Expression Of Cell Cycle Regulatory Genes Research Articles

#2529 The role of the circadian clock system in the transition from acute kidney injury to chronic kidney disease

Abstract Background and Aims Recent studies suggest that circadian rhythms are an important factor in maintaining healthy kidney functions; however, its role in acute kidney injury (AKI) remains unclear. Method To investigate the role of tubule-specific clock gene disturbance in hypoxic tubular injury, we created human proximal tubule-specific BMAL1 KO cells using CRISPR-Cas9 gene editing. In mice, we also determined whether time-restricted feeding (TRF), a dietary strategy to enhance circadian rhythm, can have beneficial effect on recovery of ischemia reperfusion injury (IRI) or its progression to chronic kidney disease (CKD). Results BMAL1 loss in tubule cells resulted in the upregulation of cell cycle regulatory gene (p21) and inflammatory genes (TNF-α and CCL4) mRNA expression, suggesting that BMAL1 is a critical regulator of these genes. Exposure to hypoxia for 48 h resulted in more increased expressions of p21 in BMAL 1 KO cells than in WT HK-2 cells, leading to higher expression of TGF-b. In addition, loss of BMAl-1 led to significantly increased vimentin and decreased E-cadherin expression upon TGF-b stimulation, suggesting an aggravated epithelial-mesenchymal transition upon BMAL1 deletion. To clarify the role of clock system in AKI, we also tested whether TRF can have a beneficial effect during recovery phase after IRI. Despite limited access to food, TRF for 4 weeks after IRI had no effect on the total intake or weight of mice. However, we observed that TRF significantly improved renal function on day 21 and reduced renal fibrosis on day 28, which suggests that enhancing clock gene oscillations through restoring fasting/feeding cycle has a protective effect in the AKI to CKD transition. Conclusion We identified an important role of clock system in hypoxic tubular injury and the effects of treatment targeting circadian rhythm in AKI. Our results can provide a new perspective for developing novel therapeutic strategies for AKI.

Effects of dietary melatonin on growth performance, antioxidant capacity, and nonspecific immunity in crayfish, Cherax destructor.

Melatonin (MT) is an indole hormone widely found in plants and animals. Many studies have shown that MT promotes the growth and immunity of mammals, fish, and crabs. However, the effect on commercial crayfish has not been demonstrated. The purpose of this study was to evaluate the effects of dietary MT on growth performance and innate immunity of Cherax destructor from three aspects of individual level, biochemical level, and molecular level after 8 weeks of culture. In this study, we found that MT supplementation increased weight gain rate, specific growth rate, and digestive enzyme activity in C. destructor compared to the control group. Dietary MT not only promoted the activity of T-AOC, SOD, and GR, increased the content of GSH, and decreased the content of MDA in the hepatopancreas, but also increased the content of hemocyanin and copper ions and AKP activity in hemolymph. Gene expression results showed that MT supplementation at appropriate doses increased the expression of cell cycle-regulated genes (CDK, CKI, IGF, and HGF) and non-specific immune genes (TRXR, HSP60, and HSP70). In conclusion, our study showed that adding MT to the diet improved growth performance, enhanced the antioxidant capacity of hepatopancreas, and immune parameters of hemolymph in C. destructor. In addition, our results showed that the optimal dietary supplementation dose of MT in C. destructor is 75-81mg/kg.

The EZH2–PRC2–H3K27me3 axis governs the endometrial cell cycle and differentiation for blastocyst invasion

Infertility occurs in 15% of couples worldwide. Recurrent implantation failure (RIF) is one of the major problems in in vitro fertilization and embryo transfer (IVF–ET) programs, and how to manage patients with RIF to achieve successful pregnancy outcomes remains unresolved. Here, a uterine polycomb repressive complex 2 (PRC2)-regulated gene network was found to control embryo implantation. Our RNA-seq analyses of the human peri-implantation endometrium obtained from patients with RIF and fertile controls revealed that PRC2 components, including its core enzyme enhancer of zeste homolog 2 (EZH2)-catalyzing H3K27 trimethylation (H3K27me3) and their target genes are dysregulated in the RIF group. Although fertility of uterine epithelium-specific knockout mice of Ezh2 (eKO mice) was normal, Ezh2-deleted mice in the uterine epithelium and stroma (uKO mice) exhibited severe subfertility, suggesting that stromal Ezh2 plays a key role in female fertility. The RNA-seq and ChIP-seq analyses revealed that H3K27me3-related dynamic gene silencing is canceled, and the gene expression of cell-cycle regulators is dysregulated in Ezh2-deleted uteri, causing severe epithelial and stromal differentiation defects and failed embryo invasion. Thus, our findings indicate that the EZH2–PRC2–H3K27me3 axis is critical to preparing the endometrium for the blastocyst invasion into the stroma in mice and humans.

