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.

Cell cycle regulation: p53-p21-RB signaling.

The retinoblastoma protein RB and the transcription factor p53 are central tumor suppressors. They are often found inactivated in various tumor types. Both proteins play central roles in regulating the cell division cycle. RB forms complexes with the E2F family of transcription factors and downregulates numerous genes. Among the RB-E2F target genes, a large number code for key cell cycle regulators. Their transcriptional repression by the RB-E2F complex is released through phosphorylation of RB, leading to expression of the cell cycle regulators. The release from repression can be prevented by the cyclin-dependent kinase inhibitor p21/CDKN1A. The CDKN1A gene is transcriptionally activated by p53. Taken together, these elements constitute the p53-p21-RB signaling pathway. Following activation of p53, for example by viral infection or induction of DNA damage, p21 expression is upregulated. High levels of p21 then result in RB-E2F complex formation and downregulation of a large number of cell cycle genes. Thus, p53-dependent transcriptional repression is indirect. The reduced expression of the many regulators leads to cell cycle arrest. Examination of the p53-p21-RB targets and genes controlled by the related p53-p21-DREAM signaling pathway reveals that there is a large overlap of the two groups. Mechanistically this can be explained by replacing RB-E2F complexes with the DREAM transcriptional repressor complex at E2F sites in target promoters. In contrast to RB-E2F, DREAM can downregulate genes also through CHR transcription factor binding sites. This results in a distinct gene set controlled by p53-p21-DREAM signaling independent of RB-E2F. Furthermore, RB has non-canonical functions without binding to E2F and DNA. Such a role of RB supporting DREAM formation may be exerted by the RB-SKP2-p27-cyclin A/E-CDK2-p130-DREAM link. In the current synopsis, the mechanism of regulation by p53-p21-RB signaling is assessed and the overlap with p53-p21-DREAM signaling is examined.

Ki-67 gene expression.

Ki-67 serves as a prominent cancer marker. We describe how expression of the MKI67 gene coding for Ki-67 is controlled during the cell cycle. MKI67 mRNA and Ki-67 protein are maximally expressed in G2 phase and mitosis. Expression is dependent on two CHR elements and one CDE site in the MKI67 promoter. DREAM transcriptional repressor complexes bind to both CHR sites and downregulate the expression in G0/G1 cells. Upregulation of MKI67 transcription coincides with binding of B-MYB-MuvB and FOXM1-MuvB complexes from S phase into G2/M. Importantly, binding of B-MYB to the two CHR elements correlates with loss of CHR-dependent MKI67 promoter activation in B-MYB-knockdown experiments. In knockout cell models, we find that DREAM/MuvB-dependent transcriptional control cooperates with the RB Retinoblastoma tumor suppressor. Furthermore, the p53 tumor suppressor indirectly downregulates transcription of the MKI67 gene. This repression by p53 requires p21/CDKN1A. These results are consistent with a model in which DREAM, B-MYB-MuvB, and FOXM1-MuvB together with RB cooperate in cell cycle-dependent transcription and in transcriptional repression following p53 activation. In conclusion, we present mechanisms how MKI67 gene expression followed by Ki-67 protein synthesis is controlled during the cell cycle and upon induction of DNA damage, as well as upon p53 activation.

DNA Affinity Purification: A Pulldown Assay for Identifying and Analyzing Proteins Binding to Nucleic Acids.

The interaction of proteins with DNA plays a central role in gene regulation. We describe a DNA affinity purification method that allows for identification and analysis of protein complex components. For example, a DNA probe carrying a transcription factor binding site is used to purify proteins from a nuclear extract. The proteins binding to the probe are then identified by mass spectrometry. In similar experiments, proteins purified by this pulldown method can be analyzed by Western blot. Employing this method, we found that the DREAM transcriptional repressor complex binds to CHR transcriptional elements in promoters of cell cycle genes. This complex is important for cell cycle-dependent repression and as part of the p53-DREAM pathway serves as a link for indirect transcriptional repression of target genes by the tumor suppressor p53. In general, the methods described can be applied for the identification and analysis of proteins binding to DNA.

