Essential Cell Cycle Regulator Research Articles

Toxicity of the model protein 3×GFP arises from degradation overload, not from aggregate formation.

Although protein aggregation can cause cytotoxicity, such aggregates can also form to mitigate cytotoxicity from misfolded proteins, although the nature of these contrasting aggregates remains unclear. We previously found that overproduction (op) of a three green fluorescent protein-linked protein (3×GFP) induces giant aggregates and is detrimental to growth. Here, we investigated the mechanism of growth inhibition by 3×GFP-op using non-aggregative 3×MOX-op as a control in Saccharomyces cerevisiae. The 3×GFP aggregates were induced by misfolding, and 3×GFP-op had higher cytotoxicity than 3×MOX-op because it perturbed the ubiquitin-proteasome system. Static aggregates formed by 3×GFP-op dynamically trapped Hsp70 family proteins (Ssa1 and Ssa2 in yeast), causing the heat-shock response. Systematic analysis of mutants deficient in the protein quality control suggested that 3×GFP-op did not cause a critical Hsp70 depletion and aggregation functioned in the direction of mitigating toxicity. Artificial trapping of essential cell cycle regulators into 3×GFP aggregates caused abnormalities in the cell cycle. In conclusion, the formation of the giant 3×GFP aggregates itself is not cytotoxic, as it does not entrap and deplete essential proteins. Rather, it is productive, inducing the heat-shock response while preventing an overload to the degradation system.

Design, synthesis, and biological evaluation of novel molecules as potent inhibitors of PLK1

Polo-like kinase 1 (PLK1) is an attractive therapeutic target for the treatment of tumors, as it is an essential cell-cycle regulator frequently overexpressed in tumor tissues. PLK1 can promote tumor invasion and metastasis, and is often associated with poor prognosis in cancer patients. However, no PLK1 inhibitor has been granted marketing approval until now. Therefore, more potentially promising PLK1 inhibitors need to be investigated. In this study, a series of novel inhibitors targeting PLK1 was designed and optimized derived from a new scaffold. After synthesis and characterization, we obtained the structure–activity relationship and led to the discovery of the most promising compound 30e for PLK1. The antiproliferative activity against HCT116 cells (IC50 = 5 nM versus 45 nM for onvansertib) and the cellular permeability and efflux ratio were significantly improved (PappA→B = 2.03 versus 0.345 and efflux ratio = 1.65 versus 94.7 for 30e and onvansertib, respectively). Further in vivo studies indicated that 30e had favorable antitumor activity with 116.2% tumor growth inhibition (TGI) in comparison with TGI of 43.0% for onvansertib. Furthermore, 30e improved volume of tumor tissue distribution in mice as compared to onvansertib. This initial study on 30e holds promise for further development of an antitumor agent.

Hdac1-deficiency affects the cell cycle axis Cdc25-Cdk1 causing impaired G2/M phase progression and reduced cardiomyocyte proliferation in zebrafish

Zebrafish have the ability to fully regenerate their hearts after injury since cardiomyocytes subsequently dedifferentiate, re-enter cell cycle, and proliferate to replace damaged myocardial tissue. Recent research identified the reactivation of dormant developmental pathways during cardiac regeneration in adult zebrafish, suggesting pro-proliferative pathways important for developmental heart growth to be also critical for regenerative heart growth after injury.Histone deacetylase 1 (Hdac1) was recently shown to control both, embryonic as well as adult regenerative cardiomyocyte proliferation in the zebrafish model. Nevertheless, regulatory pathways controlled by Hdac1 are not defined yet. By analyzing RNA-seq-derived transcriptional profiles of the Hdac1-deficient zebrafish mutant baldrian, we here identified DNA damage response (DDR) pathways activated in baldrian mutant embryos. Surprisingly, although the DDR signaling pathway was transcriptionally activated, we found the complete loss of protein expression of the known DDR effector and cell cycle inhibitor p21. Consequently, we observed an upregulation of the p21-downstream target Cdk2, implying elevated G1/S phase transition in Hdac1-deficient zebrafish hearts. Remarkably, Cdk1, another p21-but also Cdc25-downstream target was downregulated. Here, we found the significant downregulation of Cdc25 protein expression, explaining reduced Cdk1 levels and suggesting impaired G2/M phase progression in Hdac1-deficient zebrafish embryos. To finally prove defective cell cycle progression due to Hdac1 loss, we conducted Cytometer-based cell cycle analyses in HDAC1-deficient murine HL-1 cardiomyocytes and indeed found impaired G2/M phase transition resulting in defective cardiomyocyte proliferation.In conclusion, our results suggest a critical role of Hdac1 in maintaining both, regular G1/S and G2/M phase transition in cardiomyocytes by controlling the expression of essential cell cycle regulators such as p21 and Cdc25.

