Role In Genomic Stability Research Articles

Identification of Functional Domains in the Cohesin Loader Subunit Scc4 by a Random Insertion/Dominant Negative Screen.

Cohesin is a multi-subunit complex that plays an essential role in genome stability. Initial association of cohesin with chromosomes requires the loader—a heterodimer composed of Scc4 and Scc2. However, very little is known about the loader’s mechanism of action. In this study, we performed a genetic screen to identify functional domains in the Scc4 subunit of the loader. We isolated scc4 mutant alleles that, when overexpressed, have a dominant negative effect on cell viability. We defined a small region in the N terminus of Scc4 that is dominant negative when overexpressed, and on which Scc2/Scc4 activity depends. When the mutant alleles are expressed as a single copy, they are recessive and do not support cell viability, cohesion, cohesin loading or Scc4 chromatin binding. In addition, we show that the mutants investigated reduce, but do not eliminate, the interaction of Scc4 with either Scc2 or cohesin. However, we show that Scc4 cannot bind cohesin in the absence of Scc2. Our results provide new insight into the roles of Scc4 in cohesin loading, and contribute to deciphering the loading mechanism.

Ubiquitin-specific Peptidase 10 (USP10) Deubiquitinates and Stabilizes MutS Homolog 2 (MSH2) to Regulate Cellular Sensitivity to DNA Damage

MSH2 is a key DNA mismatch repair protein, which plays an important role in genomic stability. In addition to its DNA repair function, MSH2 serves as a sensor for DNA base analogs-provoked DNA replication errors and binds to various DNA damage-induced adducts to trigger cell cycle arrest or apoptosis. Loss or depletion of MSH2 from cells renders resistance to certain DNA-damaging agents. Therefore, the level of MSH2 determines DNA damage response. Previous studies showed that the level of MSH2 protein is modulated by the ubiquitin-proteasome pathway, and histone deacetylase 6 (HDAC6) serves as an ubiquitin E3 ligase. However, the deubiquitinating enzymes, which regulate MSH2 remain unknown. Here we report that ubiquitin-specific peptidase 10 (USP10) interacts with and stabilizes MSH2. USP10 deubiquitinates MSH2 in vitro and in vivo Moreover, the protein level of MSH2 is positively correlated with the USP10 protein level in a panel of lung cancer cell lines. Knockdown of USP10 in lung cancer cells exhibits increased cell survival and decreased apoptosis upon the treatment of DNA-methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and antimetabolite 6-thioguanine (6-TG). The above phenotypes can be rescued by ectopic expression of MSH2. In addition, knockdown of MSH2 decreases the cellular mismatch repair activity. Overall, our results suggest a novel USP10-MSH2 pathway regulating DNA damage response and DNA mismatch repair.

TALEored Epigenetics: A DNA-Binding Scaffold for Programmable Epigenome Editing and Analysis.

Epigenetic modification of the cytosine 5-position is an important regulator of gene expression with essential roles in genome stability, development, and disease. In addition to 5-methylcytosine (mC), the oxidized mC derivatives 5-hydroxymethyl-, 5-formyl-, and 5-carboxylcytosine (hmC, fC, and caC) have recently been discovered. These are intermediates of an active demethylation pathway but might also represent new epigenetic marks with individual biological roles. This increase in chemical complexity of DNA-encoded information has created a pressing need for new approaches that allow reading and editing of this information. Transcription-activator-like effectors (TALEs) are DNA-binding domains with programmable sequence selectivity that enable the direct reading of epigenetic cytosine modifications but can also guide enzymatic editing domains to genomic loci of choice. Here, we review recent advances in employing TALEs for these applications.

OsSRT1 is involved in rice seed development through regulation of starch metabolism gene expression

OsSRT1 is a NAD+-dependent histone deacetylase, closely related to the human SIRT6 that plays key roles in genome stability and metabolic homeostasis. In this work, we investigated the role of OsSRT1 in rice seed development. Down-regulation of OsSRT1 induced higher expression of Rice Starch Regulator1 (RSR1) and amylases genes in developing seeds, which resulted in a decrease of starch synthesis and an increase of starch degradation, leading to abnormal seed development. ChIP assay showed that OsSRT1 was required to reduce histone H3K9 acetylation on starch metabolism genes and transposons in developing seeds. In addition, OsSRT1 was detected to directly bind to starch metabolism genes such as OsAmy3B, OsAmy3E, OsBmy4, and OsBmy9. Our results suggested that OsSRT1-mediated histone deacetylation is involved in starch accumulation and transposon repression to regulate normal seed development.

