Chromatid Cohesion Research Articles

Structural maintenance of chromosomes and associated genetic disorders

Structural maintenance of chromosomes (SMC), including cohesin, condensin and the SMC5/6 complex, are protein complexes which maintain the higher structure and dynamic stability of chromatin. Such circular complexes, with similar structures, play pivotal roles in chromatid cohesion, chromosomal condensation, DNA replication and repair, as well as gene transcription. Despite extensive research on the functions of the SMCs, our understanding of the SMC5/6 complex has remained limited compared with the other two complexes. This article has reviewed the architecture and crucial physiological roles of the SMCs, and explored the associated phenotypes resulting from mutations of the SMC components such as Cornelia de Lange syndrome (CdLS) and microcephaly, with an aim to provide insights into their functions in eukaryotic cells and implications for human diseases.

NamiRNA-mediated high expression of KNSTRN correlates with poor prognosis and immune infiltration in hepatocellular carcinoma.

Mutations of kinetochore-localized astrin/sperm-associated antigen 5 (KNSTRN) can interfere with chromatid cohesion, increase aneuploidy in tumours, and enhance tumourigenesis. However, the role of the KNSTRN-binding protein in hepatocellular carcinoma (HCC) remains unclear. Using The Cancer Genome Atlas databases, we investigated the potential oncogenic functions of KNSTRN in HCC along with R and various computational tools. Detailed results revealed that elevated expression of KNSTRN was considerably associated with poor overall survival (HR = 1.48, 95% CI: 1.05-2.09, p = 0.027) and progress-free interval (HR = 1.41, 95% CI: 1.05-1.89, p = 0.021) in HCC. Gene ontology/Kyoto Encyclopedia of Genes and Genomes functional enrichment analysis showed that KNSTRN is closely related to organelle fission, chromosomal region, tubulin binding, and cell cycle signalling pathway. TIMER database analysis showed the correlations between KNSTRN expression and tumour-infiltrating immune cells, biomarkers of immune cells, and immune checkpoint expression. Moreover, the KNSTRN level was significantly positively associated with immunosuppressive cells in the tumour microenvironment, including regulatory T-cells, myeloid-derived suppressor cells, and cancer-associated fibrocytes. Finally, a possible nuclear activating miRNA (NamiRNA)-enhancer network of hsa-miR-107, which activates the KNSTRN expression in liver hepatocellular carcinoma, was constructed by correlation analysis. NamiRNA-mediated upregulation of KNSTRN correlated with poor prognosis and tumour immune infiltration in HCC. KNSTRN could serve as an effective biomarker for the diagnosis and prognosis of HCC and support the development of novel therapeutic strategies.

Abstract 786: STAG2 acts as a transcriptional repressor and promotes invasion of muscle invasive bladder cancer cells