N- and O-glycosylation patterns and functional testing of CGB7 versus CGB3/5/8 variants of the human chorionic gonadotropin (hCG) beta subunit.

The classical function of human chorionic gonadotropin (hCG) is its role in supporting pregnancy. hCG is a dimer consisting of two highly glycosylated subunits, alpha (CGA) and beta (CGB). The beta-hCG protein is encoded by CGB3, CGB5, CGB7 and CGB8 genes. CGB3, 5 and 8 code for an identical protein, CGB3/5/8, whereas CGB7 differs in three amino acids from CGB3/5/8. We had observed earlier that CGB7 and CGB3/5/8 display very distinct tissue expression patterns and that the tumor suppressor and transcription factor p53 can activate expression of CGB7 but not of CGB3/5/8 genes. Here, we investigate the glycan structures and possible functional differences of the two CGB variants. To this end, we established a system to produce and isolate recombinant CGA, CGB7 and CGB3/5/8 proteins. We found that N- and O-glycosylation patterns of CGB7 and CGB3/5/8 are quite similar. Functional assays were performed by testing activation of the ERK1/2 pathway and demonstrated that CGB7 and CGB5/5/8 appear to be functionally redundant isoforms, although a slight difference in the kinetics of ERK1/2 pathway activation was observed. This is the first time that biological activity of CGB7 is shown. In summary, the results lead to the hypothesis that CGB7 and CGB3/5/8 do not hold significant functional differences but that timing and cell type of their expression is the key for understanding their divergent evolution.

The Role of lncRNAs TAPIR-1 and -2 as Diagnostic Markers and Potential Therapeutic Targets in Prostate Cancer.

In search of new biomarkers suitable for the diagnosis and treatment of prostate cancer, genome-wide transcriptome sequencing was carried out with tissue specimens from 40 prostate cancer (PCa) and 8 benign prostate hyperplasia patients. We identified two intergenic long non-coding transcripts, located in close genomic proximity, which are highly expressed in PCa. Microarray studies on a larger cohort comprising 155 patients showed a profound diagnostic potential of these transcripts (AUC~0.94), which we designated as tumor associated prostate cancer increased lncRNA (TAPIR-1 and -2). To test their therapeutic potential, knockdown experiments with siRNA were carried out. The knockdown caused an increase in the p53/TP53 tumor suppressor protein level followed by downregulation of a large number of cell cycle- and DNA-damage repair key regulators. Furthermore, in radiation therapy resistant tumor cells, the knockdown leads to a renewed sensitization of these cells to radiation treatment. Accordingly, in a preclinical PCa xenograft model in mice, the systemic application of nanoparticles loaded with siRNA targeting TAPIR-1 significantly reduced tumor growth. These findings point to a crucial role of TAPIR-1 and -2 in PCa.

DREAM and RB cooperate to induce gene repression and cell-cycle arrest in response to p53 activation.

Most human cancers acquire mutations causing defects in the p53 signaling pathway. The tumor suppressor p53 becomes activated in response to genotoxic stress and is essential for arresting the cell cycle to facilitate DNA repair or to initiate apoptosis. p53-induced cell cycle-arrest is mediated by expression of the CDK inhibitor p21WAF1/Cip1, which prevents phosphorylation and inactivation of the pocket proteins RB, p130, and p107. In a hypophosphorylated state, pocket proteins bind to E2F factors forming RB-E2F and DREAM transcriptional repressor complexes. Here, we analyze the influence of RB and DREAM on p53-induced gene repression and cell-cycle arrest. We show that abrogation of DREAM function by knockout of the DREAM component LIN37 results in a reduced repression of cell-cycle genes. We identify the genes repressed by the p53-DREAM pathway and describe a set of genes that is downregulated by p53 independent of LIN37/DREAM. Most strikingly, p53-dependent repression of cell-cycle genes is completely abrogated in LIN37-/-;RB-/- cells leading to a loss of the G1/S checkpoint. Taken together, we show that DREAM and RB are key factors in the p53 signaling pathway to downregulate a large number of cell-cycle genes and to arrest the cell cycle at the G1/S transition.

The Special AT-rich Sequence Binding Protein 1 (SATB1) and its role in solid tumors.

