresponding proteins are central regulators of the cell division cycle. In summary, we demonstrate that A-MYB is an activator of the mitotic cell cycle by activating late cell cycle genes.

A genome-wide long noncoding RNA CRISPRi screen identifies PRANCR as a novel regulator of epidermal homeostasis.

Genome-wide association studies indicate that many disease susceptibility regions reside in non-protein-coding regions of the genome. Long noncoding RNAs (lncRNAs) are a major component of the noncoding genome, but their biological impacts are not fully understood. Here, we performed a CRISPR interference (CRISPRi) screen on 2263 epidermis-expressed lncRNAs and identified nine novel candidate lncRNAs regulating keratinocyte proliferation. We further characterized a top hit from the screen, progenitor renewal associated non-coding RNA (PRANCR), using RNA interference–mediated knockdown and phenotypic analysis in organotypic human tissue. PRANCR regulates keratinocyte proliferation, cell cycle progression, and clonogenicity. PRANCR-deficient epidermis displayed impaired stratification with reduced expression of differentiation genes that are altered in human skin diseases, including keratins 1 and 10, filaggrin, and loricrin. Transcriptome analysis showed that PRANCR controls the expression of 1136 genes, with strong enrichment for late cell cycle genes containing a CHR promoter element. In addition, PRANCR depletion led to increased levels of both total and nuclear CDKN1A (also known as p21), which is known to govern both keratinocyte proliferation and differentiation. Collectively, these data show that PRANCR is a novel lncRNA regulating epidermal homeostasis and identify other lncRNA candidates that may have roles in this process as well.

Abstract 2538: p53 activation induces cell cycle arrest by promoting DREAM and RB repression of cell cycle genes

Abstract p53 activation results cell cycle arrest at the G1/S checkpoint and the repression of early (G1/S) and late (G2/M) cell cycle gene expression in a p21 dependent manner. The mechanism how p53 and p21 repress cell cycle gene expression is unclear. p21 is known to repress CDK2 which is a key inhibitor of the pocket protein family members RB and p130. During a normal cell cycle, p130 as part of the DREAM complex (DP1, RB-like p130, E2F4, and MuvB) and RB cooperate to repress expression of early (G1/S) and late (G2/M) cell cycle gene expression. DREAM binds and represses late (G2/M) cell cycle promoters through MuvB binding and early (G1/S) cell cycle promoters though repressor E2F binding. RB is only able to bind and repress early (G1/S) cell cycle promoters through its interaction with activator E2Fs. The requirement for DREAM and RB for control of cell cycle gene expression after p53 activation was unknown. We hypothesized that DREAM and RB would cooperate to repress cell cycle gene expression after DNA damage. Using primary human foreskin fibroblast cells lacking RB and/or p130, we found repression of early and late cell cycle gene expression is differentially regulated after p53 activation. We found that while RB is required for repression of early (G1/S) cell cycle genes after p53 activation, DREAM is required for repression of expression of late (G2/M) cell cycle genes. Surprisingly, DREAM was unable to repress early cell cycle genes after p53 activation, suggesting that RB is the key regulator of G1/S gene expression during cellular stress like DNA damage. Further, the requirement for DREAM to repress late cell cycle gene expression underlies the importance of DREAM to prevent expression of mitosis inducing genes in a state like DNA damage when mitosis could result in propagation of mutated DNA. We found that p107, a protein reported be able to replace p130 in DREAM, does not potently repress early cell cycle genes in HFFs under contact arrest and serum starvation. However, we found that p107 strongly represses expression of late cell cycle genes during G0 in cells lacking p130 alone or in combination with RB. These data provide evidence for a specific role for p107-DREAM not previously appreciated. Together, our data show that control of cell cycle gene expression after p53 activation is split in two: RB represses early cell cycle genes while DREAM represses late cell cycle genes. Citation Format: Amy E. Schade, Martin Fischer, James A. DeCaprio. p53 activation induces cell cycle arrest by promoting DREAM and RB repression of cell cycle genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2538.

Activation of the oncogenic transcription factor B-Myb via multisite phosphorylation and prolyl cis/trans isomerization.