MiR-34a negatively regulates cell cycle factor Cdt2/DTL in HPV infected cervical cancer cells

MicroRNAs have emerged as an important regulator of cell cycle and various other cellular processes. Aberration in microRNAs has been linked with development of several cancers and other diseases but still very little is known about the mechanism by which they regulate these cellular events. High risk human papilloma virus (HR HPV) is the causative agent of 99% of cervical cancer cases which attenuates multiple tumor suppressors and checkpoint factors of the host cell. The viral proteins also stabilize many oncogenic factors, including an essential cell cycle regulator Cdt2/DTL which in turn promotes cell transformation and proliferation. In this study, we report that a micro-RNA, miR-34a by suppressing HPV E6 protein, destabilizes Cdt2/DTL protein level in HPV infected cervical cancer cell lines. Destabilization of Cdt2 stabilizes pro-apoptotic and onco-suppressor proteins like p21 and Set8 and suppresses cell proliferation, invasion and migration capabilities of the HPV positive cervical cancer cells. Overexpression of either HPV E6 or Cdt2 genes along with miR-34a restored back the suppressed proliferation rate. This study is the first-ever report to show that miR-34a regulates cell cycle factor Cdt2 by suppressing viral E6 protein level, thus opening up the possibility of exploring miR-34a as a specific therapy for cervical cancer treatment.

Rice Cell Division Cycle 20s are required for faithful chromosome segregation and cytokinesis during meiosis.

Chromosome segregation must be under strict regulation to maintain chromosome euploidy and stability. Cell Division Cycle 20 (CDC20) is an essential cell cycle regulator that promotes the metaphase-to-anaphase transition and functions in the spindle assembly checkpoint, a surveillance pathway that ensures the fidelity of chromosome segregation. Plant CDC20 genes are present in multiple copies, and whether CDC20s have the same functions in plants as in yeast and animals is unclear, given the potential for divergence or redundancy among the multiple copies. Here, we studied all three CDC20 genes in rice (Oryza sativa) and constructed two triple mutants by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated genome editing to explore their roles in development. Knocking out all three CDC20 genes led to total sterility but did not affect vegetative development. Loss of the three CDC20 proteins did not alter mitotic division but severely disrupted meiosis as a result of asynchronous and unequal chromosome segregation, chromosome lagging, and premature separation of chromatids. Immunofluorescence of tubulin revealed malformed meiotic spindles in microsporocytes of the triple mutants. Furthermore, cytokinesis of meiosis I was absent or abnormal, and cytokinesis II was completely prevented in all mutant microsporocytes; thus, no tetrads or pollen formed in either cdc20 triple mutant. Finally, the subcellular structures and functions of the tapetum were disturbed by the lack of CDC20 proteins. These findings demonstrate that the three rice CDC20s play redundant roles but are indispensable for faithful meiotic chromosome segregation and cytokinesis, which are required for the production of fertile microspores.