Nuclear Factor AP2X-4 Governs the Expression of Cell Cycle- and Life Stage-Regulated Genes and is Critical for Toxoplasma Growth.

ABSTRACTToxoplasma gondii is a ubiquitous pathogen infecting one third of the world’s population and diverse animals. It has a complex life cycle alternating among different developmental stages, which contributes to its transmission and pathogenesis. The parasite has a sophisticated gene regulation network that enables timely expression of genes at designated stages. However, little is known about the underlying regulatory mechanisms. Here, we identified an AP2 family transcription factor named TgAP2X-4, which was crucial for parasite growth during the acute infection stage. TgAP2X-4 deletion leads to reduced expression of many genes that are normally upregulated during the M phase of the cell cycle. These include genes that encode rhoptry neck proteins that are key for parasite invasion. As a result, the Δap2X-4 mutant displayed significantly decreased efficiency of host cell invasion. Transcriptomic analyses suggested that TgAP2X-4 also regulates a large group of genes that are typically induced during chronic infection, such as BAG1 and LDH2. Given the diverse impacts on gene expression, TgAP2X-4 inactivation results in severely impaired parasite growth, as well as drastic attenuation of parasite virulence and complete inability to form chronic infection. Therefore, TgAP2X-4 represents a candidate for antitoxoplasmic drug and vaccine designs.IMPORTANCE Toxoplasma gondii has a complicated gene regulation network that allows “just in time” expression of genes to cope with the physiological needs at each stage during the complex life cycle. However, how such regulation is achieved is largely unknown. Here, we identified a transcription factor named TgAP2X-4 that is critical for the growth and life cycle progression of the parasite. Detailed analyses found that TgAP2X-4 regulated the expression of many cell cycle-regulated genes, including a subset of rhoptry genes that were essential for the parasites to enter host cells. It also regulated the expression of many genes involved in the development of chronic infection. Because of the diverse impacts on gene expression, TgAP2X-4 inactivation caused reduced parasite growth in vitro and attenuated virulence in vivo. Therefore, it is a potential target for drug or vaccine designs against Toxoplasma infections.

Ascorbic acid and all-trans retinoic acid promote proliferation of chicken blastoderm cells (cBCs) by mediating DNA demethylation.

Chicken blastoderm cells (cBCs) obtained from stage X (EG&K) embryos are easily available materials for the study of cell development. However, cBCs are not widely used because they are hard to maintain in long-term culture in vitro. To solve this problem, ascorbic acid (AA; also known as vitamin C (VC)) and all-trans retinoic acid (ATRA) were added into basic culture medium to promote cell growth. Results suggested that cultured cBCs possessed strongly proliferative activity and maintained their pluripotency on the support of chicken embryonic fibroblast (CEF) feeder. Moreover, when VC or/and ATRA was added, the number and area of cBC colonies increased significantly compared with the control group. The expression of pluripotency genes (Sox2 and Nanog) and cell cycle-regulated genes (CCND1 and CDK6) was upregulated obviously. Furthermore, results showed that 5hmC levels in VC and RA groups increased significantly by DNA dot blot and immunofluorescence staining. These results provide strong evidence that VC and ATRA induced DNA demethylation and enhanced 5hmC level. The level of H3K27me3 was raised, while the level of H3K9me2 was reduced by addition of VC and ATRA. Finally, the expression of Tet1 and Dnmt3b was upregulated remarkably. Therefore, these results indicated that VC and ATRA enhanced DNA demethylation and then promoted cBC survival and proliferation in vitro.

DREAM On: Cell Cycle Control in Development and Disease.