The oncogenic transcription factor B-Myb is an essential regulator of late cell cycle genes whose activation by phosphorylation is still poorly understood. We describe a stepwise phosphorylation mechanism of B-Myb, which involves sequential phosphorylations mediated by cyclin-dependent kinase (Cdk) and Polo-like kinase 1 (Plk1) and Pin1-facilitated peptidyl-prolyl cis/trans isomerization. Our data suggest a model in which initial Cdk-dependent phosphorylation of B-Myb enables subsequent Pin1 binding and Pin1-induced conformational changes of B-Myb. This, in turn, initiates further phosphorylation of Cdk-phosphosites, enabling Plk1 docking and subsequent Plk1-mediated phosphorylation of B-Myb to finally allow B-Myb to stimulate transcription of late cell cycle genes. Our observations reveal novel mechanistic hierarchies of B-Myb phosphorylation and activation and uncover regulatory principles that might also apply to other Myb family members. Strikingly, overexpression of B-Myb and of factors mediating its activation strongly correlates with adverse prognoses for tumor patients, emphasizing B-Myb's role in tumorigenesis.

Abstract 4288: The cell cycle gene regulatory DREAM complex is disrupted by oncogenic B-Myb

Abstract The oncogene MYBL2 (encoding B-Myb) is a poor prognostic biomarker in many cancers. B-Myb interacts with the MuvB core of five proteins (LIN9, LIN37, LIN52, LIN53/RBBP4, and LIN54) to form the MMB (Myb-MuvB) complex and promotes expression of late cell cycle genes necessary for progression through mitosis. Both MYBL2 amplification and over-expression are associated with deregulation of the cell cycle and increased cell proliferation. Alternatively, by interacting with E2F4-DP1 and p130/p107, the MuvB core becomes part of the DREAM complex (DP, RB-like, E2F, and MuvB). The DREAM complex opposes MMB by globally repressing cell cycle genes in G0/G1, maintaining the cell in a quiescent state. However, the specific mechanism by which B-Myb alters the cell cycle is not well understood. Herein, we show that B-Myb disrupts the DREAM complex by sequestration and stabilization of LIN52. Analysis of The Cancer Genome Atlas data revealed significant upregulation of DREAM and MMB target genes in breast and ovarian cancer with MYBL2 gain. Given that most of the DREAM target genes are not directly regulated by B-Myb, we investigated the effects of B-Myb on DREAM formation. We found that depletion of B-Myb results in increased DREAM formation in cancer cell lines, while its overexpression inhibits DREAM formation in non-transformed cells. Since the MuvB core subunit LIN52 is essential for assembly of both the DREAM and MMB complexes, we tested whether B-Myb disrupts DREAM by sequestering LIN52. Overexpression of LIN52 did not increase either DREAM or MMB formation, but instead increased the turnover rate of the endogenous LIN52 protein. Interestingly, co-expression of B-Myb increased the expression of both endogenous and overexpressed LIN52 while knockdown of B-Myb had an opposite effect. We found that regulation of LIN52 occurs at the protein level, and that activity of DYRK1A kinase, the enzyme that triggers DREAM complex formation by phosphorylating LIN52, is required for this regulation. These findings are the first to implicate B-Myb in the disassembly of the DREAM complex and offer further mechanistic insights for cancers with MYBL2 amplification. We conclude that B-Myb's oncogenic effects are not only secondary to increased mitotic gene expression by the MMB complex, but also broad disruption of cell cycle gene regulatory programs through compromised DREAM formation. Citation Format: Audra N. Iness, Varsha Ananthapadmanabhan, Fatmata Sesay, Mikhail Dozmorov, Larisa Litovchick. The cell cycle gene regulatory DREAM complex is disrupted by oncogenic B-Myb [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4288.