USP48 Governs Cell Cycle Progression by Regulating the Protein Level of Aurora B

Deubiquitinating enzymes play key roles in the precise modulation of Aurora B—an essential cell cycle regulator. The expression of Aurora B increases before the onset of mitosis and decreases during mitotic exit; an imbalance in these levels has a severe impact on the fate of the cell cycle. Dysregulation of Aurora B can lead to aberrant chromosomal segregation and accumulation of errors during mitosis, eventually resulting in cytokinesis failure. Thus, it is essential to identify the precise regulatory mechanisms that modulate Aurora B levels during the cell division cycle. Using a deubiquitinase knockout strategy, we identified USP48 as an important candidate that can regulate Aurora B protein levels during the normal cell cycle. Here, we report that USP48 interacts with and stabilizes the Aurora B protein. Furthermore, we showed that the deubiquitinating activity of USP48 helps to maintain the steady-state levels of Aurora B protein by regulating its half-life. Finally, USP48 knockout resulted in delayed progression of cell cycle due to accumulation of mitotic defects and ultimately cytokinesis failure, suggesting the role of USP48 in cell cycle regulation.

PLK1 Induces Chromosomal Instability and Overrides Cell-Cycle Checkpoints to Drive Tumorigenesis.

Polo-like kinase 1 (PLK1) is an essential cell-cycle regulator that is frequently overexpressed in various human cancers. To determine whether Plk1 overexpression drives tumorigenesis, we established transgenic mouse lines that ubiquitously express increased levels of Plk1. High Plk1 levels were a driving force for different types of spontaneous tumors. Increased Plk1 levels resulted in multiple defects in mitosis and cytokinesis, supernumerary centrosomes, and compromised cell-cycle checkpoints, allowing accumulation of chromosomal instability (CIN), which resulted in aneuploidy and tumor formation. Clinically, higher expression of PLK1 positively associated with an increase in genome-wide copy-number alterations in multiple human cancers. This study provides in vivo evidence that aberrant expression of PLK1 triggers CIN and tumorigenesis and highlights potential therapeutic opportunities for CIN-positive cancers. SIGNIFICANCE: These findings establish roles for PLK1 as a potent proto-oncogene and a CIN gene and provide insights for the development of effective treatment regimens across PLK1-overexpressing and CIN-positive cancers.

Transcriptional repression of p21 by EIF1AX promotes the proliferation of breast cancer cells

ObjectiveDysregulation of the cell cycle is associated with the progression of malignant cancer, but its precise functional contribution is unknown.Materials and MethodsThe expression of EIF1AX in breast cancer tissues was detected by qRT‐PCR and immunohistochemistry staining. Colony formation and tumour xenograft assays were used to examine the tumorigenesis‐associated function of EIF1AX in vitro and in vivo. RNA‐Seq analysis was used to select the downstream target genes of EIF1AX. Flow cytometry, ChIP and luciferase assays were used to investigate the molecular mechanisms by which EIF1AX regulates p21 in breast cancer cells.ResultsEIF1AX promoted breast cancer cell proliferation by promoting the G1/S cell cycle transition. A mechanistic investigation showed that EIF1AX inhibited the expression of p21, which is an essential cell cycle regulator. We identified that the transcriptional regulation of p21 by EIF1AX was p53‐independent. Clinically, EIF1AX levels were significantly elevated in breast cancer tissues, and the high level of EIF1AX was associated with lower survival rates in breast cancer patients.ConclusionsOur results imply that EIF1AX may play a key role in the incidence and promotion of breast cancer and may, thus, serve as a valuable target for breast cancer therapy.

Investigation of essential cell cycle regulator genes as candidates for immortalized shrimp cell line establishment based on the effect of in vitro culturing on gene expression of shrimp primary cells