Abstract STAG2 (Stromal Antigen 2) functions in chromatid cohesion, DNA damage repair and genome organization, but its role in muscle invasive bladder cancer (MIBC) remains unknown. We have previously found that in MIBC, loss of STAG2 protein expression is associated with better overall survival (n=169; p=0.049) and progression free survival (n=169; p=0.016). Based on these retrospective analyses, we hypothesized that STAG2 promotes an aggressive cell phenotype through its genomic interactions and transcriptomic regulation in MIBC. First, to study the effects of STAG2 on gene expression, we stably knocked down (KD) STAG2 in T24 MIBC cells using two short hairpin RNAs. Scrambled shRNA served as a control in all experiments. Second, using KD and control T24 cell lines, we performed RNA and chromatin-immunoprecipitation sequencing, then integrated these results utilizing the Cistrome analysis algorithm (Wang, S. et al., 2013) to determine how STAG2 regulates gene expression at its genomic binding sites. STAG2 KD led to the differential expression of 2158 genes, with 648 overlapping between the two shRNA cell lines. Through Cistrome analysis, we discovered that genes with increased expression after STAG2 KD were enriched for STAG2 binding sites (p=0.000503), yet genes with decreased expression did not show significant enrichment (p=0.873). This suggests that STAG2 functions as a transcriptional repressor, and subsequently we focused on genes with increased expression after STAG2 KD. We identified the significantly upregulated gene Reelin (RELN) (log2FC=2.89, 1.86; p<1*10-42, 1*10-17 for each shRNA, respectively), which has a well-established role in cell migration and invasion. To investigate this in vitro in MIBC cells, we performed time lapse microscopy and invasion assays to quantitatively determine cell movement over time. Compared to controls, T24 cells with STAG2 KD had reduced displacement (78 vs 114 µm, p<0.05), speed (0.30 vs 0.41 µm/min, p<0.05) and invasion (137 vs 190 cells/field, p<0.001) in vitro. Altogether, our results indicate that STAG2 functions as a transcriptional repressor and promotes movement and invasion of MIBC cells. This may explain why STAG2 protein loss leads to better outcomes for MIBC patients, and points to STAG2 protein expression as a potential prognostic biomarker. In our current work, we are utilizing a large drug screen of FDA-approved agents to identify vulnerabilities of tumor cells lacking STAG2. These studies will help to identify drugs that augment current standard of care and benefit the large proportion of patients with STAG2-mutant tumors. Citation Format: Sarah R. Athans, Nithya Krishnan, Swathi Ramakrishnan, Eduardo Cortes Gomez, Sofia Lage-Vickers, Monika Rak, Zara Kazmierczak, Aimee Stablewski, Kristopher Attwood, Jianmin Wang, Anna Woloszynska. STAG2 acts as a transcriptional repressor and promotes invasion of muscle invasive bladder cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 786.

174-OR: ADA Presidents’ Select Abstract: Epigenetic Regulation of Functional Beta-Cell Mass by Cohesin Smc3

Both type 1 and type 2 diabetes are caused by insulin insufficiency due to inadequate functional beta-cell mass. The demise of beta-cells in diabetes is preceded by progressive impairment of beta-cell function. Several studies have shown that the regulation of beta-cell function and survival is tightly linked. However, the precise molecular mechanisms that link beta-cell function and survival remain far from clear. In this study, we report that the cohesin Smc3 is a novel epigenetic regulator of functional beta-cell mass. Smc3 is an ATPase that regulates the 3D chromatin architecture and chromatid cohesion, and plays an important role in cellular self-renewal, differentiation, and survival. Smc3 exists in a tripartite ring complex with proteins Smc1 and Rad21, and the association of this complex to the chromatin is regulated by Stag1. Our chromatin immunoprecipitation data suggest that the cohesin complex regulates beta-cell maturation and survival programs. Using transcriptomic profiling combined with histological and physiological analyses, we show that that the beta-cell specific Smc3 knockout mice (Smc3-BKO) become diabetic by 3 months of age, due to a progressive loss of beta-cell maturity and viability. Islets from the Smc3-BKO mice display impaired glucose stimulated insulin secretion (GSIS) around 1-2 months of age, with gene expression signatures that resemble functionally immature neonatal beta-cells. This is followed by progressive decline of beta-cell mass. At a molecular level, disruption of Smc3 leads to reduced protein levels of the other two subunits of the cohesin ring complex, Smc1 and Rad21. On the other hand, the mRNA levels of Stag1 are increased in the Smc3-BKO islets. Finally, we show that the cohesin regulatory program is disrupted in islets from humans with type 2 diabetes. Taken together, these studies provide a novel insight into the integrative epigenetic regulation of functional beta-cell mass. Disclosure J. K. Wang: None. A. Pandey: None. A. P. Ham: None. N. Parveen: None. S. Dhawan: None.

