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Background:. Age-related macular degeneration (AMD) is the leading cause of vision loss worldwide. However, the mechanisms involved in the development and progression of AMD are poorly delineated. We aimed to explore the critical genes involved in the progression of AMD. Methods:. The differentially expressed genes (DEGs) in AMD retinal pigment epithelial (RPE)/choroid tissues were identified using the microarray datasets GSE99248 and GSE125564, which were downloaded from the gene expression omnibus database. The overlapping DEGs from the two datasets were screened to identify DEG-related biological pathways using gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses. The hub genes were identified from these DEGs through protein-protein interaction network analyses. The expression levels of hub genes were evaluated by quantitative real-time polymerase chain reaction following the induction of senescence in ARPE-19 with FK866. Following the identification of AMD-related key genes, the potential small molecule compounds targeting the key genes were predicted by PharmacoDB. Finally, a microRNA-gene interaction network was constructed. Results:. Microarray analyses identified 174 DEGs in the AMD RPE compared to the healthy RPE samples. These DEGs were primarily enriched in the pathways involved in the regulation of DNA replication, cell cycle, and proteasome-mediated protein polyubiquitination. Among the top ten hub genes, HSP90AA1, CHEK1, PSMA4, PSMD4, and PSMD8 were upregulated in the senescent ARPE-19 cells. Additionally, the drugs targeting HSP90AA1, CHEK1, and PSMA4 were identified. We hypothesize that Hsa-miR-16-5p might target four out of the five key DEGs in the AMD RPE. Conclusions:. Based on our findings, HSP90AA1 is likely to be a central gene controlling the DNA replication and proteasome-mediated polyubiquitination during the RPE senescence observed in the progression of AMD. Targeting HSP90AA1, CHEK1, PSMA4, PSMD4, and/or PSMD8 genes through specific miRNAs or small molecules might potentially alleviate the progression of AMD through attenuating RPE senescence.

The cyclic GMP-AMP (cGAMP) synthase (cGAS) has been identified as a cytosolic double stranded DNA sensor that plays a pivotal role in the type I interferon and inflammation responses via the STING-dependent signaling pathway. In the past several years, a growing body of evidence has revealed that cGAS is also localized in the nucleus where it is associated with distinct nuclear substructures such as nucleosomes, DNA replication forks, the double-stranded breaks, and centromeres, suggesting that cGAS may have other functions in addition to its role in DNA sensing. However, while the innate immune function of cGAS is well established, the non-canonical nuclear function of cGAS remains poorly understood. Here, we review our current understanding of the complex nature of nuclear cGAS and point to open questions on the novel roles and the mechanisms of action of this protein as a key regulator of cell nuclear function, beyond its well-established role in dsDNA sensing and innate immune response.

Motivation: The de novo identification of the initiation and termination zones—regions that replicate earlier or later than their upstream and downstream neighbours, respectively—remains a key challenge in DNA replication. Results: Building on advances in deep learning, we developed a novel hybrid architecture combining a pre-trained, deep neural network and a hidden Markov model (DNN-HMM) for the de novo identification of replication domains using replication timing profiles. Our results demonstrate that DNN-HMM can significantly outperform strong, discriminatively trained Gaussian mixture model–HMM (GMM-HMM) systems and other six reported methods that can be applied to this challenge. We applied our trained DNN-HMM to identify distinct replication domain types, namely the early replication domain (ERD), the down transition zone (DTZ), the late replication domain (LRD) and the up transition zone (UTZ), using newly replicated DNA sequencing (Repli-Seq) data across 15 human cells. A subsequent integrative analysis revealed that these replication domains harbour unique genomic and epigenetic patterns, transcriptional activity and higher-order chromosomal structure. Our findings support the ‘replication-domain’ model, which states (1) that ERDs and LRDs, connected by UTZs and DTZs, are spatially compartmentalized structural and functional units of higher-order chromosomal structure, (2) that the adjacent DTZ-UTZ pairs form chromatin loops and (3) that intra-interactions within ERDs and LRDs tend to be short-range and long-range, respectively. Our model reveals an important chromatin organizational principle of the human genome and represents a critical step towards understanding the mechanisms regulating replication timing. Availability and implementation: Our DNN-HMM method and three additional algorithms can be freely accessed at https://github.com/wenjiegroup/DNN-HMM. The replication domain regions identified in this study are available in GEO under the accession ID GSE53984. Contact: shuwj@bmi.ac.cn or boxc@bmi.ac.cn Supplementary information: Supplementary data are available at Bioinformatics online.