Continuous shrimp cell cultures offer opportunities for studying shrimp pathogens at the cellular and molecular level and developing diagnostic tools. However, no continuous shrimp cell lines have yet been successfully established. This might be due to the lack of information on the molecular mechanisms that control shrimp cell proliferation and cell arrest under culture conditions. In this study, differentially expressed genes (DEGs) in shrimp primary culture cells were determined by comparison with normal tissue (in vivo). This study aimed to comprehensively identify key regulator genes that control cell proliferation and cell cycle arrest in culture conditions. The primary shrimp cells were derived from testicular tissue and cultured in vitro. RNA sequencing (RNA-Seq) was performed to investigate change in gene expression level across the entire transcriptome between shrimp primary cells and normal tissue (in vivo). RNA-seq results revealed over 100 genes with distinct gene expression patterns between primary cells and normal tissue. The DEG results and functional gene analysis showed a clear difference in gene expression patterns of cell cycle-related genes between primary cells and normal tissue. LRWD1, TMEM127, CDCA3, PPP2R1A, and GOLGA2, which are cell cycle-related genes, were downregulated in shrimp primary cells. These genes are required for G1/S phase transition, entry into mitosis, and cell proliferation control. However, a gene that induces cell cycle arrest, ARAF, was upregulated in culture conditions. The results demonstrated that these genes might play essential roles in cell proliferation and cell arrest under culture conditions. These candidate genes may be alternative potential targets for genetic manipulation to maintain cell proliferation and establishment of a shrimp continuous cell line.

2047-P: Possible Implication of Cyclin D2 in Beta-Cell Proliferation of Human Perinatal Islet

Cyclin D2 (CCND2) is an essential cell cycle regulator of rodent pancreatic β cells, while its expression in human islets is still controversial in the past studies. It may be because few proliferative β cell is generally present in human adult islets. In contrast, expansion of β cell population mainly depends on a cell proliferation in human perinatal islets. Therefore, CCND2 is expected to be implicated in β cell proliferation in human perinatal islets. The aim of this study is to explore the implication of CCND2 in proliferation of human perinatal β cells. We evaluated pancreata of 39 autopsy cases from prenatal 24 weeks old to postnatal 19 years old. The subjects were divided into fetus (F: prenatal 24-31 weeks), infant (I: 0-1 years), child (C: 2-12 years) and adolescence (A: 13-19 years). Quintuple-immunohistochemistry (IHC) for glucagon, insulin, somatostatin, pp and Ki-67 was performed for the measurement of islet cell volume density (V) and cell replication status. Double-IHC for insulin with CCND2, 5-hydroxy tryptophan (5-HT) and p16 were performed. Double positive cells for each staining were counted. Morphometric analysis revealed that Vα and Vβ were increased with maturation. Ki-67 positivity for α and Δ cells was the highest in F (p<0.01), whereas that of β cells was the highest in I (p<0.01). CCND2 was robustly expressed in the nucleus of β cells in F (>90%) and I (>90%). Thereafter, its expression was dramatically declined in C and A (2.25±1.4%). 5-HT was highly expressed in perinatal subjects but not in C and A. Conversely, p16 expression was minimum before adolescence and its expression was evident in A. Our results clearly suggested that CCND2 could be implicated in the development of human β cell population in perinatal stage. The expression of CCND2 might be independently regulated by 5-HT and p16, while other factors may be implicated in the expression mechanism. To elucidate the regulation mechanism of CCND2 in human islets may lead to develop a novel strategy for diabetes treatments. Disclosure S. Osonoi: None. H. Ichinohe: None. K. Kudo: None. S. Yagihashi: None. H. Mizukami: None.

Ribophorin II is upregulated in myelodysplastic syndromes and prevents apoptosis and cell cycle progression.

This study explored the role of ribophorin II (RPN2) in myelodysplastic syndromes (MDSs) cell proliferation and growth and revealed that RPN2 knockdown suppressed OCI-AML3 cell growth and proliferation and triggered cell cycle arrest and elicited apoptosis in OCI-AML3 cells. In addition, it shed light on the etiology of RPN2's role in MDS cell proliferation that RPN2 can negatively impact enhancer of zeste homolog-2 (EZH2) expression, which in turn is able to modulate the cell cycle location and death in OCI-AML3 cells. Hence, RPN2 expression could be a latent predictor of prognosis in patients with MDS.

Periplasmic protein EipA determines envelope stress resistance and virulence in Brucella abortus.