Sequence-dependent but not sequence-specific piRNA adhesion traps mRNAs to the germ plasm.

The conserved Piwi family of proteins and piwi-interacting RNAs (piRNAs) play a central role in genomic stability, which is inextricably tied with germ cell formation, by forming ribonucleoproteins (piRNPs) that silence transposable elements (TEs)1. In Drosophila melanogaster and other animals, primordial germ cell (PGC) specification in the developing embryo is driven by maternal mRNAs and proteins that assemble into specialized mRNPs localized in the germ (pole) plasm at the posterior of the oocyte2,3. Maternal piRNPs, especially those loaded on Aubergine (Aub), a Piwi protein, are transmitted to the germ plasm to initiate transposon silencing in the offspring germline4–7. Transport of mRNAs to the oocyte by midoogenesis is an active, microtubule-dependent process8; mRNAs necessary for PGC formation are enriched in the germ plasm at late oogenesis via a diffusion and entrapment mechanism, whose molecular identity remains unknown8,9. Aub is a central component of germ granule RNPs, which house mRNAs in the germ plasm10–12 and interactions between Aub and Tudor are essential for the formation of germ granules13–16. Here we show that Aub-loaded piRNAs use partial base pairing characteristic of Argonaute RNPs to bind mRNAs randomly, acting as an adhesive trap that captures mRNAs in the germ plasm, in a Tudor-dependent manner. Strikingly, germ plasm mRNAs in Drosophilids are generally longer and more abundant than other mRNAs, suggesting that they provide more target sites for piRNAs to promote their preferential tethering in germ granules. Thus complexes containing Tudor, Aub piRNPs and mRNAs couple piRNA inheritance with germline specification. Our findings reveal an unexpected function for Piwi ribonucleoprotein complexes in mRNA trapping that may be generally relevant to the function of animal germ granules.

Deletion of sirtuin 6 accelerates endothelial dysfunction and atherosclerosis in apolipoprotein E-deficient mice

Sirtuin 6 (SIRT6) is a chromatin-associated deacetylase that plays a leading role in genomic stability and aging. However, the precise role of SIRT6 in atherosclerosis, an aging-associated cardiovascular disease, remains elusive. This study aims at defining the role of SIRT6 in atherosclerotic lesion development. SIRT6 messenger RNA and protein expression are markedly decreased in atherosclerotic aortas ofapolipoprotein E-deficient (ApoE(-/-)) mice fed a high-cholesterol diet. SIRT6 wasknocked down in ApoE(-/-) mice using small hairpin RNAs (shRNAs) lentivirus injection. SIRT6-shRNA-treated ApoE(-/-) mice showed impaired endothelium-dependent vasodilation, increased plaque size (in aortic sinus, aortic root and en face aorta), and augmented plaque vulnerability (evidenced by increased necrotic core areas and macrophage accumulation and reduced collagen content). At the cellular level, SIRT6 depletion by RNA interference in human umbilical vein endothelial cells significantly increased monocyte adhesion to endothelial cells by inducing the expression of intracellular adhesion molecule-1. Consistently, intracellular adhesion molecule-1 expression was significantly upregulated in aortic endothelium of SIRT6-shRNA-treated ApoE(-/-) mice compared with controls. In sum, the aforementioned findings suggest that SIRT6 is a primary negative regulation factor in endothelial dysfunction and atherosclerosis development. As a result, SIRT6 is a promising therapeutic target for treating atherosclerosis and its cardiovascular complications.