Perfectly orchestrated periodic gene expression during cell cycle progression is essential for maintaining genome integrity and ensuring that cell proliferation can be stopped by environmental signals. Genetic and proteomic studies during the past two decades revealed remarkable evolutionary conservation of the key mechanisms that control cell cycle-regulated gene expression, including multisubunit DNA-binding DREAM complexes. DREAM complexes containing a retinoblastoma family member, an E2F transcription factor and its dimerization partner, and five proteins related to products of Caenorhabditis elegans multivulva (Muv) class B genes lin-9, lin-37, lin-52, lin-53, and lin-54 (comprising the MuvB core) have been described in diverse organisms, from worms to humans. This review summarizes the current knowledge of the structure, function, and regulation of DREAM complexes in different organisms, as well as the role of DREAM in human disease.

A single-cell analysis of the Arabidopsis vegetative shoot apex.

The shoot apical meristem allows for reiterative formation of new aerial structures throughout the life cycle of a plant. We use single-cell RNA sequencing to define the cellular taxonomy of the Arabidopsis vegetative shoot apex at the transcriptome level. We find that the shoot apex is composed of highly heterogeneous cells, which can be partitioned into 7 broad populations with 23 transcriptionally distinct cell clusters. We delineate cell-cycle continuums and developmental trajectories of epidermal cells, vascular tissue, and leaf mesophyll cells and infer transcription factors and gene expression signatures associated with cell fate decisions. Integrative analysis of shoot and root apical cell populations further reveals common and distinct features of epidermal and vascular tissues. Our results, thus, offer a valuable resource for investigating the basic principles underlying cell division and differentiation in plants at single-cell resolution.

Yin Yang 1 is critical for mid-hindbrain neuroepithelium development and involved in cerebellar agenesis

The highly conserved and ubiquitously expressed transcription factor Yin Yang 1 (Yy1), was named after its dual functions of both activating and repressing gene transcription. Yy1 plays complex roles in various fundamental biological processes such as the cell cycle progression, cell proliferation, survival, and differentiation. Patients with dominant Yy1 mutations suffer from central nervous system (CNS) developmental defects. However, the role of Yy1 in mammalian CNS development remains to be fully elucidated. The isthmus organizer locates to the mid-hindbrain (MHB) boundary region and serves as the critical signaling center during midbrain and cerebellar early patterning. To study the function of Yy1 in mesencephalon/ rhombomere 1 (mes/r1) neuroepithelium development, we utilized the tissue-specific Cre-LoxP system and generated a conditional knockout mouse line to inactivate Yy1 in the MHB region. Mice with Yy1 deletion in the mes/r1 region displayed cerebellar agenesis and dorsal midbrain hypoplasia. The Yy1 deleted neuroepithelial cells underwent cell cycle arrest and apoptosis, with the concurrent changes of cell cycle regulatory genes expression, as well as activation of the p53 pathway. Moreover, we found that Yy1 is involved in the transcriptional activation of Wnt1 in neural stem cells. Thus, our work demonstrates the involvement of Yy1 in cerebellar agenesis and the critical function of Yy1 in mouse early MHB neuroepithelium maintenance and development.

The coordinate actions of calcineurin and Hog1 mediate the stress response through multiple nodes of the cell cycle network.

Upon exposure to environmental stressors, cells transiently arrest the cell cycle while they adapt and restore homeostasis. A challenge for all cells is to distinguish between stress signals and coordinate the appropriate adaptive response with cell cycle arrest. Here we investigate the role of the phosphatase calcineurin (CN) in the stress response and demonstrate that CN activates the Hog1/p38 pathway in both yeast and human cells. In yeast, the MAPK Hog1 is transiently activated in response to several well-studied osmostressors. We show that when a stressor simultaneously activates CN and Hog1, CN disrupts Hog1-stimulated negative feedback to prolong Hog1 activation and the period of cell cycle arrest. Regulation of Hog1 by CN also contributes to inactivation of multiple cell cycle-regulatory transcription factors (TFs) and the decreased expression of cell cycle-regulated genes. CN-dependent downregulation of G1/S genes is dependent upon Hog1 activation, whereas CN inactivates G2/M TFs through a combination of Hog1-dependent and -independent mechanisms. These findings demonstrate that CN and Hog1 act in a coordinated manner to inhibit multiple nodes of the cell cycle-regulatory network. Our results suggest that crosstalk between CN and stress-activated MAPKs helps cells tailor their adaptive responses to specific stressors.

The functions of azurin of Pseudomonas aeruginosa and human mammaglobin-A on proapoptotic and cell cycle regulatory genes expression in the MCF-7 breast cancer cell line.