Abstract 4353: Integrated molecular analysis of upper urinary tract urothelial carcinoma

Abstract Backgrounds Upper urinary tract urothelial carcinoma (UTUC) is a rare cancer accounting for 10% of all urothelial malignancies, whose molecular pathology, however, is poorly understood. To clarify diagnostic and prognostic impact of molecular profiles, we comprehensively investigated genetic alterations in UTUC. Materials & methods Surgical specimens of UTUC and matched normal samples were obtained from 102 patients with various stages, and were subjected to whole exome/RNA sequencing and array-based methylation analysis. Results UTUC cases displayed as many as 5.3 mutations per megabase (Mb). Seven cases showed hypermutation (>20/Mb), 6 of which harbored biallelic defects of mismatch repair (MMR) genes, suggesting the presence of Lynch syndrome in these. Genetic alterations were most frequently observed in TERT promoter (51% of cases), followed by KMT2D (48%), FGFR3 (46%, including fusion genes), CDKN2A (homozygous deletion) (42%), TP53 (31%), and RAS family genes (HRAS/KRAS/NRAS) (21%). More than 95% of cases harbored either TP53, MDM2, FGFR3, or RAS alterations in almost mutually exclusive manner. Hierarchical clustering on the basis of commonly altered genes in 95 non-hypermutated cases identified 3 distinct subgroups. Group 1 cases were frequently affected by TP53/MDM2 alterations and complex copy number alterations (CNAs), characterized by high stage, high grade, and poor prognosis. In Group2, FGFR3 and CDKN2A alterations and simple CNAs were enriched, which were associated with low stage/grade and favorable prognosis. All cases in Group3 harbored RAS-family gene alterations, showing intermediate prognosis. In mutation signature analysis, we extracted age-related, APOBEC, Transcriptional coupled repair (TCR), and MMR associated signatures. TCR signature was more frequently found in patients with an ALDH2 polymorphism implicated in low alcohol metabolism (rs671, GG/AG). MMR signature was observed only in the hypermutated cases with MMR deficiency. Gene expression analysis using unsupervised clustering identified 3 clusters. Cluster 1 was characterized by high expression of late cell-cycle genes, basal markers, and notably, immune blockade targets, CD274 and PDCD1LG2. Cluster 1 also harbored frequent TP53 alterations. In contrast, cases in cluster 2 and 3 showed high expression of early cell cycle genes and luminal markers. Cluster 2 were associated with FGFR3 mutations (positive in 75%), while almost all (92%) of RAS-mutated cases were classified in cluster 3. Finally, hierarchical analysis of methylation data revealed that approximately 60% of examined samples showed CpG island methylator phenotype (CIMP), which was positive in all the groups defined by mutational landscape. Conclusion UTUC showed distinct genetic landscape, associated with various clinical features. Our findings of molecular characteristics in UTUC will contribute to the development of novel diagnostics and therapeutics. Citation Format: Yoichi Fujii, Yusuke Sato, Hiromichi Suzuki, Tetsuichi Yoshizato, Yusuke Shiozawa, Kenichi Yoshida, Yuichi Shiraishi, Tohru Nakagawa, Hiroaki Nishimatsu, Toshikazu Okaneya, Masashi Sanada, Hideki Makishima, Satoru Miyano, Haruki Kume, Seishi Ogawa. Integrated molecular analysis of upper urinary tract urothelial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4353.