Analysis of Aggressive Behavior in Young Russian Males Using 250 SNP Markers

Until not so long ago, the main area of genetic studies of aggressive behavior was represented by associative analysis of candidate genes, which were identified according to the relationship of phenotypic manifestations of aggression with the functioning of neuromediator and reproductive systems. Recent years have been marked by development of a new direction of genome-wide associative studies of aggression, which makes it possible to detect new genes that previously were not the object of interest to specialists. The present study is an analysis of aggressive behavior in Russian males living in the Moscow metropolis, using a panel of 250 SNP marker loci. In addition to SNP markers of known candidate genes, the panel contained single base substitutions in genes involved in the development and functioning of the brain, in the processes of neuronal development and synaptic plasticity, and in the formation of interneuronal connections, as well as in genes associated with various brain pathologies. The panel of SNP markers also included control genes that were in no way associated with aggressive behavior or behavior in general. These are primarily housekeeping genes, as well as genes encoding proteins associated with chromatid cohesion, etc. Aggressive behavior was self-assessed using the Bass–Perry and reactive–proactive aggression questionnaires. After applying a number of filters, 35 males were included in the final sample. Fragments containing 250 single-nucleotide polymorphic sites of interest were sequenced on the Ion PGM System using the Ion 318™ Chip. The principal component analysis and clustering based on the Bayesian a posteriori probability did not identify subdivisions in the analyzed sample of Russian males. For each aggression scale, a statistically significant association with a specific set of SNP markers was obtained, and only one polymorphic locus rs1047788 was associated with both physical and reactive aggression. Bioinformatic analysis revealed that most of the identified markers are associated with neuropeptides involved in the development and functioning of the nervous system as a whole and its regeneration and in the development of sections of the brain responsible for stress reactions, regulation of the humoral system, and intercellular signaling. For a number of markers from this set, it was possible to identify possible mechanisms of relationship to behavioral traits. The list of identified genes is as follows: corticotropin-releasing hormone, CRH; semenogelin-1 protein, SEMG1; intercellular interaction proteins, LAMC2 and ITGA2; DNA repair endonuclease, ERCC5; cohesin complex protein that provides conjugation of sister chromosomes, ESCO1; transmembrane serine protease, TMPRSS15; apoptosis inhibitor, BIRC5; interferon beta-1, IFNB1; scaffold protein, XRCC1; heat shock protein, HSP90AA1.

Efficacy of Allicin against Plant Pathogenic Fungi and Unveiling the Underlying Mode of Action Employing Yeast Based Chemogenetic Profiling Approach

Allicin (diallylthiosulfinate) is the principal organosulfur compound present in freshly damaged garlic tissue which exhibits a wide range of biological actions including antibacterial, antifungal, antiviral and anticancer properties. The antifungal activities of allicin were investigated against plant pathogenic fungi of agriculture importance. Furthermore, a yeast genome haploinsufficiency screening was also employed to decipher the antifungal mode of action of allicin. Wildtype and 1152 yeast mutant strains (each deprived of one specific allele of an essential gene in a diploid strain) were screened against allicin. Allicin exhibited promising antifungal properties against all the tested plant pathogens. Haploinsufficiency screening revealed three hypersensitive yeast mutants with gene deletions coding for proteins involved in DNA replication, mitochondrial translation and chromatids cohesion. These processes play a vital role in the cell cycle, growth and viability of yeast cells. Taken together, the results of the present study unravel the excellent antifungal activities and mechanisms and modes of action of allicin. These findings also indicate the potential use of allicin as an alternative “green” fungicide (fumigant) in agriculture.

Loss of ISWI ATPase SMARCA5 (SNF2H) in Acute Myeloid Leukemia Cells Inhibits Proliferation and Chromatid Cohesion.