Azurin protein of Pseudomonas aeruginosa is an anti-tumor agent against breast cancer and mammaglobin-A (MAM-A) protein is a specific antigen on the surface of MCF-7 for induction of cellular immune. The purpose of the present study was to investigate the effects of simultaneous expression of azurin and human MAM-A genes on the mRNA expression level of apoptosis-related and cell cycle genes in MCF-7 breast cancer cell line. The recombinant or empty plasmids were separately transferred into MCF-7 cells using Lipofectamine reagent. Flow cytometry was done to detect cell death and apoptosis. The expression of azurin and MAM-A genes were evaluated by IF assay, RT-PCR and western blot methods. Finally, apoptosis-related and cell cycle genes expression was examined in transformed and non-transformed MCF-7 cells by qPCR method. The successful expression of azurin and MAM-A genes in the MCF-7 cell were confirmed by RT-PCR, IF and western blotting. The apoptosis assay was showed a statistically significant (p < 0.05) difference after transfection. The expression of BAK, FAS, and BAX genes in transformed cells compare with non-transformed and transformed MCF-7 by pBudCE4.1 were increased statistically significant (p < 0.05) increases. Although, the increase of SURVIVIN and P53 expressions in transformed cells were not statistically significant (p > 0.05). Co-expression of azurin and MAM-A genes could induce apoptosis and necrosis in human MCF-7 breast cancer cells by up-regulation of BAK, FAS, and BAX genes. In future researches, it must be better the immune stimulation of pBudCE4.1-azurin-MAM-A recombinant vector in animal models and therapeutic approaches will be evaluated.

Impact of spaceflight on the murine thymus and mitigation by exposure to artificial gravity during spaceflight

The environment experienced during spaceflight may impact the immune system and the thymus appears to undergo atrophy during spaceflight. However, molecular aspects of this thymic atrophy remain to be elucidated. In this study, we analysed the thymi of mice on board the international space station (ISS) for approximately 1 month. Thymic size was significantly reduced after spaceflight. Notably, exposure of mice to 1 × g using centrifugation cages in the ISS significantly mitigated the reduction in thymic size. Although spaceflight caused thymic atrophy, the global thymic structure was not largely changed. However, RNA sequencing analysis of the thymus showed significantly reduced expression of cell cycle-regulating genes in two independent spaceflight samples. These reductions were partially countered by 1 × g exposure during the space flights. Thus, our data suggest that spaceflight leads to reduced proliferation of thymic cells, thereby reducing the size of the thymus, and exposure to 1 × g might alleviate the impairment of thymus homeostasis induced by spaceflight.

Abstract C035: A FoxM1-autoregulation motif serves as an inhibitor to suppress cellular vitality of NSCLC

Abstract Elevated expression level of Forkhead box M1 (FoxM1) transcription factor is commonly found in cancers, which is highly associated with tumor malignancy and poor prognosis of cancer patients. Molecular mechanism and biological function studies showed that FoxM1 regulates tumor initiation and progression in many aspects. In cell proliferation, FoxM1 activates transcription network of cell-cycle regulatory genes, including Cdc25B, Cyclin B1, Skp2, JNK1 and TOPOIIa. Inhibition of FoxM1 reduces expression of cell-cycle regulatory genes, leading to cell cycle blockade in G1/S and G2/M transition. In tumor malignant conversion, FoxM1 enhances epithelial-mesenchymal transition (EMT) as well as invasion and migration of cancer cells by activating SNAIL, MMP-9 expression. Moreover, FoxM1 activates VEGF transcription, which may contribute to tumor survival and proliferation via angiogenesis. Prior research showed that nuclear translocation and activation of FoxM1 depends on subsequential phosphorylations on the C-terminal transactivation domain (TAD) by Ras/Raf/MAPK signaling and Cyclin E/A-CdK complex through S phase to G2 phase respectively. In addition, FoxM1 reduced its transactivity while the TAD interacts with its N-terminus sequence as deletion of N-terminus results in a constitutively active form of FoxM1 (FoxM1-ΔN). These suggest that an intrinsic autoregression of FoxM1 may be relieved through dissociation of N-terminal Inhibitory domain (NID) from C-terminus after phosphorylation on TAD. However, the underlying mechanism of how this interaction occurs and affects biological function remains elusive. To determine the biological functions of respective FoxM1 protein domains, we performed promoter activity assays and demonstrated that a putative ββαβ motif on FoxM1 N-terminus is the region essential for the inhibitory function, which depends on hydrophobic residues within the structure. Simultaneously, the inhibitory function of the ββαβ peptide can be counteracted by expression of PLK1 phosphomimetic mutations on C-terminus (S715D/S724D). Yet, FoxM1S715D/S724D mutant displayed a weaker transactivity than FoxM1-ΔN, indicating that PLK1-mediated phosphorylation on C-terminus is insufficient to relieve N-terminus inhibition. Protein co-immunoprecipitation and yeast-two hybrid assays proved that both ββαβ and αβα motifs are required for the interaction between FoxM1 N- and C-termini, underlying the intramolecular interaction based-autorepression of FoxM1. Additionally, expression of ββαβ peptide suppresses NSCLC A549 and H23 cell growth by inducing cell apoptosis and senescence, suggesting targeting FoxM1 by ββαβ peptide as a potential therapeutic strategy for cancer inhibition. Citation Format: Chia-Chan Hsu, I-Ching Wang. A FoxM1-autoregulation motif serves as an inhibitor to suppress cellular vitality of NSCLC [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C035. doi:10.1158/1535-7163.TARG-19-C035