The Special AT-rich Sequence Binding Protein 1 (SATB1) exerts multiple functions, by influencing the structural organization of chromatin and interacting with several co-activators and co-repressors of transcription. Thus, SATB1 affects the expression of various genes by multiple mechanisms of action, involving three-dimensional chromatin architecture. More recently, SATB1 has been connected with solid tumors, tumorigenesis, tumor progression and tumor immunity. On the diagnostic side, SATB1 levels were found to correlate with clinicopathological features like increased TNM stage, reduced tumor differentiation, and a shorter overall survival. SATB1 expression was also identified as an independent prognostic marker in various cancers. Moreover, different gene knockdown or ectopic overexpression strategies in cancer cells have identified SATB1 to affect proliferation, cell cycle, apoptosis, cell morphology / cell polarity, EMT and multidrug-resistance as well as tumor formation, growth, invasion and metastasis in vivo. These processes are mediated through a great multitude of SATB1 target genes, including many (proto-) oncogenes. Functional and molecular studies on SATB1 in various cancers are comprehensively summarized, and the prospects and caveats of SATB1 as tumor marker and as putative target molecule are discussed.

Cell cycle arrest through indirect transcriptional repression by p53: I have a DREAM.

Activation of the p53 tumor suppressor can lead to cell cycle arrest. The key mechanism of p53-mediated arrest is transcriptional downregulation of many cell cycle genes. In recent years it has become evident that p53-dependent repression is controlled by the p53-p21-DREAM-E2F/CHR pathway (p53-DREAM pathway). DREAM is a transcriptional repressor that binds to E2F or CHR promoter sites. Gene regulation and deregulation by DREAM shares many mechanistic characteristics with the retinoblastoma pRB tumor suppressor that acts through E2F elements. However, because of its binding to E2F and CHR elements, DREAM regulates a larger set of target genes leading to regulatory functions distinct from pRB/E2F. The p53-DREAM pathway controls more than 250 mostly cell cycle-associated genes. The functional spectrum of these pathway targets spans from the G1 phase to the end of mitosis. Consequently, through downregulating the expression of gene products which are essential for progression through the cell cycle, the p53-DREAM pathway participates in the control of all checkpoints from DNA synthesis to cytokinesis including G1/S, G2/M and spindle assembly checkpoints. Therefore, defects in the p53-DREAM pathway contribute to a general loss of checkpoint control. Furthermore, deregulation of DREAM target genes promotes chromosomal instability and aneuploidy of cancer cells. Also, DREAM regulation is abrogated by the human papilloma virus HPV E7 protein linking the p53-DREAM pathway to carcinogenesis by HPV. Another feature of the pathway is that it downregulates many genes involved in DNA repair and telomere maintenance as well as Fanconi anemia. Importantly, when DREAM function is lost, CDK inhibitor drugs employed in cancer treatment such as Palbociclib, Abemaciclib and Ribociclib can compensate for defects in early steps in the pathway upstream from cyclin/CDK complexes. In summary, the p53-p21-DREAM-E2F/CHR pathway controls a plethora of cell cycle genes, can contribute to cell cycle arrest and is a target for cancer therapy.

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.

Human papilloma virus E7 oncoprotein abrogates the p53-p21-DREAM pathway.

High risk human papilloma viruses cause several types of cancer. The HPV oncoproteins E6 and E7 are essential for oncogenic cell transformation. E6 mediates the degradation of the tumor suppressor p53, and E7 can form complexes with the retinoblastoma pRB tumor suppressor. Recently, it has been shown that HPV E7 can also interfere with the function of the DREAM transcriptional repressor complex. Disruption of DREAM-dependent transcriptional repression leads to untimely early expression of central cell cycle regulators. The p53-p21-DREAM pathway represents one important means of cell cycle checkpoint activation by p53. By activating this pathway, p53 can downregulate transcription of genes controlled by DREAM. Here, we present a genome-wide ranked list of genes deregulated by HPV E7 expression and relate it to datasets of cell cycle genes and DREAM targets. We find that DREAM targets are generally deregulated after E7 expression. Furthermore, our analysis shows that p53-dependent downregulation of DREAM targets is abrogated when HPV E7 is expressed. Thus, p53 checkpoint control is impaired by HPV E7 independently of E6. In summary, our analysis reveals that disruption of DREAM through the HPV E7 oncoprotein upregulates most, if not all, cell cycle genes and impairs p53's control of cell cycle checkpoints.