ISWI chromatin remodeling ATPase SMARCA5 (SNF2H) is a well-known factor for its role in regulation of DNA access via nucleosome sliding and assembly. SMARCA5 transcriptionally inhibits the myeloid master regulator PU.1. Upregulation of SMARCA5 was previously observed in CD34+ hematopoietic progenitors of acute myeloid leukemia (AML) patients. Since high levels of SMARCA5 are necessary for intensive cell proliferation and cell cycle progression of developing hematopoietic stem and progenitor cells in mice, we reasoned that removal of SMARCA5 enzymatic activity could affect the cycling or undifferentiated state of leukemic progenitor-like clones. Indeed, we observed that CRISPR/cas9-mediated SMARCA5 knockout in AML cell lines (S5KO) inhibited the cell cycle progression. We also observed that the SMARCA5 deletion induced karyorrhexis and nuclear budding as well as increased the ploidy, indicating its role in mitotic division of AML cells. The cytogenetic analysis of S5KO cells revealed the premature chromatid separation. We conclude that deleting SMARCA5 in AML blocks leukemic proliferation and chromatid cohesion.

Microtubule End-Clustering Maintains a Steady-State Spindle Shape

Each time a cell divides, the microtubule cytoskeleton self-organizes into the metaphase spindle: anellipsoidal steady-state structure that holds its stereotyped geometry despite microtubule turnover and internal stresses [1-6]. Regulation of microtubule dynamics, motor proteins, microtubule crosslinking, and chromatid cohesion can modulate spindle size and shape, and yet modulated spindles reach and hold a new steady state [7-11]. Here, we ask what maintains any spindle steady-state geometry. We report that clustering of microtubule ends by dynein and NuMA is essential for mammalian spindles to hold a steady-state shape. After dynein or NuMA deletion, the mitotic microtubule network is "turbulent"; microtubule bundles extend and bend against the cell cortex, constantly remodeling network shape. We find that spindle turbulence is driven by the homotetrameric kinesin-5 Eg5, and that acute Eg5 inhibition in turbulent spindles recovers spindle geometry and stability. Inspired by invitro work on activeturbulent gels of microtubules and kinesin [12, 13], we explore the kinematics of this invivo turbulent network. We find that turbulent spindles display decreased nematic order and that motile asters distort the nematic director field. Finally, we see that turbulent spindles can drive both flow of cytoplasmic organelles and whole-cell movement-analogous to the autonomous motility displayed by droplet-encapsulated turbulent gels [12]. Thus, end-clustering by dynein and NuMA is required for mammalian spindles to reach a steady-state geometry, and in their absence Eg5 powers a turbulent microtubule network inside mitotic cells.

Genome Wide Association Analyses Identify Pleiotropic Variants Associated with Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS) Susceptibility