Cypermethrin Induced Liver Toxicity: Altered Gene Expression and DNA Methylation

Cypermethrin, a type II pyrethroid, is a widely used insecticide worldwide. It is considered a safe insecticide to be used in pet shampoos and household cleaning agents thus ignoring the possible health hazards caused by it. Cypermethrin may contaminate food, water, fruits or get deposited on furniture, mats or floors leading to acute or chronic disorders in non‐target organisms. Very limited studies with low and non‐frequent dose mimicking the unintentional cypermethrin intake in non‐target organisms including human beings have been carried out. The present study was aimed at evaluating the toxic effect of cypermethrin in the liver of Balb/c mice exposed to a low and once in a week oral dose (1/10 LD50) for 28 days. Serum was extracted from blood after the dosing period and utilized for Liver Function Tests. The gene regulatory studies were performed by amplifying the cDNA, employing specific primers for p21 and p53 and other cell cycle regulatory genes through PCR (Polymerase Chain Reaction). DNA methylation studies were carried out by bisulfite treatment of DNA followed by Bisulphite specific PCR. The methylation status was found out by analyzing the gene promoter regions through sequencing.The changes in liver enzyme activities and histo‐architecture of liver cells show that sub‐chronic exposure to cypermethrin causes liver damage. It also induces a significant increase in hepatic markers enzymes (ALT, AST, ALP) along with necrosis, vacuolar degeneration, increased kupffer cells and a decrease in the number of hepatocytes. A significant change in the gene expression of p21 and p53 is seen in the test animals pointing towards the relation of liver injury by cypermethrin with cell cycle regulatory mechanisms. The gene promoter region of p53 gene show alteration in methylation status in the test animal sample as compared to the control.Sub‐chronic exposure to cypermethrin causes significant changes in liver enzyme activities, cell architecture, cell cycle regulatory gene expression and methylation status of the concerned genes. These genes along with the other cell cycle regulatory genes can be used as biomarkers for liver injury caused by low doses of cypermethrin.Support or Funding Information1. University Grant Commission, New Delhi, India.2. DST‐PURSE grant, New Delhi, India.3. Institute of Forensic Science and Criminology, Panjab University, India.4. Centre for Stem cell and Tissue Engineering, Panjab University, India.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Postnatal liver functional maturation requires Cnot complex-mediated decay of mRNAs encoding cell cycle and immature liver genes.

ABSTRACTLiver development involves dramatic gene expression changes mediated by transcriptional and post-transcriptional control. Here, we show that the Cnot deadenylase complex plays a crucial role in liver functional maturation. The Cnot3 gene encodes an essential subunit of the Cnot complex. Mice lacking Cnot3 in liver have reduced body and liver masses, and they display anemia and severe liver damage. Histological analyses indicate that Cnot3-deficient (Cnot3−/−) hepatocytes are irregular in size and morphology, resulting in formation of abnormal sinusoids. We observe hepatocyte death, increased abundance of mitotic and mononucleate hepatocytes, and inflammation. Cnot3−/− livers show increased expression of immune response-related, cell cycle-regulating and immature liver genes, while many genes relevant to liver functions, such as oxidation-reduction, lipid metabolism and mitochondrial function, decrease, indicating impaired liver functional maturation. Highly expressed mRNAs possess elongated poly(A) tails and are stabilized in Cnot3−/− livers, concomitant with an increase of the proteins they encode. In contrast, transcription of liver function-related mRNAs was lower in Cnot3−/− livers. We detect efficient suppression of Cnot3 protein postnatally, demonstrating the crucial contribution of mRNA decay to postnatal liver functional maturation.