Non-catalytic Roles for XPG with BRCA1 and BRCA2 in Homologous Recombination and Genome Stability

XPG is a structure-specific endonuclease required for nucleotide excision repair, and incision-defective XPG mutations cause the skin cancer-prone syndrome xeroderma pigmentosum. Truncating mutations instead cause the neurodevelopmental progeroid disorder Cockayne syndrome, but little is known about how XPG loss results in this devastating disease. We identify XPG as a partner of BRCA1 and BRCA2 in maintaining genomic stability through homologous recombination (HRR). XPG depletion causes DNA double-strand breaks, chromosomal abnormalities, cell-cycle delays, defective HRR, inability to overcome replication fork stalling, and replication stress. XPG directly interacts with BRCA2, RAD51, and PALB2, and XPG depletion reduces their chromatin binding and subsequent RAD51 foci formation. Upstream in HRR, XPG interacts directly with BRCA1. Its depletion causes BRCA1 hyper-phosphorylation and persistent chromatin binding. These unexpected findings establish XPG as an HRR protein with important roles in genome stability and suggest how XPG defects produce severe clinical consequences including cancer and accelerated aging.

Abstract A1-04: The role of chromatin remodeler CHD1 in genomic alterations and prostate cancer progression

Abstract The chromodomain helicase DNA (CHD) binding proteins have been implicated to play a role in genome stability. CHD1 is second only to PTEN as the most frequent homozygously deleted gene in the tumor genome of prostate cancer (PCa). To assess the role of CHD1 in genomic integrity and PCa progression, we performed a comprehensive survey of DNA copy number alterations (CNAs) in the tumor genome among a total of 611 patients from 5 independent cohorts and an association analysis of CHD1 deletion with cancer progression from low (Gleason 6) to high (Gleason ≥ 8) grade tumors. Our data revealed that the frequency of CHD1 deletion significantly increased as cancer progressing from low to intermediate and to high grades, suggesting a role of this CNA in cancer progression. Loss of CHD1 was significantly associated with homozygous deletions at other genomic locations in the tumor genome, and with deletion of MAP3K7. We next evaluated the in vitro and in vivo joint effects of knockdown of CHD1 and the collaborative gene MAP3K7 on tumorigenesis, cell proliferation (Ki67), tumor size, histopathological characteristics, and cancer progression, using shRNA and tissue recombination with rat urogenital mesenchyme and mouse prostate epithelial stem/progenitor cells grafted under the renal capsule and subcutaneous xenografts in nude mice. While suppressing CHD1 expression, in general, resulted in deceased growth of tumor cells vs. shControl cells, concurrently suppressing expression of both CHD1 and MAP3K7 promoted aggressive tumor growth and reduced survival. We finally explored the effect of knockdown of CHD1/MAP3K7 expression in stable cell lines derived human normal prostate epithelial cells and cancer cells on genomic rearrangement via CNA analysis. Although a wide range of CNAs was observed in different cell lines, some of them recapitulated the CNAs that were commonly observed in the tumors of human PCa. Collectively, these data suggest that loss of CHD1 leads to further evolution of cancer cells via genomic instability and combined loss of CHD1 and MAP3K7 drives the progression of PCa. Note: This abstract was not presented at the conference. Citation Format: Wennuan Liu, Lindsey Ulkus Rodrigues, Tao Li, Zheng Zhang, Leah Rider, Lina Romero, Lilly S. Zheng, William B. Isaacs, Scott Cramer, Jianfeng Xu. The role of chromatin remodeler CHD1 in genomic alterations and prostate cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-04.

Abstract 12622: The Modeling of Werner Syndrome by Induced Pluripotent Stem Cells