MP58-09 COMPREHENSIVE ANALYSIS OF UPPER URINARY TRACT UROTHELIAL CARCINOMA

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology II1 Apr 2018MP58-09 COMPREHENSIVE ANALYSIS OF UPPER URINARY TRACT UROTHELIAL CARCINOMA Yoichi Fujii, Yusuke Sato, Hiromichi Suzuki, Tetsuichi Yoshizato, Yusuke Shiozawa, Kenichi Yoshida, Yuichi Shiraishi, Tohru Nakagawa, Hiroaki Nishimatsu, Masashi Sanada, Hideki Makishima, Satoru Miyano, Seishi Ogawa, and Haruki Kume Yoichi FujiiYoichi Fujii More articles by this author , Yusuke SatoYusuke Sato More articles by this author , Hiromichi SuzukiHiromichi Suzuki More articles by this author , Tetsuichi YoshizatoTetsuichi Yoshizato More articles by this author , Yusuke ShiozawaYusuke Shiozawa More articles by this author , Kenichi YoshidaKenichi Yoshida More articles by this author , Yuichi ShiraishiYuichi Shiraishi More articles by this author , Tohru NakagawaTohru Nakagawa More articles by this author , Hiroaki NishimatsuHiroaki Nishimatsu More articles by this author , Masashi SanadaMasashi Sanada More articles by this author , Hideki MakishimaHideki Makishima More articles by this author , Satoru MiyanoSatoru Miyano More articles by this author , Seishi OgawaSeishi Ogawa More articles by this author , and Haruki KumeHaruki Kume More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.1841AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Upper urinary tract urothelial carcinoma (UTUC) is a relatively rare disease which accounts for 5-10% of all urothelial malignancies, and its molecular biology is poorly understood. We comprehensively investigated the genetic alterations of this disease. METHODS Surgical specimens of UTUC and matched normal samples were obtained from 102 patients with various stages who underwent nephroureterectomy, and were subjected to whole exome/RNA sequencing and methylation array. RESULTS UTUC displayed a large number of mutations with 5.3 mutations per megabase (Mb). 7 cases showed hypermutation (>20/Mb), 6 of which harbored biallelic mismatch repair (MMR) gene alterations, supporting UTUC is a Lynch syndrome-associated cancer. Genetic alterations were most frequently observed in TERT promoter (51% of cases), followed by KMT2D (48%), FGFR3 (46%, including fusion genes), CDKN2A (homozygous deletion) (42%), TP53 (31%), and RAS family genes (HRAS/KRAS/NRAS, 21%). More than 95% cases harbored either TP53, MDM2, FGFR3, or RAS alterations in almost mutually exclusive manner. Hierarchical clustering of frequently altered genes in 95 non-hypermutated cases identified 3 distinct subgroups. Group 1 is characterized by frequent TP53/MDM2 alterations with complex copy number alterations (CNAs), high stage/grade, and poor prognosis. Group2 is enriched in FGFR3 and CDKN2A alterations, showing simple CNAs, low stage/grade, and favorable prognosis. All cases in Group3 harbored RAS-family gene alterations. Gene expression analysis with unsupervised clustering identified 3 clusters. Cluster 1 was enriched for TP53 defective cases, showing high expression of late cell-cycle genes and basal markers. On the other hand, cluster 2 and 3 showed high expression of early cell cycle genes and luminal markers. Cases in cluster 2 were mainly FGFR3-mutanted cases (75%) while almost all of the RAS-mutated cases were classified in cluster 3 (92%). Notably, cluster 1, which showed poor prognosis, had significantly high expression of the immune blockade target CD274 and PDCD1LG2.Hierarchical analysis of methylation data revealed that approximately 60% of examined samples showed extensive hypermethylation (CIMP+). However, there was no significant correlation with the mutation subtypes. CONCLUSIONS UTUC showed distinct genetic landscape in terms of mutations, which was also associated with prognosis, clinical features, and expression pattern. Our findings of molecular characteristics of UTUC will contribute to the development of new potential diagnostics and therapeutics. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e776 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Yoichi Fujii More articles by this author Yusuke Sato More articles by this author Hiromichi Suzuki More articles by this author Tetsuichi Yoshizato More articles by this author Yusuke Shiozawa More articles by this author Kenichi Yoshida More articles by this author Yuichi Shiraishi More articles by this author Tohru Nakagawa More articles by this author Hiroaki Nishimatsu More articles by this author Masashi Sanada More articles by this author Hideki Makishima More articles by this author Satoru Miyano More articles by this author Seishi Ogawa More articles by this author Haruki Kume More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Non-Small Cell Lung Cancer Radiogenomics Map Identifies Relationships between Molecular and Imaging Phenotypes with Prognostic Implications.

Purpose To create a radiogenomic map linking computed tomographic (CT) image features and gene expression profiles generated by RNA sequencing for patients with non-small cell lung cancer (NSCLC). Materials and Methods A cohort of 113 patients with NSCLC diagnosed between April 2008 and September 2014 who had preoperative CT data and tumor tissue available was studied. For each tumor, a thoracic radiologist recorded 87 semantic image features, selected to reflect radiologic characteristics of nodule shape, margin, texture, tumor environment, and overall lung characteristics. Next, total RNA was extracted from the tissue and analyzed with RNA sequencing technology. Ten highly coexpressed gene clusters, termed metagenes, were identified, validated in publicly available gene-expression cohorts, and correlated with prognosis. Next, a radiogenomics map was built that linked semantic image features to metagenes by using the t statistic and the Spearman correlation metric with multiple testing correction. Results RNA sequencing analysis resulted in 10 metagenes that capture a variety of molecular pathways, including the epidermal growth factor (EGF) pathway. A radiogenomic map was created with 32 statistically significant correlations between semantic image features and metagenes. For example, nodule attenuation and margins are associated with the late cell-cycle genes, and a metagene that represents the EGF pathway was significantly correlated with the presence of ground-glass opacity and irregular nodules or nodules with poorly defined margins. Conclusion Radiogenomic analysis of NSCLC showed multiple associations between semantic image features and metagenes that represented canonical molecular pathways, and it can result in noninvasive identification of molecular properties of NSCLC. Online supplemental material is available for this article.