The p53-p21-DREAM-CDE/CHR pathway regulates G2/M cell cycle genes.

The tumor suppressor p53 functions predominantly as a transcription factor by activating and downregulating gene expression, leading to cell cycle arrest or apoptosis. p53 was shown to indirectly repress transcription of the CCNB2, KIF23 and PLK4 cell cycle genes through the recently discovered p53-p21-DREAM-CDE/CHR pathway. However, it remained unclear whether this pathway is commonly used. Here, we identify genes regulated by p53 through this pathway in a genome-wide computational approach. The bioinformatic analysis is based on genome-wide DREAM complex binding data, p53-depedent mRNA expression data and a genome-wide definition of phylogenetically conserved CHR promoter elements. We find 210 target genes that are expected to be regulated by the p53-p21-DREAM-CDE/CHR pathway. The target gene list was verified by detailed analysis of p53-dependent repression of the cell cycle genes B-MYB (MYBL2), BUB1, CCNA2, CCNB1, CHEK2, MELK, POLD1, RAD18 and RAD54L. Most of the 210 target genes are essential regulators of G2 phase and mitosis. Thus, downregulation of these genes through the p53-p21-DREAM-CDE/CHR pathway appears to be a principal mechanism for G2/M cell cycle arrest by p53.

Indirect p53-dependent transcriptional repression of Survivin, CDC25C, and PLK1 genes requires the cyclin-dependent kinase inhibitor p21/CDKN1A and CDE/CHR promoter sites binding the DREAM complex.

The transcription factor p53 is central to cell cycle control by downregulation of cell cycle-promoting genes upon cell stress such as DNA damage. Survivin (BIRC5), CDC25C, and PLK1 encode important cell cycle regulators that are repressed following p53 activation. Here, we provide evidence that p53-dependent repression of these genes requires activation of p21 (CDKN1A, WAF1, CIP1). Chromatin immunoprecipitation (ChIP) data indicate that promoter binding of B-MYB switches to binding of E2F4 and p130 resulting in a replacement of the MMB (Myb-MuvB) by the DREAM complex. We demonstrate that this replacement depends on p21. Furthermore, transcriptional repression by p53 requires intact DREAM binding sites in the target promoters. The CDE and CHR cell cycle promoter elements are the sites for DREAM binding. These elements as well as the p53 response of Survivin, CDC25C, and PLK1 are evolutionarily conserved. No binding of p53 to these genes is detected by ChIP and mutation of proposed p53 binding sites does not alter the p53 response. Thus, a mechanism for direct p53-dependent transcriptional repression is not supported by the data. In contrast, repression by DREAM is consistent with most previous findings and unifies models based on p21-, E2F4-, p130-, and CDE/CHR-dependent repression by p53. In conclusion, the presented data suggest that the p53-p21-DREAM-CDE/CHR pathway regulates p53-dependent repression of Survivin, CDC25C, and PLK1.

Cyclin F suppresses B-Myb activity to promote cell cycle checkpoint control.

Cells respond to DNA damage by activating cell cycle checkpoints to delay proliferation and facilitate DNA repair. Here, to uncover new checkpoint regulators, we perform RNA interference screening targeting genes involved in ubiquitylation processes. We show that the F-box protein cyclin F plays an important role in checkpoint control following ionizing radiation. Cyclin F-depleted cells initiate checkpoint signalling after ionizing radiation, but fail to maintain G2 phase arrest and progress into mitosis prematurely. Importantly, cyclin F suppresses the B-Myb-driven transcriptional programme that promotes accumulation of crucial mitosis-promoting proteins. Cyclin F interacts with B-Myb via the cyclin box domain. This interaction is important to suppress cyclin A-mediated phosphorylation of B-Myb, a key step in B-Myb activation. In summary, we uncover a regulatory mechanism linking the F-box protein cyclin F with suppression of the B-Myb/cyclin A pathway to ensure a DNA damage-induced checkpoint response in G2.