The first two authors and last two authors contributed equally.Genome-wide association studies (GWAS) have identified risk loci for Acute Lymphoblastic Leukemia (ALL), Chronic Lymphoblastic Leukemia (CLL) and Non-Hodgkin Lymphoma, however an Acute Myeloid Leukemia (AML) GWAS has not been published to date. We performed a GWAS to identify AML and Myelodysplastic Syndrome (MDS) risk loci using a nested case-control study design in the DISCOVeRY-BMT cohorts which includes almost 2000 AML and MDS patients as cases and 2813 unrelated donors as controls.Genotyping was performed using the Illumina Human OmniExpress BeadChip and imputed using the Haplotype Reference Consortium, yielding > 8 million high-quality variants for statistical analysis. Logistic regression models with AML (de novo AML with normal cytogenetics, de novo AML with abnormal cytogenetics and therapy-related AML) and MDS cases and European American healthy donor controls adjusted for age and sex were used to test the association of each SNP with disease status. To identify the strongest association signal with disease we conducted a summary statistic SNP-based association analysis (ASSET) using non-overlapping AML and MDS cases implemented in R statistical software. ASSET uses an exhaustive search for SNPs with small but common pleiotropic effects across groups of traits while accounting for the multiple tests required by the subset search, as well as any shared controls between groups. This approach allowed us to further investigate the heterogeneity within AML subtypes and to gain increased power by pooling subtypes that show pleiotropic effects. ASSET genome wide (GW) significance is defined as P<5.0x10-8, however, we present results of each subset analysis.ASSET analyses identified an association of T allele at rs12203592, Interferon Regulatory Factor 4 (IRF4), with increased risk of de novo AML and MDS (Figure 1) (OR=1.37; 95% CI, 1.25-1.51, Pmeta=1.59x10-10). The variant has been reported significant in GWAS of skin pigmentation, hair color, non-melanoma skin cancer, squamous cell carcinoma, actinic keratosis, and childhood ALL; GW case-control studies of neuroblastoma and breast cancer show suggestive association signals (P<5 x 10-6).IRF4 belongs to the IRF family of transcription factors and is a key regulator of differentiation stages in hematopoiesis. The T allele at rs12203592 is associated with significantly increased expression of IRF4 in whole blood and lung tissue and in vitro mouse studies have shown the T allele to be associated with higher levels of IRF4 transcription. rs12203592 is <80bp from an IRF4 transcription start site and in an important position within NF-κB motifs in multiple blood and immune cell lines, supporting the hypothesis that this SNP modulates NF-κB repression of IRF4 expression.Another variant in the IRF4 regulatory region, rs62389423 (A allele), showed a putative association with subsets of de novo AML and MDS (OR=1.36; 95% CI,1.21-1.52, Pmeta=1.2x10-7). Although not in linkage disequilibrium with rs12203592, the A allele has also been previously associated with susceptibility to skin cancer and melanoma in multiple GWAS. Analysis of 596 UKbiobank CLL cases and >300,000 controls, shows the A allele correlates with a 50% increased risk of CLL (95% CI, 1.32-1.73, P= 2.4x10-9).Several other GWAS also show an association between additional IRF4 variants and CLL.Analyses in individual subtypes also revealed an intronic variant, rs10098598, in RAD21 to be associated with de novo AML (OR=2.18; 95% CI, 1.60-2.97; Pmeta=8.9x10-7). RAD21, on8q24, a region known to be associated with multiple cancer types, is involved in DNA double-stranded break repair and in chromatid cohesion in mitotic cells. Loss of function would theoretically lead to chromosomal instability and tumorigenesis. Components of the cohesion complex (including RAD21) are somatically mutated in ~12% of de novo AML and MDS patients, where it is frequently an early event.We provide the first GW evidence of association between a common variant and AML susceptibility. This SNP has been shown to be associated with multiple phenotypes, suggesting that there are pleiotropic effects at work. Our RAD21 finding is consistent with the role of cohesion in leukemogenesis and provides some evidence of its role in de novo AML susceptibility. Replication and further subset analyses with genome-wide data in >2000 AML cases are ongoing. [Display omitted] DisclosuresGriffiths:Alexion Inc.: Honoraria, Research Funding; Astex/Otsuka Pharmaceuticals: Honoraria, Research Funding; Celgene, Inc: Honoraria, Research Funding; Novartis, Inc.: Research Funding; Pfizer, Inc.: Research Funding. McCarthy:Bristol Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Janssen: Consultancy, Honoraria.

CALR mutational status identifies different disease subtypes of essential thrombocythemia showing distinct expression profiles

Polycythemia vera (PV) and essential thrombocythemia (ET) are Philadelphia-negative myeloproliferative neoplasms (MPNs) characterized by erythrocytosis and thrombocytosis, respectively. Approximately 95% of PV and 50–70% of ET patients harbor the V617F mutation in the exon 14 of JAK2 gene, while about 20–30% of ET patients carry CALRins5 or CALRdel52 mutations. These ET CALR-mutated subjects show higher platelet count and lower thrombotic risk compared to JAK2-mutated patients. Here, we showed that CALR-mutated and JAK2V617F-positive CD34+ cells display different gene and miRNA expression profiles. Indeed, we highlighted several pathways differentially activated between JAK2V617F- and CALR-mutated progenitors, i.e., mTOR, MAPK/PI3K, and MYC pathways. Furthermore, we unveiled that the expression of several genes involved in DNA repair, chromatin remodeling, splicing, and chromatid cohesion are decreased in CALR-mutated cells. According to the low risk of thrombosis in CALR-mutated patients, we also found the downregulation of several genes involved in thrombin signaling and platelet activation. As a whole, these data support the model that CALR-mutated ET could be considered as a distinct disease entity from JAK2V617F-positive MPNs and may provide the molecular basis supporting the different clinical features of these patients.