FOXM1 predicts overall and disease specific survival in muscle-invasive urothelial carcinoma and presents a differential expression between bladder cancer subtypes.

Forkhead box M1 (FOXM1) is a late cell cycle gene that plays a crucial role in carcinogenesis and chemotherapeutic drug resistance. In this study, the impact of FOXM1 expression on patient outcome was investigated for the first time in formalin fixed and paraffin embedded (FFPE) samples of chemotherapy naïve muscle-invasive bladder cancer (MIBC) patients. Expression analyses were performed on the Mannheim cohort (n=84) and validated on the independent Chungbuk cohort (n=61). In a Cox’ proportional hazards model, a distinct FOXM1 expression cut-off dividing both cohorts in a ‘high-risk’ and ‘low-risk’ group has been determined. Multivariate analyses showed that FOXM1 is an independent risk factor for outcome prediction superior to the TNM system. The FOXM1 ‘high-risk’ group had a 4- to 7-fold increased risk of death (p<0.03) and presented further an overexpression of MKI67. Recent studies showed that MIBCs can be subclassified in breast cancer-like subtypes: basal, luminal and p53-like. Here we demonstrated that FOXM1 was differentially expressed between MIBC subtypes concordant to its subtype specific expression in breast cancer. Since the proto-oncogene FOXM1 is known to play an important role in cisplatin resistance and to be a promising drug target, this study supports FOXM1 as a crucial biomarker in the personalization of MIBC therapy and urges prospective translational studies.

AURKA Overexpression Is Driven by FOXM1 and MAPK/ERK Activation in Melanoma Cells Harboring BRAF or NRAS Mutations: Impact on Melanoma Prognosis and Therapy

The cell cycle-related genes AURKA and FOXM1 are overexpressed in melanoma. We show here that AURKA overexpression is associated with poor prognosis in three independent cohorts of melanoma patients and correlates with the presence of genomic amplification of AURKA locus and BRAFV600E mutation. AURKA overexpression may also be driven by increased promoter activation through elements such as ETS and FOXM1 found within the 5' proximal promoter region. Activated MAPK/ERK signaling pathway mediates robust AURKA promoter activation, thereby knockdown of BRAFV600E and ERK inhibition results in reduced AURKA transcription and expression. We show a positive correlation between FOXM1 and AURKA expression in three independent cohorts of melanoma patients. FOXM1 silencing decreases expression of AURKA and late cell cycle genes in melanoma cells. We further found that FOXM1 expression levels are significantly higher in tumors carrying the BRAFV600E mutation compared with the wild-type BRAF (BRAFwt). Accordingly, the knockdown of BRAFV600E also reduces the expression of FOXM1 in BRAFV600E cells. Moreover, Aurora kinase A and FOXM1 inhibition by either genetic knockdown or pharmacologic inhibitors impair melanoma growth and survival both in culture and invivo, underscoring their therapeutic value for melanoma patients who fail to benefit from BRAF/MEK signaling inhibition.