Molecular components of the Brucella abortus cell envelope play a major role in its ability to infect, colonize and survive inside mammalian host cells. In this study, we have defined a role for a conserved gene of unknown function in B. abortus envelope stress resistance and infection. Expression of this gene, which we name eipA, is directly activated by the essential cell cycle regulator, CtrA. eipA encodes a soluble periplasmic protein that adopts an unusual eight-stranded β-barrel fold. Deletion of eipA attenuates replication and survival in macrophage and mouse infection models, and results in sensitivity to treatments that compromise the cell envelope integrity. Transposon disruption of genes required for LPS O-polysaccharide biosynthesis is synthetically lethal with eipA deletion. This genetic connection between O-polysaccharide and eipA is corroborated by our discovery that eipA is essential in Brucella ovis, a naturally rough species that harbors mutations in several genes required for O-polysaccharide production. Conditional depletion of eipA expression in B. ovis results in a cell chaining phenotype, providing evidence that eipA directly or indirectly influences cell division in Brucella. We conclude that EipA is a molecular determinant of Brucella virulence that functions to maintain cell envelope integrity and influences cell division.

CENP-W inhibits CDC25A degradation by destabilizing the SCFβ-TrCP-1 complex at G2/M.

Skp, Cullin, F-box (SCF)β-TrCP-1 ubiquitin ligases play a central role in cell cycle regulation and tumorigenesis via proteolytic cleavage of many essential cell cycle regulators. In this study, we propose that centromere protein (CENP)-W, a newly identified kinetochore component, is a novel negative regulator of the SCFβ-TrCP-1 complex. CENP-W interacts with Cullin (CUL)-1 and β-Transducin repeat-containing protein (β-TrCP)-1 through highly overlapped binding sites with S-phase kinase-associated protein (SKP)-1. CENP-W is incorporated into the SCFβ-TrCP-1 complex to promote complex disassembly. Unlike other known regulators that increase SCFβ-TrCP-1 ubiquitin ligase activity by promoting complex reassociation, CENP-W-mediated complex disorganization induced β-TrCP1 degradation and consequently decreased its activity. The association between CENP-W and the SCFβ-TrCP-1 complex was prominent during the G2/M transition in the nucleus. Especially, CENP-W knockdown decreased the cell division cycle-25A protein level, leading to a delay in mitotic progression. We propose that CENP-W participates in cell cycle regulation by modulating SCFβ-TrCP-1 ubiquitin ligase activity.-Cheon, Y., Lee, S. CENP-W inhibits CDC25A degradation by destabilizing the SCFβ-TrCP-1 complex at G2/M.

CtrA Is Nonessential for Cell Cycle Regulation in <i>Rhodobacter sphaeroides</i>

The bacterial cell cycle consists of a series of genetically coordinated biochemical and biophysical events. In Caulobacter crescentus, CtrA is an essential cell cycle regulator that modulates many cell cycle processes. In the present study, the role of the CtrA was investigated in Rhodobacter sphaeroides 2.4.1 by employing genetic, molecular, and bioinformatic approaches. Examination of the ctrA-null mutant revealed that the loss of CtrA did not affect growth characteristics and cell morphology in R. sphaeroides when grown under aerobic or photosynthetic growth conditions but slower growth was noticed in the anaerobic-dark-DMSO condition. Phylogenetic analyses demonstrated that CtrA has diversified its role in major lineages of α-Proteobacteria and has possibly been involved in adaptation to variable lifestyles. Analysis of the CtrA binding sites in the R. sphaeroides genome suggests that CtrA may regulate 127 genes involving different cellular processes. Protein homology searches revealed that only a small number of ctrA-regulated genes are homologous across C. crescentus, R. capsulatus, and R. sphaeroides. Comparison of the functions of putative ctrA-regulated genes in C. crescentus, R. capsulatus, and R. sphaeroides revealed that all three species possessed broad pathway control across a variety of cluster of orthologous gene functions (COGs). However, interestingly, it seems that the essentiality of CtrA in C. crescentus may depend more on the selective control that it exerts on a few critical cell cycle genes and pathways that are not controlled by CtrA in a similar fashion in R. capsulatus and R. sphaeroides.