Backgrounds: Werner syndrome (WS) is a rare autosomal recessive disorder characterized by premature onset of several aging-associated diseases, such as atherosclerosis, diabetes, cancer, and early death. The aging phenotypes of WS is resembling to those of normal aging. To uncover the mechanism of aging, we tried to model WS by patient-specific induced pluripotent stem cells (iPSCs). WS is caused by mutations in WRN gene belonging to the RecQ DNA helicase family which plays a role in genomic stability. But some of WS phenotypes are hardly explained by genomic instability. Thus, we aimed to model WS by patient-specific iPSCs to elucidate the mechanisms. Methods and Results: We sampled T lymphocytes from a patient with WS. Then we transduced with Yamanaka factors (OCT4, SOX2, KLF4, and MYC) by sendai virus, and iPSC colonies were derived. We confirmed that WS-iPSCs expressed pluripotent markers, could differentiate into all three germ-layer derived tissues, and retained a normal karyotype. We could culture WS-iPSCs over 2 years with pluripotent status. Then, we differentiated WS-iPSCs into fibroblasts-like cells. The proliferation rate of WS-iPSC-derived fibroblast-like cells (WS-iPSC-fibroblasts) was significantly decreased. WS-iPSC-fibroblasts showed a vulnerability to cellular stress and resulted in increased cell population which is positive for senescence associated β-galactosidase activity and γ-H2AX foci. Singled WS-iPSC-fibroblasts showed excessive blebbing of plasma membrane and increased apoptosis compared with control-iPSC-fibroblasts. To compare global gene expression profiles, we performed microarray analysis in WS-iPSC-fibroblasts and control-iPSC-fibroblasts. Interestingly, WS-iPSC-fibroblasts reproduced the global gene expression pattern of physiological aging. To confirm whether the phenotypes of WS-iPSCs are induced by WRN mutation, we generated isogenic control of WS-iPSC (corrected-WS-iPSC) by homologous recombination using helper-dependent adenovirus vector. Corrected-WS-iPSCs lost the aging-associated phenotypes but showed the phenotypes resembling to control-iPSCs. Conclusion: We modeled aging phenotypes by WS-specific iPSCs. This model would be utilize for uncovering the aging mechanisms.

ZSCAN4 and TRF1: A functionally indirect interaction in cancer cells independent of telomerase activity

PurposeRecently, the newly identified embryonic stem cell marker, Zinc finger and SCAN domain containing 4 gene (ZSCAN4), which plays a key role in genomic stability by regulating telomere elongation, was shown to co-localize with TRF1 foci. This suggests that the interaction of ZSCAN4 with TRF1 functions in regulation of telomere elongation in ESC. Based on these studies, we hypothesized that ZSCAN4 binds to TRF1 in cancer cells to function in regulating telomere length. The purpose of this study was to determine whether this interaction occurred across different cell lineage-derived cancers and whether telomerase status impacted this relationship. To that end, telomerase positive cervical cancer cells (HeLa) and breast cancer cells (MCF7), and telomerase negative osteosarcoma cells (SaOS2), were analyzed for ZSCAN4 and TRF1 interactions. ResultsImmunocytochemistry demonstrated co-localization of ZSCAN4 and TRF1 to the nucleus. This functional relationship was confirmed using BiFC imaging analysis based on distance in situ. Co-immunoprecipitation and pull-down assay results demonstrated that ZSCAN4 binds with TRF1 in vitro indirectly. All three cell types showed similar results. ConclusionsIn this study, we revealed, for the first time, that ZSCAN4 indirectly interacts with TRF1 (functional association protein) in cancer cells. Furthermore, we show that ZSCAN4 plays an important role independent of telomere maintenance pathways (telomerase positive and ALT) or cell lineage.

Human POLD1 modulates cell cycle progression and DNA damage repair.

BackgroundThe activity of eukaryotic DNA polymerase delta (Pol δ) plays an essential role in genome stability through its effects on DNA replication and repair. The p125 catalytic subunit of Pol δ is encoded by POLD1 gene in human cells. To clarify biological functions of POLD1, we investigated the effects of POLD1 overexpression or downregulation on cell proliferation, cell cycle progression, DNA synthesis and oxidative DNA damage induced by H2O2.MethodsHEK293 cells were transfected with POLD1 expression plasmid or shRNA, cell proliferation, cell cycle progression, and DNA synthesis in HEK293 cells were analyzed.ResultsHEK293 cells were transfected with POLD1 expression plasmid or shRNA. POLD1 downregulation by shRNA suppressed cell proliferation, cell cycle progression, and DNA synthesis in HEK293 cells. However, POLD1 overexpression had no significant effects on these processes. Finally, comet assay showed that POLD1 downregulation led to increased DNA damage.ConclusionsOur results suggest that human POLD1 plays important role in the regulation of cell cycle progression and DNA damage repair.