The active role of the transcription factor Sp1 in NFATc2-mediated gene regulation in pancreatic cancer

BackgroundAdenocarcinoma of the pancreas is one of the most aggressive tumor diseases affecting the human body. The oncogenic potential of pancreatic cancer is mainly characterized by extremely rapid growth triggered by the activation of oncogenic signaling cascades, which suggests a change in the regulation of important transcription factors. Amongst others, NFAT transcription factors are assumed to play a central role in the carcinogenesis of pancreatic cancer. Recent research has shown the importance of the transcription factor Sp1 in the transcriptional activity of NFATc2 in pancreatic cancer. However, the role of the interaction between these two binding partners remains unclear. The current study investigated the role of Sp1 proteins in the expression of NFATc2 target genes and identified new target genes and their function in cells. A further objective was the domain of the Sp1 protein that mediates interaction with NFATc2.The involvement of Sp1 proteins in NFATc2 target genes was shown by means of a gene expression profile analysis, and the results were confirmed by quantitative RT-PCR. The functional impact of this interaction was shown in a thymidine incorporation assay. A second objective was the physical interaction between NFATc2 and different Sp1 deletion mutants that was investigated by means of immunoprecipitation.ResultsIn pancreatic cancer, the proto-oncogene c-Fos, the tumor necrosis factor TNF-alpha, and the adhesion molecule integrin beta-3 are target genes of the interaction between Sp1 and NFATc2. Loss of just one transcription factor inhibits oncogenic complex formation and expression of cell cycle-regulating genes, thus verifiably decreasing the carcinogenic effect. The current study also showed the interaction between the transcription factor NFATc2 and the N-terminal domain of Sp1 in pancreatic cancer cells. Sp1 increases the activity of NFATc2 in the NFAT-responsive promoter.ConclusionsThe regulation of gene promotors during transcription is a rather complex process because of the involvement of many proteins that – as transcription factors or co-factors – regulate promotor activity as required and control cell function. NFATc2 and Sp1 seem to play a key role in the progression of pancreatic cancer.

Loss of CDKN2A and CDKN2B expression is associated with disease recurrence in oral cancer.

Background:Loco-regional recurrence is one of the major reasons for poor prognosis of oral squamous cell carcinoma (OSCC). However, till date, no feasible molecular marker is available to predict the risk of recurrence in OSCC patients.Aim:To evaluate the cell cycle regulatory genes expression and its association with the risk of recurrence in oral cancer patients.Materials and Methods:Transcript level expressions of 47 cell cycle regulatory genes were analyzed in 73 OSCC tumors from buccal mucosa and tongue, 26 adjacent normal samples using real-time polymerase chain reaction. TaqMan low-density array data were analyzed using the DataAssist™ v 3.01. Significantly altered genes within the tumor samples and samples showing recurrence (re-appearance of disease during the follow-up in cases having complete response to initial treatment assessed after 3 months of the treatment) were identified. Further, Kyoto Encyclopedia of Genes and Genomes pathway analysis and The Cancer Genome Atlas (TCGA) online data analysis portal were used to analyze interacting protein and pathways significantly associated with the altered gene.Results:CCNA1, CCNB2, CCND2, CCNE1, CCNF, CDC2, CDK6, CHEK1, and TGFA found to significantly alter in the tumor sample of oral cancer patients, and down-expression of CDKN2A and CDKN2B found to associate with the recurrence of disease in oral cancer patients. TCGA data also showed the loss of CDKN2A and CDKN2B significantly associated with recurrence in head and neck cancer patients.Conclusion:CDKN2A and CDKN2B expression analysis can be used as the prognostic marker for the oral cancer patients. The present method of data analysis helps overcome the limitations and complications of high throughput techniques and thereby increases the opportunity of employing molecular markers in routine clinical management of OSCC.