Abstract A17: Cell cycle control through DREAM and MMB complexes

Abstract The loss of cell cycle control is the most recognized trait of cancer cells. The ability of cells to exit the cell cycle has recently been shown to be dependent upon a large multi-protein repressor complex called DREAM. This complex represses the expression of all cell cycle genes during reversible cell cycle exit. In cycling cells, DREAM is disassembled and some of DREAM associated proteins form a new complex to promote cell cycle gene expression. These proteins that can act together both as repressors and activators of cell cycle gene expression are called the MuvB core. The MuvB core interacts with B-Myb during S phase to promote late cell cycle gene expression. This activator complex is called MMB and its integrity is required for proper cell division. The purpose of this project is to determine how the MuvB core interacts with B-Myb, and if MuvB can be targeted by therapeutics to restore proper cell cycle control in cancer. We have structurally and functionally characterized the mechanism of DREAM assembly and regulation, however MMB regulation remains poorly understood. DREAM has been suggested as a therapeutic target in cancer cells that arrest in response to drug treatment to evade apoptosis. Disruption of DREAM could force evasive cancer cells back into the cell cycle and elicit a response to chemotherapeutic treatment. Until recently, a DREAM interface to target was unavailable. I solved the x-ray crystal structure of MuvB core protein LIN52 complexed with RB tumor suppressor protein p107 and functionally characterized how this interaction is regulated in DREAM assembly. This is a suitable DREAM interface to target given disruption of this interface drives cell cycle entry. I have found that LIN52 also interacts with B-Myb and have obtained crystals of this subcomplex that diffract to 2.4 Å resolution. Therefore, I hypothesize that the MuvB core protein LIN52 mediates interactions with various transcription factors throughout the cell cycle to drive gene specificity through cooperative binding. I will test this hypothesis with two aims: 1) solve the x-ray crystal structure of B-Myb complexed with the MuvB core and investigate the cooperative DNA binding of MuvB and B-Myb; 2) Disrupt LIN52 interactions to disassemble DREAM in drug resistant cancer cells. Completion of these aims will unveil the mechanism of B-Myb activation in cancer and determine if LIN52 is a viable cancer therapeutic target. Citation Format: Keelan Guiley, Seth M. Rubin. Cell cycle control through DREAM and MMB complexes. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr A17.

Timing of transcription during the cell cycle: Protein complexes binding to E2F, E2F/CLE, CDE/CHR, or CHR promoter elements define early and late cell cycle gene expression.

A central question in cell cycle control is how differential gene expression is regulated. Timing of expression is important for correct progression through the cell cycle. E2F, CDE, and CHR promoter sites have been linked to transcriptional repression in resting cells and activation during the cell cycle. Further, the DREAM complex binds CHR or CDE/CHR elements of G2/M genes resulting in repression during G0/G1. Here, we show that DREAM also binds to E2F sites of S phase genes in quiescence and upon p53 activation. Furthermore, we describe a novel class of promoter sites, the CHR-like elements (CLE), which can support binding of DREAM to E2F elements. Activation of such S phase genes is achieved through binding of E2F1-3/DP complexes to E2F sites. In contrast, the activating MuvB complexes MMB and FOXM1-MuvB bind to CHR elements and mediate peak expression in G2/M. In conclusion, data presented here in combination with earlier results leads us to propose a model that explains how DREAM can repress early cell cycle genes through E2F or E2F/CLE sites and late genes through CHR or CDE/CHR elements. Also p53-dependent indirect transcriptional repression through the p53-p21-Cyclin/CDK-DREAM-E2F/CLE/CDE/CHR pathway requires DREAM binding to E2F or E2F/CLE sites in early cell cycle genes and binding of DREAM to CHR or CDE/CHR elements of late cell cycle genes. Specific timing of activation is achieved through binding of E2F1-3/DP to E2F sites and MMB or FOXM1-MuvB complexes to CHR elements.

MEIS2 is essential for neuroblastoma cell survival and proliferation by transcriptional control of M-phase progression.

MEIS2 has an important role in development and organogenesis, and is implicated in the pathogenesis of human cancer. The molecular basis of MEIS2 action in tumorigenesis is not clear. Here, we show that MEIS2 is highly expressed in human neuroblastoma cell lines and is required for neuroblastoma cell survival and proliferation. Depletion of MEIS2 in neuroblastoma cells leads to M-phase arrest and mitotic catastrophe, whereas ectopic expression of MEIS2 markedly enhances neuroblastoma cell proliferation, anchorage-independent growth, and tumorigenicity. Gene expression profiling reveals an essential role of MEIS2 in maintaining the expression of a large number of late cell-cycle genes, including those required for DNA replication, G2-M checkpoint control and M-phase progression. Importantly, we identify MEIS2 as a transcription activator of the MuvB-BMYB-FOXM1 complex that functions as a master regulator of cell-cycle gene expression. Further, we show that FOXM1 is a direct target gene of MEIS2 and is required for MEIS2 to upregulate mitotic genes. These findings link a developmentally important gene to the control of cell proliferation and suggest that high MEIS2 expression is a molecular mechanism for high expression of mitotic genes that is frequently observed in cancers of poor prognosis.