Deciphering Epigenetic Cytosine Modifications by Direct Molecular Recognition.

Epigenetic modification at the 5-position of cytosine is a key regulatory element of mammalian gene expression with important roles in genome stability, development, and disease. The repertoire of cytosine modifications has long been confined to only 5-methylcytosine (mC) but has recently been expanded by the discovery of 5-hydroxymethyl-, 5-formyl-, and 5-carboxylcytosine. These are key intermediates of active mC demethylation but may additionally represent new epigenetic marks with distinct biological roles. This leap in chemical complexity of epigenetic cytosine modifications has not only created a pressing need for analytical approaches that enable unraveling of their functions, it has also created new challenges for such analyses with respect to sensitivity and selectivity. The crucial step of any such approach that defines its analytic potential is the strategy used for the actual differentiation of the cytosine 5-modifications from one another, and this selectivity can in principle be provided either by chemoselective conversions or by selective, molecular recognition events. While the former strategy has been particularly successful for accurate genomic profiling of cytosine modifications in vitro, the latter strategy provides interesting perspectives for simplified profiling of natural, untreated DNA, as well as for emerging applications such as single cell analysis and the monitoring of cytosine modification in vivo. We here review analytical techniques for the deciphering of epigenetic cytosine modifications with an emphasis on approaches that are based on the direct molecular recognition of these modifications in DNA.

RNA-processing proteins regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA.

Eukaryotic cells respond to DNA double-strand breaks (DSBs) by activating a checkpoint that depends on the protein kinases Tel1/ATM and Mec1/ATR. Mec1/ATR is activated by RPA-coated single-stranded DNA (ssDNA), which arises upon nucleolytic degradation (resection) of the DSB. Emerging evidences indicate that RNA-processing factors play critical, yet poorly understood, roles in genomic stability. Here, we provide evidence that the Saccharomyces cerevisiae RNA decay factors Xrn1, Rrp6 and Trf4 regulate Mec1/ATR activation by promoting generation of RPA-coated ssDNA. The lack of Xrn1 inhibits ssDNA generation at the DSB by preventing the loading of the MRX complex. By contrast, DSB resection is not affected in the absence of Rrp6 or Trf4, but their lack impairs the recruitment of RPA, and therefore of Mec1, to the DSB. Rrp6 and Trf4 inactivation affects neither Rad51/Rad52 association nor DSB repair by homologous recombination (HR), suggesting that full Mec1 activation requires higher amount of RPA-coated ssDNA than HR-mediated repair. Noteworthy, deep transcriptome analyses do not identify common misregulated gene expression that could explain the observed phenotypes. Our results provide a novel link between RNA processing and genome stability.

Abstract 11059: Role of Sirtuin 6 in Macrophage Polarization and Cardiac Repair in Diabetes

Clinical and experimental studies provide evidence that metabolic and inflammatory pathways are functionally interconnected to cardiovascular diseases. Dynamic changes in macrophage activation [classical M1 activation (promote inflammation) or alternative M2 activation (promote wound healing)], in response to various stress signals, modulate cardiac physiopathology in diabetes. Sirtuin 6 (SIRT6), a NAD-dependent nuclear deacetylase plays an important role in genomic stability, cellular metabolism, stress response and aging. However, the mechanism by which SIRT6 activity affects macrophage phenotype and cardiac function in diabetes is still unexplored. Mouse bone marrow-derived macrophages (BMM) exposed to high glucose (HG, 25mM D-glucose) showed reduced expression of SIRT6 as compared to low glucose (LG, 5mM D-glucose)- and osmotic control (OC, 5mM D-glucose+20mM D-mannitol)-treated cells, associated with increased expression of proinflammatory cytokine and transcription factors (NFkb, c-JUN, FOXO, SP1 and STAT1). In addition, SIRT6 level was reduced in peritoneal macrophages of both diabetic models (streptozotocin-induced and db/db mice) as compared to non-diabetic mice. SIRT6 knockdown in RAW 264.7 cells exaggerated inflammatory response when exposed to HG. In contrast, IL-4-induced increase in mRNA expression of macrophage M2 phenotype markers like Arg1, Chi4l4, Retnla and IRS-2, but not IRS-1 expression was repressed suggesting that alternative macrophage (M2) phenotype was defective in SIRT6 deficient BM-macrophages under HG condition. SIRT6 protein expression was low in myocardial infarction-induced (MI) and diabetes-affected hearts. Interestingly, mice receiving intramyocardial injection of SIRT6-deficient macrophages showed further deterioration in left ventricular function, post-MI. Taken together, these data highlight a role for SIRT6 in regulating the balance of M1/M2 polarization, therefore, modulate macrophage mediated cardiac repair and regeneration in numerous inflammatory disease states including diabetes