Functions and regulation of the Polo-like kinase Cdc5 in the absence and presence of DNA damage.

Polo-like kinases are essential cell cycle regulators that are conserved from yeast to humans. Unlike higher eukaryotes, who express multiple Polo-like kinase family members that perform many important functions, budding yeast express only a single Polo-like kinase, Cdc5, which is the homolog of mammalian cell cycle master regulator Polo-like kinase 1. Cdc5 is a fascinating multifaceted protein that is programmed to target its many substrates in a timely, sequential manner to ensure proper cell cycle progression. Over the years, many lessons about Polo-like kinase 1 have been learned by studying Cdc5 in budding yeast. Cdc5 has been well documented in regulating mitotic entry, chromosome segregation, mitotic exit, and cytokinesis. Cdc5 also plays important roles during cell division after DNA damage. Here, we briefly review the many functions of Cdc5 and its regulation in the absence and presence of DNA damage.

Differential Regulation of Cyclin E by Yorkie-Scalloped Signaling in Organ Development.

Tissue integrity and homeostasis are accomplished through strict spatial and temporal regulation of cell growth and proliferation during development. Various signaling pathways have emerged as major growth regulators across metazoans; yet, how differential growth within a tissue is spatiotemporally coordinated remains largely unclear. Here, we report a role of a growth modulator Yorkie (Yki), the Drosophila homolog of Yes-associated protein (YAP), that differentially regulates its targets in Drosophila wing imaginal discs; whereby Yki interacts with its transcriptional partner, Scalloped (Sd), the homolog of the TEAD/TEF family transcription factor in mammals, to control an essential cell cycle regulator Cyclin E (CycE). Interestingly, when Yki was coexpressed with Fizzy-related (Fzr), a Drosophila endocycle inducer and homolog of Cdh1 in mammals, surrounding hinge cells displayed larger nuclear size than distal pouch cells. The observed size difference is attributable to differential regulation of CycE, a target of Yki and Sd, the latter of which can directly bind to CycE regulatory sequences, and is expressed only in the pouch region of the wing disc starting from the late second-instar larval stage. During earlier stages of larval development, when Sd expression was not detected in the wing disc, coexpression of Fzr and Yki did not cause size differences between cells along the proximal–distal axis of the disc. We show that ectopic CycE promoted cell proliferation and apoptosis, and inhibited transcriptional activity of Yki targets. These findings suggest that spatiotemporal expression of transcription factor Sd induces differential growth regulation by Yki during wing disc development, highlighting coordination between Yki and CycE to control growth and maintain homeostasis.

The Essential Cell Cycle Regulator Setd8 Drives Chromatin Condensation during Terminal Erythroid Maturation