The CHR site: definition and genome-wide identification of a cell cycle transcriptional element.

The cell cycle genes homology region (CHR) has been identified as a DNA element with an important role in transcriptional regulation of late cell cycle genes. It has been shown that such genes are controlled by DREAM, MMB and FOXM1-MuvB and that these protein complexes can contact DNA via CHR sites. However, it has not been elucidated which sequence variations of the canonical CHR are functional and how frequent CHR-based regulation is utilized in mammalian genomes. Here, we define the spectrum of functional CHR elements. As the basis for a computational meta-analysis, we identify new CHR sequences and compile phylogenetic motif conservation as well as genome-wide protein-DNA binding and gene expression data. We identify CHR elements in most late cell cycle genes binding DREAM, MMB, or FOXM1-MuvB. In contrast, Myb- and forkhead-binding sites are underrepresented in both early and late cell cycle genes. Our findings support a general mechanism: sequential binding of DREAM, MMB and FOXM1-MuvB complexes to late cell cycle genes requires CHR elements. Taken together, we define the group of CHR-regulated genes in mammalian genomes and provide evidence that the CHR is the central promoter element in transcriptional regulation of late cell cycle genes by DREAM, MMB and FOXM1-MuvB.

Regulation of a transcription factor network by Cdk1 coordinates late cell cycle gene expression

To maintain genome stability, regulators of chromosome segregation must be expressed in coordination with mitotic events. Expression of these late cell cycle genes is regulated by cyclin-dependent kinase (Cdk1), which phosphorylates a network of conserved transcription factors (TFs). However, the effects of Cdk1 phosphorylation on many key TFs are not known. We find that elimination of Cdk1-mediated phosphorylation of four S-phase TFs decreases expression of many late cell cycle genes, delays mitotic progression, and reduces fitness in budding yeast. Blocking phosphorylation impairs degradation of all four TFs. Consequently, phosphorylation-deficient mutants of the repressors Yox1 and Yhp1 exhibit increased promoter occupancy and decreased expression of their target genes. Interestingly, although phosphorylation of the transcriptional activator Hcm1 on its N-terminus promotes its degradation, phosphorylation on its C-terminus is required for its activity, indicating that Cdk1 both activates and inhibits a single TF. We conclude that Cdk1 promotes gene expression by both activating transcriptional activators and inactivating transcriptional repressors. Furthermore, our data suggest that coordinated regulation of the TF network by Cdk1 is necessary for faithful cell division.

The MuvB complex sequentially recruits B-Myb and FoxM1 to promote mitotic gene expression

Cell cycle progression is dependent on two major waves of gene expression. Early cell cycle gene expression occurs during G1/S to generate factors required for DNA replication, while late cell cycle gene expression begins during G2 to prepare for mitosis. Here we demonstrate that the MuvB complex-comprised of LIN9, LIN37, LIN52, LIN54, and RBBP4-serves an essential role in three distinct transcription complexes to regulate cell cycle gene expression. The MuvB complex, together with the Rb-like protein p130, E2F4, and DP1, forms the DREAM complex during quiescence and represses expression of both early and late genes. Upon cell cycle entry, the MuvB complex dissociates from p130/DREAM, binds to B-Myb, and reassociates with the promoters of late genes during S phase. MuvB and B-Myb are required for the subsequent recruitment of FoxM1 to late gene promoters during G2. The MuvB complex remains bound to FoxM1 during peak late cell cycle gene expression, while B-Myb binding is lost when it undergoes phosphorylation-dependent, proteasome-mediated degradation during late S phase. Our results reveal a novel role for the MuvB complex in recruiting B-Myb and FoxM1 to promote late cell cycle gene expression and in regulating cell cycle gene expression from quiescence through mitosis.