Pokemon is a transcription regulator involved in embryonic development, cellular differentiation and oncogenesis. It is aberrantly overexpressed in multiple human cancers including Hepatocellular carcinoma (HCC) and is considered as a promising biomarker for HCC. In this work, the isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics strategy was used to investigate the proteomic profile associated with Pokemon in human HCC cell line QGY7703 and human hepatocyte line HL7702. Samples were labeled with four-plex iTRAQ reagents followed by two-dimensional liquid chromatography coupled with tandem mass spectrometry analysis. A total of 24 differentially expressed proteins were selected as significant. Nine proteins were potentially up-regulated by Pokemon while 15 proteins were potentially down-regulated and many proteins were previously identified as potential biomarkers for HCC. Gene ontology (GO) term enrichment revealed that the listed proteins were mainly involved in DNA metabolism and biosynthesis process. The changes of glucose-6-phosphate 1-dehydrogenase (G6PD, up-regulated) and ribonucleoside-diphosphate reductase large sub-unit (RIM1, down-regulated) were validated by Western blotting analysis and denoted as Pokemon's function of oncogenesis. We also found that Pokemon potentially repressed the expression of highly clustered proteins (MCM3, MCM5, MCM6, MCM7) which played key roles in promoting DNA replication. Altogether, our results may help better understand the role of Pokemon in HCC and promote the clinical applications.

The early stages of head and neck cancer are presumed to require a senes of genetic alterations that are not represented by a distinct clinical phenotype. Therefore, genes with altered expression in the preneoplasia may be useful for the early detection of this highly recurrent cancer. In this study, we immortalized normal human oral keratinocytes (NHOK) by retroviral-mediated infection of HPV 16 transforming oncogenes, E6 and E7 (HOK16E6E7). Using the Affymetrix gene chip (U95Av2), we identified 177 known genes and EST that were overexpressed at least 3-fold or above in the immortalized cells, while 133 were down-regulated compared to NHOK. Northern blot analysis showed elevated levels of p55CDC in the immortalized cells, while NHOK showed high basal expression of small proline rich protein (SPRR2). The altered expression of these genes maybe associated with cellular proliferation or differentiation and the early stages of oral carcinogenesis.

The effects of Fibroblast Growth Factor-2 (FGF-2) on breast cancer cell DNA synthesis are controversial. To elucidate the mechanisms by which FGF-2 stimulates or inhibits DNA synthesis, we analysed FGF-2 signaling pathways in breast cancer MCF-7 and MCF-7 cells overexpressing Ha-Ras (MCF-7ras). We found that FGF-2-induction of DNA synthesis correlates with Ras transient activation, FRS-2 tyrosine phosphorylation and low level of expression of p66Shc. In addition, Nck-associated proteins are highly tyrosine phosphorylated and JNK reaches a higher level of activation when FGF-2 triggers DNA synthesis. Interestingly upon FGF-2 treatment, JNK activation and DNA synthesis are dependent on Rac-1 activity. These results confirm that in MCF-7 cells, induction of DNA synthesis by FGF-2 requires a transient activation of the Ras/MAPK cascade and demonstrates for the first time that intact Rac-1 and Nck signaling networks are required.