Chromatin condensation culminating in enucleation is a hallmark of erythropoiesis, however the mechanisms driving this process are incompletely understood. Setd8 is the sole enzyme that can mono-methylate histone H4, lysine 20 (H4K20me1) and is an important regulator of cell cycle progression, higher order chromatin structure, and genome stability. (Reviewed in Beck, Genes and Development, 2010) Setd8 and H4K20me1 are unique among epigenetic regulators in that their expression is dynamically regulated during the cell cycle. Setd8 expression peaks during G2/M, where it promotes mitotic chromatin condensation, and becomes undetectable during S-phase due to ubiquitin dependent destruction. (Oda, Mol Cell, 2010) The presence of H4K20me1 mirrors that of Setd8, peaking in G2/M, and reaching a nadir during S-phase due to removal by the histone demethylase PHF8. (Liu, Nature, 2010) Interestingly, Setd8 is expressed at levels 8- to 10- fold higher in CD71+ erythroblasts than in any other cell type, (Wu Genome Biology,2009) suggesting that it has an erythroid-specific function. We hypothesize that Setd8 drives chromatin condensation in maturing erythroblasts.In cell lines, forced accumulation of H4K20me1 during S-phase due to perturbation of either Setd8 or PHF8 results in pre-mitotic chromatin condensation (Centore Mol Cell 2010; Liu Nature 2010). We demonstrate that primary erythroblasts express Setd8 and accumulate H4K20me1 throughout the cell cycle, suggesting that Setd8 and H4K20me1 in promote chromatin condensation during terminal maturation. We further demonstrate that Setd8 is essential for erythropoiesis, with erythroid-specific Setd8 deletion resulting in profound anemia that is lethal by E12.5. The early onset of anemia indicates a defect in the primitive erythroid lineage, which emerges from the yolk sac at E8.5, and proliferates, matures, and enucleates in the circulation as a semi-synchronous cohort. (Kingsley, Blood, 2004) Detailed analyses of Setd8-null erythroblasts revealed severe defects in cell cycle progression, increased DNA content suggesting loss of genomic integrity, accumulation of DNA damage, and a modest increase in the rate of apoptosis. Global transcriptome analyses demonstrated that Setd8-null erythroblasts had activation of checkpoint genes such as CDKN1a and Gene Set Enrichment Analyses identified significant enrichment of cell cycle and p53 signaling pathways. Despite evidence of p53 activation, concomitant p53 deletion was not able to rescue the Set8-null phenotype, indicating that Setd8 has an essential role in promoting erythroid proliferation and survival that is independent of the p53 pathway.Consistent with our hypothesis that Setd8 drives chromatin condensation in maturing erythroblasts, the nuclear area of Setd8-null cells was nearly twice that of controls at E11.5 (119 and 69 um2, respectively p<0.0003). Transmission electron microscopy confirmed a profound defect in global chromatin condensation in the Setd8-null cells. Unexpectedly, heterochromatin was nearly absent from the Setd8-null cells, with the Setd8-null cells containing only a small amount of heterochromatin localized to the nuclear periphery. To determine the impact of Setd8 deletion on local chromatin structure, we performed ATAC-seq (Assay for Transposase Accessible Chromatin) on sorted populations of Setd8-null and control erythroblasts. Preliminary analyses of ATAC-seq data identified 364 ATAC peaks present in the Setd8-null cells but not in controls (p<0.001). Intriguingly, Gene Ontogeny analyses of the genes nearest to those regions was significant for multiple terms associated with higher order chromatin structure including "regulation of chromatin organization" and "positive regulation of histone deacetylation." Taken together, our results indicate that erythroblasts have adapted an essential cell cycle regulator to drive chromatin condensation during terminal maturation. DisclosuresNo relevant conflicts of interest to declare.

Down-regulation of hTERT and Cyclin D1 transcription via PI3K/Akt and TGF-β pathways in MCF-7 Cancer cells with PX-866 and Raloxifene

Human telomerase reverse transcriptase (hTERT) is the catalytic and limiting component of telomerase and also a transcription factor. It is critical to the integrity of the ends of linear chromosomes and to the regulation, extent and rate of cell cycle progression in multicellular eukaryotes. The level of hTERT expression is essential to a wide range of bodily functions and to avoidance of disease conditions, such as cancer, that are mediated in part by aberrant level and regulation of cell cycle proliferation. Value of a gene in regulation depends on its ability to both receive input from multiple sources and transmit signals to multiple effectors. The expression of hTERT and the progression of the cell cycle have been shown to be regulated by an extensive network of gene products and signaling pathways, including the PI3K/Akt and TGF-β pathways. The PI3K inhibitor PX-866 and the competitive estrogen receptor ligand raloxifene have been shown to modify progression of those pathways and, in combination, to decrease proliferation of estrogen receptor positive (ER+) MCF-7 breast cancer cells. We found that combinations of modulators of those pathways decreased not only hTERT transcription but also transcription of additional essential cell cycle regulators such as Cyclin D1. By evaluating known expression profile signatures for TGF-β pathway diversions, we confirmed additional genes such as heparin-binding epidermal growth factor-like growth factor (HB EGF) by which those pathways and their perturbations may also modify cell cycle progression.