Germinal Center B Cell-Like (GCB) and Activated B Cell-Like (ABC) Type of Diffuse Large B Cell Lymphoma (DLBCL): Analysis of Molecular Predictors, Signatures, Cell Cycle State and Patient Survival

Aiming to find key genes and events, we analyze a large data set on diffuse large B-cell lymphoma (DLBCL) gene-expression (248 patients, 12196 spots). Applying the loess normalization method on these raw data yields improved survival predictions, in particular for the clinical important group of patients with medium survival time. Furthermore, we identify a simplified prognosis predictor, which stratifies different risk groups similarly well as complex signatures. We identify specific, activated B cell-like (ABC) and germinal center B cell-like (GCB) distinguishing genes. These include early (e.g. CDKN3) and late (e.g. CDKN2C) cell cycle genes. Independently from previous classification by marker genes we confirm a clear binary class distinction between the ABC and GCB subgroups. An earlier suggested third entity is not supported. A key regulatory network, distinguishing marked over-expression in ABC from that in GCB, is built by: ASB13, BCL2, BCL6, BCL7A, CCND2, COL3A1, CTGF, FN1, FOXP1, IGHM, IRF4, LMO2, LRMP, MAPK10, MME, MYBL1, NEIL1 and SH3BP5. It predicts and supports the aggressive behaviour of the ABC subgroup. These results help to understand target interactions, improve subgroup diagnosis, risk prognosis as well as therapy in the ABC and GCB DLBCL subgroups.

Expression of Late Cell Cycle Genes and an Increased Proliferative Capacity Characterize Very Early Relapse of Childhood Acute Lymphoblastic Leukemia

In childhood acute lymphoblastic leukemia (ALL), approximately 25% of patients suffer from relapse. In recurrent disease, despite intensified therapy, overall cure rates of 40% remain unsatisfactory and survival rates are particularly poor in certain subgroups. The probability of long-term survival after relapse is predicted from well-established prognostic factors (i.e., time and site of relapse, immunophenotype, and minimal residual disease). However, the underlying biological determinants of these prognostic factors remain poorly understood. Aiming at identifying molecular pathways associated with these clinically well-defined prognostic factors, we did gene expression profiling on 60 prospectively collected samples of first relapse patients enrolled on the relapse trial ALL-REZ BFM 2002 of the Berlin-Frankfurt-Münster study group. We show here that patients with very early relapse of ALL are characterized by a distinctive gene expression pattern. We identified a set of 83 genes differentially expressed in very early relapsed ALL compared with late relapsed disease. The vast majority of genes were up-regulated and many were late cell cycle genes with a function in mitosis. In addition, samples from patients with very early relapse showed a significant increase in the percentage of S and G(2)-M phase cells and this correlated well with the expression level of cell cycle genes. Very early relapse of ALL is characterized by an increased proliferative capacity of leukemic blasts and up-regulated mitotic genes. The latter suggests that novel drugs, targeting late cell cycle proteins, might be beneficial for these patients that typically face a dismal prognosis.

Inhibition of lung epithelial cell proliferation by hyperoxia. Posttranscriptional regulation of proliferation-related genes.

The alveolar surface of the lung is a major target for oxidant injury. After injury, repair of the alveolar epithelium is dependent on the ability of epithelial type 2 (T2) cells to proliferate. The regulation of T2 cell proliferation and the effect of reactive oxygen (O2) species on this lung cell proliferation have not been well defined. To investigate this process we focused on the regulation of two late cell cycle genes, histone and thymidine kinase, in T2 cells and fibroblasts exposed in vitro to varying periods of hyperoxia (95% O2). Hyperoxia for 24 to 48 h arrested cell proliferation in a SV40T-immortalized T2 cell line we have developed and in primary and SV40T-immortalized lung fibroblasts. Despite the cessation of proliferation, histone and TK mRNA continued to be expressed at high levels; mRNA half-lives were markedly prolonged but neither protein was translated. Thus proliferation arrest induced by hyperoxia was associated with posttranscriptional control of at least two late cell cycle-related genes. This form of proliferation arrest is also seen when primary and SV40T-T2 cells but not fibroblasts are serum deprived, suggesting that T2 cells in vitro may be uniquely sensitive to alterations in their redox state and that these alterations in turn affect translational control of a subset of proliferation-related genes.