Background: In the hope of understanding how chromosomes condense at mitosis, we took advantage of a subdivision of the cell cycle into 11 stages to examine the changes in DNA taking place during the stages preceding the emergence of metaphase chromosomes. Methods: To identify DNA changes, pieces of mouse duodenum were fixed in formaldehyde, and sections of the rapidly dividing cells of the crypts were stained by the osmium-ammine method, which is specific for the detection of DNA in the electron microscope. Results: Throughout the cell cycle, DNA is present in nucleofilaments composed of rows of 11-nm-wide nucleosomes. At stage I, during which the DNA-synthesizing or S phase of the cell cycle begins, some of the nucleofilaments are compacted in the heterochromatin accumulations associated with nuclear envelope and nucleoli, while the others are scattered in the nucleoplasm where they appear either “free” or “attached” to the heterochromatin. This DNA distribution is similar to that observed in the noncycling cells examined. After the beginning of the S phase, “free” nucleofilaments are seen to assemble into structures composed of compacted nucleofilaments and referred to as “aggregates”; these make their appearance at stage II and increase in size through stage III up to the end of S during stage IV. Meanwhile, the heterochromatin associated with nuclear envelope and nucleoli expands toward the nucleoplasm in the form of protrusions referred to as “bulges,” which gradually enlarge during stages III and IV, while the heterochromatin shrinks and eventually vanishes. On average, a total of 1,171 aggregates and bulges are formed in the nucleus during the S phase. At the apparition of stage V, which corresponds approximately to prophase, aggregates and bulges are rapidly gathered into an average of 288 spheroidal bodies referred to as “chromomeres.” These are connected to one another by nucleofilamentous bridges in such a way as to be lined up in rows. The formation of rows of chromomeres represents in the electron microscope the prophasic condensation observed in the light microscope. Finally, during stage VIa, which corresponds to prometaphase, the chromomeres approach one another within each row, make contact, and coalesce to become the 40 chromosomes of the mouse, which during stage VIb are organized in the equatorial plate of metaphase. Conclusions: The condensation of metaphase chromosomes occurs in three main steps. The first and longest takes place during the S phase, as nucleofilaments are assembled into aggregates, while the heterochromatin give rise to bulges. The brief second step occurs toward the beginning of prophase, when the numerous aggregates and bulges are congregated into a limited number of chromomeres, which are lined up in rows. The third step takes place during the brief prometaphase, when the chromomeres of a row coalesce into a mitotic chromosome. © 1995 Wiley-Liss, Inc.

Background: Since it has been found that new chromatin structures make their appearance in the nucleus during the DNA-synthesizing or S phase of the cell cycle, the question arises as to how these structures are related to the nascent DNA. Methods: DNA-containing structures were detected in sections of mouse duodenal crypt cells by the DNA-specific osmium-ammine procedure. In the same sections, the nascent or newly-replicated DNA was localized during stages I–IV of the cell cycle (corresponding to four successive parts of the S phase) by immunogold labeling of the DNA precursor bromodeoxyuridine (BrdU) in mice sacrificed 10 min after its injection. Moreover, the fate of the nascent DNA with time was traced up to 6 hr after the injection. (The nomenclature of the DNA-containing structures is that proposed by El-Alfy et al., 1995.) Results: Ten minutes after BrdU injection, the gold particles indicative of nascent DNA are associated with discrete nucleofilaments scattered in the nucleoplasm, but not with the compacted nucleofilaments making up the heterochromatin or the new S phase structures named “aggregates.” The gold-particle-associated discrete nucleofilaments are classified into three types: a) The “free” nucleofilaments have been given this name, since they appear to be independent of heterochromatin and aggregates; nearly all gold particles are over these at stage I; but the numbers of particles over them decreases from stage I to IV. b) The “aggregate-attached” nucleofilaments project from the surface of the aggregates; the number of particles over these is high at stages II and III but decreases at stage IV. c) The “heterochromatin-attached” nucleofilaments project from the surface of the heterochromatin; the number of particles over these increases from stage II to IV. By 1 hr after BrdU injection, gold particles can be over loose clumps of nucleofilaments at stages I and II, but are mostly over small aggregates at stage II, midsized aggregates and small heterochromatin-associated “bulges” at stage III and large aggregates and large bulges at stage IV. By 2–6 hr, virtually all particles are over aggregates and bulges, frequently deep within them. Conclusions: The distribution of the gold particles at 10 min reveals that DNA is synthesized in discrete nucleofilaments that are “free” or “aggregate-attached” or “heterochromatin-attached.” In contrast, by one and especially two hours, the gold particles are present over aggregates and bulges, indicating that, after discrete nucleofilaments acquire nascent DNA, they are displaced to become part of these structures. More precisely, the aggregates arise from the repeated addition of replicated portions of “free” nucleofilaments, while the bulges arise from the repeated addition of replicated portions, of “heterochromatin-attached” nucleofilaments. Aggregates and bulges are the two initial building stones from which mitotic chromosomes are eventually formed. © 1995 Wiley-Liss, Inc.