EG-17 * SUV420-MEDIATED HETEROCHROMATIN CHANGES IN PEDIATRIC BRAIN CANCERS

Silencing mechanisms play a role in genomic stability by maintaining condensed, non-active regions of the genome. SUV420 enzymes contain a SET domain conferring methyltransferase activity toward histones. The Histone H4 lysine 20 trimethylation (H4K20me3) mark maintained by SUV420H2 is associated with heterochromatin formation and gene silencing, whereas the dimethylated mark (H4K20me2) is associated with DNA repair. In studies of epigenetic factors in large patient cohorts with ependymoma, it was found that SUV420H2 expression was lost or diminished in patients with reciprocal increases in prognostic markers such as hTERT. To better understand the normal function of Suv4-20H1/H2 enzyme in neural progenitors, and pathological changes in cancers, a variety of differentiation paradigms were used. The NT2D1 neurally restricted cell line, and BGO1V and H9 human embryonic stem cells (ESCs), and differentiated progeny, were used alongside tumors to better understand enzyme targets and functional outcomes (e.g.,lineage, differentiation, regional chromatin modifications). Lineage stages were verified with stage-specific markers by immunofluorescence and qPCR. Suv4-20 H1 and H2 were present in ESCs and neural progenitors and decreased thereafter. RNAi knockdown of SUV420 enzymes led to decreased H4K20 methylation in cancer cells. DNA methylation microarrays and ChIP-PCR suggest 1) that SUV420 is not regulated by DNA methylation in ependymomas; 2) that active chromatin marks such as H3K4 dimethylation are enriched near the transcriptional start site in the SUV420H2 gene, and 3) that hTERT is hyper-methylated at specific CpG islands and histones in a tumor sub-group-specific manner. This data supports the hypothesis that Suv4-20H2 is highly active in progenitor cells and functionally lost in some brain cancers. These studies begin to elucidate coincident mechanisms of gene silencing active in neural progenitors that may be altered in a subset of pediatric brain cancers.

Strategy for "detoxification" of a cancer-derived histone mutant based on mapping its interaction with the methyltransferase PRC2.

The histone methyltransferase PRC2 plays a central role in genomic stability and cellular development. Consequently, its misregulation has been implicated in several cancers. Recent work has shown that a histone H3 mutant, where the PRC2 substrate residue Lys27 is replaced by methionine, is also associated with cancer phenotypes and functions as an inhibitor of PRC2. Here we investigate the mechanism of this PRC2 inhibition through kinetic studies and photo-cross-linking. Efficient inhibition is dependent on (1) hydrophobic lysine isosteres blocking the active site, (2) proximal residues, and (3) the H3 tail forming extensive contacts with the EZH2 subunit of PRC2. We further show that naturally occurring post-translational modifications of the same H3 tail, both proximal and distal to K27M, can greatly diminish the inhibition of PRC2. These results suggest that this potent gain of function mutation may be “detoxified” by modulating alternate chromatin modification pathways.