The transcription factor p53: not a repressor, solely an activator.

The predominant function of the tumor suppressor p53 is transcriptional regulation. It is generally accepted that p53-dependent transcriptional activation occurs by binding to a specific recognition site in promoters of target genes. Additionally, several models for p53-dependent transcriptional repression have been postulated. Here, we evaluate these models based on a computational meta-analysis of genome-wide data. Surprisingly, several major models of p53-dependent gene regulation are implausible. Meta-analysis of large-scale data is unable to confirm reports on directly repressed p53 target genes and falsifies models of direct repression. This notion is supported by experimental re-analysis of representative genes reported as directly repressed by p53. Therefore, p53 is not a direct repressor of transcription, but solely activates its target genes. Moreover, models based on interference of p53 with activating transcription factors as well as models based on the function of ncRNAs are also not supported by the meta-analysis. As an alternative to models of direct repression, the meta-analysis leads to the conclusion that p53 represses transcription indirectly by activation of the p53-p21-DREAM/RB pathway.

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.

Cell cycle, oncogenic and tumor suppressor pathways regulate numerous long and macro non-protein-coding RNAs.

BACKGROUND: The genome is pervasively transcribed but most transcripts do not code for proteins, constituting non-protein-coding RNAs. Despite increasing numbers of functional reports of individual long non-coding RNAs (lncRNAs), assessing the extent of functionality among the non-coding transcriptional output of mammalian cells remains intricate. In the protein-coding world, transcripts differentially expressed in the context of processes essential for the survival of multicellular organisms have been instrumental in the discovery of functionally relevant proteins and their deregulation is frequently associated with diseases. We therefore systematically identified lncRNAs expressed differentially in response to oncologically relevant processes and cell-cycle, p53 and STAT3 pathways, using tiling arrays. RESULTS: We found that up to 80% of the pathway-triggered transcriptional responses are non-coding. Among these we identified very large macroRNAs with pathway-specific expression patterns and demonstrated that these are likely continuous transcripts. MacroRNAs contain elements conserved in mammals and sauropsids, which in part exhibit conserved RNA secondary structure. Comparing evolutionary rates of a macroRNA to adjacent protein-coding genes suggests a local action of the transcript. Finally, in different grades of astrocytoma, a tumor disease unrelated to the initially used cell lines, macroRNAs are differentially expressed. CONCLUSIONS: It has been shown previously that the majority of expressed non-ribosomal transcripts are non-coding. We now conclude that differential expression triggered by signaling pathways gives rise to a similar abundance of non-coding content. It is thus unlikely that the prevalence of non-coding transcripts in the cell is a trivial consequence of leaky or random transcription events.

Polo-like kinase 4 transcription is activated via CRE and NRF1 elements, repressed by DREAM through CDE/CHR sites and deregulated by HPV E7 protein.

Infection by oncogenic viruses is a frequent cause for tumor formation as observed in cervical cancer. Viral oncoproteins cause inactivation of p53 function and false transcriptional regulation of central cell cycle genes. Here we analyze the regulation of Plk4, serving as an example of many cell cycle- and p53-regulated genes. Cell cycle genes are often repressed via CDE and CHR elements in their promoters and activated by NF-Y binding to CCAAT-boxes. In contrast, general activation of Plk4 depends on NRF1 and CRE sites. Bioinformatic analyses imply that NRF1 and CRE are central elements of the transcriptional network controlling cell cycle genes. We identify CDE and CHR sites in the Plk4 promoter, which are necessary for binding of the DREAM (DP, RB-like, E2F4 and MuvB) complex and for mediating repression in G0/G1. When cells progress to G2 and mitosis, DREAM is replaced by the MMB (Myb-MuvB) complex that only requires the CHR element for binding. Plk4 expression is downregulated by the p53-p21(WAF1/CIP1)-DREAM signaling pathway through the CDE and CHR sites. Cell cycle- and p53-dependent repression is abrogated by HPV E7 oncoprotein. Together with genome-wide analyses our results imply that many cell cycle genes upregulated in tumors by viral infection are bound by DREAM through CDE/CHR sites.