The bacterial cell cycle regulator GcrA is a σ70 cofactor that drives gene expression from a subset of methylated promoters

Cell cycle progression in most organisms requires tightly regulated programs of gene expression. The transcription factors involved typically stimulate gene expression by binding specific DNA sequences in promoters and recruiting RNA polymerase. Here, we found that the essential cell cycle regulator GcrA in Caulobacter crescentus activates the transcription of target genes in a fundamentally different manner. GcrA forms a stable complex with RNA polymerase and localizes to almost all active σ(70)-dependent promoters in vivo but activates transcription primarily at promoters harboring certain DNA methylation sites. Whereas most transcription factors that contact σ(70) interact with domain 4, GcrA interfaces with domain 2, the region that binds the -10 element during strand separation. Using kinetic analyses and a reconstituted in vitro transcription assay, we demonstrated that GcrA can stabilize RNA polymerase binding and directly stimulate open complex formation to activate transcription. Guided by these studies, we identified a regulon of ∼ 200 genes, providing new insight into the essential functions of GcrA. Collectively, our work reveals a new mechanism for transcriptional regulation, and we discuss the potential benefits of activating transcription by promoting RNA polymerase isomerization rather than recruitment exclusively.

Abstract LB-081: Elucidating the function of MCM3 ubiquitination by KEAP1: crosstalk between redox-sensing and cell cycle progression

Abstract While the KEAP1-NRF2 axis is essential for maintaining redox homeostasis, whether KEAP1 has alternative functions and how this pathway crosstalks with other important cellular processes remains unknown. KEAP1 targets the NRF2 transcription factor for proteasomal degradation in a redox-sensitive manner. Thus, this pathway serves as the cell's primary response to elevated reactive oxygen species. Importantly, KEAP1-NRF2 are frequently mutated in cancer, most strikingly in non-small cell lung cancer, where KEAP1 or NRF2 are mutated in 20-30% of patient tumors. Though regulation of NRF2 has long been considered the only physiologically important role for the E3 ligase KEAP1, we have determined that KEAP1 binds the master cell cycle regulator, MCM3, a subunit of the hexameric DNA replication licensing complex, MCM2-7. Excitingly, our ubiquitination assay data establish MCM3 as a new substrate for KEAP1; however, KEAP1 intriguingly does not regulate total cellular levels of MCM3. Consistent with this, we determined that only a small pool of cellular MCM3 is bound to KEAP1, suggesting that KEAP1 may bind and ubiquitinate a highly specified pool of MCM3. To determine the function of KEAP1-dependent ubiquitination of MCM3, we recently applied a new proteomics technique to map the ubiquitinated residues within MCM3 and identify these lysines within the larger MCM2-7 helicase. This mapping and protein modeling has provided new insight into the structure-function relationship of ubiquitinated MCM3. As MCM2-7 chromatin loading is a highly coordinated, cell cycle-dependent process, we tested whether KEAP1 loaded concurrently onto chromatin. Strikingly, we found that KEAP1 indeed loads onto chromatin during G1 and unloads in late S phase in a similar fashion as the MCM complex, further suggesting KEAP1 regulates the function of this essential cell cycle regulator on chromatin. Given the role of MCM3 in cell cycle progression, we tested whether KEAP1 was required for normal G1 to S phase progression and saw that loss of KEAP1 retards S phase DNA synthesis, which is an MCM-dependent process. Intriguingly, primary, untransformed KEAP1 knockout fibroblasts show decreased growth and aberrant cell cycle patterns consistent with a defect in the G1 to S transition. Overall, these data suggest a novel function for KEAP1 in regulating the MCM complex and cell cycle progression. We postulate that KEAP1 promotes cell cycle progression in a redox-sensitive manner through its association with MCM3 and that this presents a novel mechanism by which cells may halt cell cycle to protect DNA from damage by reactive oxygen species. Citation Format: Kathleen M. Mulvaney, Jacob Matson, Feng Yan, Dennis Goldfarb, Jeannette Cook, Michael Benjamin Major. Elucidating the function of MCM3 ubiquitination by KEAP1: crosstalk between redox-sensing and cell cycle progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-081. doi:10.1158/1538-7445.AM2015-LB-081