Nucleosome Binding Protein HMGN1 Promotes HSPC Competitive Advantage with Decreased Quiescence and Cooperates with AML Oncogenes to Block Differentiation and Increase Clonogenicity

Acute myeloid leukemia (AML) is characterized by rapid growth and differentiation arrest of immature myeloblasts. The AML blast is defined by having “open” chromatin, thus, we hypothesized that alterations of chromatin compaction may promote AML. The nucleosome binding protein HMGN1 is known to decompact chromatin and is located on chr21q22 in one of the most recurrently amplified regions in AML. We previously presented in vitro data showing that HMGN1 overexpression delayed myeloid differentiation in association with an increase in global levels of Histone H3K27 acetylation downstream of the histone acetyltransferases (HATs) CBP and p300. The goal of this study was to define the effect of HMGN1 overexpression on hematopoiesis in vivo and to identify leukemia oncogenes that cooperate with HMGN1 in primary hematopoietic cells.We analyzed native WT and OE-HMGN1 (transgenic overexpressing HMGN1) hematopoiesis at steady state in young and older mice. OE-HMGN1 transgenic mice had a significant increase in lineage-negative cells with age (Fig A, *p=0.02). We next performed competitive bone marrow transplantation with CD45.1 (WT) vs CD45.2 (WT or OE-HMGN1) donors measuring the relative contribution of CD45.1 and 45.2 cells to hematopoiesis over time after 3 consecutive serial transplants, each at 16 weeks. OE-HMGN1 stem and progenitor cells (HSPCs) had a progressive clonal advantage during serial transplantation (Fig B, *p<0.0002). This clonal advantage was observed across multiple HSPC populations and in all mature lineages. Lin-Sca1+Kit+ (LSK) and Lin-Kit+ (LK) HSPCs were sorted 4 months after transplantation for cell cycle analysis, ex vivo differentiation assays, and RNA-sequencing. OE-HMGN1 HSPCs were less quiescent in vivo, with fewer cells in G0 (Fig C, LK p= 0.0007, LSK p=0.01), and had a relative defect in differentiation capacity in vitro compared to WT. Gene set enrichment analysis was consistent with these phenotypes, showing enrichment of senescence and cell cycle regulatory gene expression programs in WT compared to OE-HMGN1 HSPCs.HMGN1 overexpression alone did not cause leukemia in mice. To screen for synergy between HMGN1 and AML oncogenes, we immortalized myeloid progenitors from WT or OE-HMGN1 mice with a HoxB8 transgene and measured growth properties after transduction with retroviruses expressing one of several AML-associated fusion proteins (BCR-ABL, MLL-AF9, PML-RAR, AML-ETO9a, MOZ-TIF2) or mutated oncogenes (FLT3, NPM1, NRAS, GATA2) with GFP. AML-ETO9a (AE9a) and MOZ-TIF2 had the most significant cooperation with HMGN1, most notably by enhancing their ability to block myeloid differentiation in vitro. This is of interest because, like we found for HMGN1, AE9a and MT2 are known promote leukemia via chromatin effects dependent on CBP/p300 and/or acetyltransferase activity. In confirmatory experiments, combination of AE9a and HMGN1 exacerbated the delay in myeloid differentiation induced by either gene alone (p<0.05), and HMGN1 enhanced serial colony formation capacity of AE9a in semi-solid media (p<0.05).In vivo transplantation of WT or OE-HMGN1 c-Kit+ HSPCs transduced with AE9a led to development of AML ~110 days after injection in both groups. However, subpopulation analysis of AE9a-expressing leukemias showed that LSK and MPP-like populations were expanded in the context of OE-HMGN1 compared to WT (p<0.05). Remarkably, global H3K27ac levels were significantly higher in LT-HSC-like HMGN1-OE AE9a leukemia cells using single cell subpopulation analysis by intracellular flow cytometry compared to WT (Fig D p=0.008), similar to what we observed in HSPCs after competitive transplant. Ex vivo clonogenic capacity was increased in AE9a leukemias that overexpressed HMGN1 in an LTC-IC assay (Fig E *p<0.05). Limiting dilution secondary transplants are ongoing to test if these differences correlate with increased leukemia-initiating capacity per cell in vivo in leukemias that overexpress HMGN1.Our study suggests that overexpression of HMGN1 provides a clonal advantage to hematopoietic populations including long-term stem cells. HMGN1 also cooperates with specific AML oncogenes, possibly those associated with H3K27 acetylation/HAT activity, to enhance leukemic phenotypes. Further study is needed to determine if HMGN1 overexpression confers specific therapeutic vulnerabilities, such as to HAT inhibitors or other chromatin-targeting agents. [Display omitted] DisclosuresLane:Stemline Therapeutics: Research Funding; N-of-one: Consultancy.