Hepatology Highlights

Hepatology Highlights

Zscan4 interacts directly with human Rap1 in cancer cells regardless of telomerase status

Telomeres are repetitive sequences at the ends of chromosomes protected by DNA binding proteins of the shelterin complex that form capping structures. Through the interaction of shelterin complex-associated proteins, telomere length maintenance is regulated. Recently, the newly identified embryonic stem cell marker, Zinc finger and SCAN domain-containing 4 gene (Zscan4), was shown to be a telomere-associated protein, co-localizing to the shelterin complex. Furthermore, it was shown to play an essential role in genomic stability by regulating telomere elongation. Although it is known that Zscan4 regulates TRF2, POT1b, and Rap1 expression in embryonic stem cells, the relationship and the exact mechanism of action for ZSscan4-mediated telomere maintenance in cancer cells is unknown. In this study, we investigated Zscan4 expression and interactions with Rap1 in telomerase positive (HeLa, MCF7) and ALT pathway (SaOS2, U2OS) cancer cells. Through western, pulldown, siRNA, and overexpression assays we demonstrate, for the first time, that Zscan4 directly associates with Rap1 (physical association protein). Furthermore, by generating truncated versions of Zscan4, we identified its zinc finger domain as the Rap1 binding site. Using bimolecular fluorescence complementation, we further validate this functional interaction in human cancer cells. Our results indicate that Zscan4 functions as a mediator of telomere length through its direct interaction with Rap1, possibly regulating shelterin complex-controlled telomere elongation in both telomerase positive and alternative lengthening of telomere pathways. This direct interaction between Zscan4 and Rap1 may explain how Zscan4 rapidly increases telomere length, yielding important information about the role of these proteins in telomere biology.

DNA polymerase ε and its roles in genome stability.

DNA Polymerase Epsilon (Pol ε) is one of three DNA Polymerases (along with Pol δ and Pol α) required for nuclear DNA replication in eukaryotes. Pol ε is comprised of four subunits, the largest of which is encoded by the POLE gene and contains the catalytic polymerase and exonuclease activities. The 3'-5' exonuclease proofreading activity is able to correct DNA synthesis errors and helps protect against genome instability. Recent cancer genome sequencing efforts have shown that 3% of colorectal and 7% of endometrial cancers contain mutations within the exonuclease domain of POLE and are associated with significantly elevated levels of single nucleotide substitutions (15-500 per Mb) and microsatellite stability. POLE mutations have also been found in other tumor types, though at lower frequency, suggesting roles in tumorigenesis more broadly in different tissue types. In addition to its proofreading activity, Pol ε contributes to genome stability through multiple mechanisms that are discussed in this review.

Kinome and mRNA expression profiling of high-grade osteosarcoma cell lines implies Akt signaling as possible target for therapy

BackgroundHigh-grade osteosarcoma is a primary malignant bone tumor mostly occurring in adolescents and young adults, with a second peak at middle age. Overall survival is approximately 60%, and has not significantly increased since the introduction of neoadjuvant chemotherapy in the 1970s. The genomic profile of high-grade osteosarcoma is complex and heterogeneous. Integration of different types of genome-wide data may be advantageous in extracting relevant information from the large number of aberrations detected in this tumor.MethodsWe analyzed genome-wide gene expression data of osteosarcoma cell lines and integrated these data with a kinome screen. Data were analyzed in statistical language R, using LIMMA for detection of differential expression/phosphorylation. We subsequently used Ingenuity Pathways Analysis to determine deregulated pathways in both data types.ResultsGene set enrichment indicated that pathways important in genomic stability are highly deregulated in these tumors, with many genes showing upregulation, which could be used as a prognostic marker, and with kinases phosphorylating peptides in these pathways. Akt and AMPK signaling were identified as active and inactive, respectively. As these pathways have an opposite role on mTORC1 signaling, we set out to inhibit Akt kinases with the allosteric Akt inhibitor MK-2206. This resulted in inhibition of proliferation of osteosarcoma cell lines U-2 OS and HOS, but not of 143B, which harbors a KRAS oncogenic transformation.ConclusionsWe identified both overexpression and hyperphosphorylation in pathways playing a role in genomic stability. Kinome profiling identified active Akt signaling, which could inhibit proliferation in 2/3 osteosarcoma cell lines. Inhibition of PI3K/Akt/mTORC1 signaling may be effective in osteosarcoma, but further studies are required to determine whether this pathway is active in a substantial subgroup of this heterogeneous tumor.