A feature-based integrated scoring scheme for cell cycle-regulated genes prioritization

Prioritization of cell cycle-regulated genes from expression time-profiles is still an open problem. The point at issue is the surprisingly poor overlap among ranked lists obtained from different experimental protocols. Instead of developing a general-purpose computational methodology for detecting periodic signals, we focus on the budding yeast mitotic cell cycle. The reason being that the current availability of a total of 12 datasets, produced by 6 independent groups using 4 different synchronization methods, permits a re-analysis and re-consideration of this problem in a more reliable and extensive data domain. Notably, budding yeast is a model organism for studying cancer and testing new drugs. Here we propose a novel multi-feature score (called PERLA, PERiodicity, Regulation and Lag-Autocorrelation) that integrates different features of cell cycle-regulated gene expression time-profiles. We obtained increased performances on a wide range of benchmarks and, most importantly, a substantially increased overlap of the top ranking genes among different datasets, thus proving the effectiveness of the proposed prioritization algorithm. Examples on how to use PERLA to gain new insight into the biology of the cell cycle, are provided in a final dedicated section.

Layers of regulation of cell-cycle gene expression in the budding yeast Saccharomyces cerevisiae.

In the budding yeast Saccharomyces cerevisiae, transcription factors (TFs) regulate the periodic expression of many genes during the cell cycle, including gene products required for progression through cell-cycle events. Experimental evidence coupled with quantitative models suggests that a network of interconnected TFs is capable of regulating periodic genes over the cell cycle. Importantly, these dynamical models were built on transcriptomics data and assumed that TF protein levels and activity are directly correlated with mRNA abundance. To ask whether TF transcripts match protein expression levels as cells progress through the cell cycle, we applied a multiplexed targeted mass spectrometry approach (parallel reaction monitoring) to synchronized populations of cells. We found that protein expression of many TFs and cell-cycle regulators closely followed their respective mRNA transcript dynamics in cycling wild-type cells. Discordant mRNA/protein expression dynamics was also observed for a subset of cell-cycle TFs and for proteins targeted for degradation by E3 ubiquitin ligase complexes such as SCF (Skp1/Cul1/F-box) and APC/C (anaphase-promoting complex/cyclosome). We further profiled mutant cells lacking B-type cyclin/CDK activity (clb1-6) where oscillations in ubiquitin ligase activity, cyclin/CDKs, and cell-cycle progression are halted. We found that a number of proteins were no longer periodically degraded in clb1-6 mutants compared with wild type, highlighting the importance of posttranscriptional regulation. Finally, the TF complexes responsible for activating G1/S transcription (SBF and MBF) were more constitutively expressed at the protein level than at periodic mRNA expression levels in both wild-type and mutant cells. This comprehensive investigation of cell-cycle regulators reveals that multiple layers of regulation (transcription, protein stability, and proteasome targeting) affect protein expression dynamics during the cell cycle.

SOX2OT knockdown derived changes in mitotic regulatory gene network of cancer cells

BackgroundSOX2 overlapping transcript (SOX2OT) is a long non-coding RNA, over-expressed in human tumor tissues and embryonic cells. Evidences support its function in the cell cycle; however there is no clear mechanism explaining its function in cell proliferation regulation. Here we investigated cancer cell response to SOX2OT knockdown by RNA sequencing.MethodsSOX2OT expression was inhibited by siRNA in two cancer cell lines (A549, U-87 MG), then the RNA of treated cells were used for the cDNA library synthesis and RNA sequencing. The differentially expressed genes were used for functional enrichment and the gene expression network was analyzed to find the most relevant biological process with SOX2OT function. Furthermore, the expression change of candidate genes was measured by qRT-PCR for more confirmation and the cell cycle was monitored by PI staining.ResultsOur findings showed that SOX2OT knockdown affects the cellular gene expression generally with enriched cell proliferation and development biological process. Particularly, the cell cycle and mitotic regulatory genes expression including: CDK2, CDK2AP2, ACTR3, and chromosome structure associated genes like SMC4, INCENP and GNL3L are changed in treated cancer cells.ConclusionOur results propound SOX2OT association with cell cycle and mitosis regulation in cancer cells.