O Cdc7 kinase where art thou?

Although Cdc7 protein kinase is important for regulating DNA replication in all eukaryotes and is a target for cancer therapy, it has never been localized in cells. Recently, a novel molecular genomic method used by our laboratory to localize Cdc7 to regions of chromosomes. Originally, mutations in the CDC7 gene were found in the classic cdc mutant collection of Hartwell et al. (Genetics 74:267-286, 1973). The CDC7 gene was found to encode a protein kinase called DDK that has been studied for many years, establishing its precise role in the initiation of DNA replication at origins. Recently, clinical studies are underway with DDK inhibitors against DDK in cancer patients. However, the conundrum is that Cdc7 has never been detected at origins of replication even though many studies have suggested it should be there. We used "Calling Card" system in which DNA binding proteins are localized to the genome via retrotransposon insertion and deep-sequencing methods. We have shown that Cdc7 localizes at many regions of the genome and was enriched at functional origins of replication. These results are consistent with DDK's role in many additional genomic processes including mutagenesis, chromatid cohesion, and meiotic recombination. Thus, the main conclusion from our studies is that Cdc7 kinase is found at many locations in the genome, but is enriched at functional origins of replication. Furthermore, we propose that application of the Calling Card system to other eukaryotes should be useful in identification of functional origins in other eukaryotic cells.

Histone Methylation by SET Domain Proteins in Fungi.

Histone-modifying enzymes are responsible for regulating transcription, recombination, DNA repair, DNA replication, chromatid cohesion, and chromosome segregation. Fungi are ideally suited for comparative chromatin biology because sequencing of numerous genomes from many clades is coupled to existing rich methodology that allows truly holistic approaches, integrating evolutionary biology with mechanistic molecular biology and ecology, promising applications in medicine or plant pathology. While genome information is rich, mechanistic studies on histone modifications are largely restricted to two yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe, and one filamentous fungus, Neurospora crassa-three species that arguably are not representative of this diverse kingdom. Here, histone methylation serves as a paradigm to illustrate the roles chromatin modifications may play in more complex fungal life cycles. This review summarizes recent advances in our understanding of histone H3 methylation at two sites associated with active transcription, lysine 4 and lysine 36 (H3K4, H3K36); a site associated with the formation of constitutive heterochromatin, lysine 9 (H3K9); and a site associated with the formation of facultative heterochromatin, lysine 27 (H3K27). Special attention is paid to differences in how methylation marks interact in different taxa.

Abstract 90: A cell cycle-related genomic and transcriptomic signature distinguish aneuploid and euploid acute myeloid leukemia

Abstract Chromosome gain or loss, which is the hallmark of aneuploidy, occurs in about 10% of adult Acute Myeloid Leukemia (AML) cases (Farag et al. IJO 2002, Breems et al. JCO 2008), despite inducing a dramatic reduction of cellular fitness in non-malignant cells (Torres et al. Science 2007). The study aimed to identify AML-specific molecular mechanisms having a causative and/or tolerogenic role towards aneuploidy. We performed 100 bp paired-end whole exome sequencing (WES, Illumina Hiseq2000) of 38 aneuploid (A) and 34 euploid (E) AML cases, identified according to cytogenetic analysis and SNP array (CytoScan HD, Affymetrix). Variants were called with GATK, MuTect and VarScan. We also compared the transcriptomic profile of leukemic bone marrow cells from 21 A-AML and 28 E-AML cases (HTA 2.0, Affymetrix). A-AML showed a significantly higher mutation load compared with E-AML (median number of variants: 25 and 15, respectively, p&amp;lt;.001) and a specific pattern of genomic lesions. Indeed, mutations in myeloid transcription factors and chromatin modifiers preferentially occurred in E-AML (p = .04 and p&amp;lt;.01, respectively), while A-AML was enriched for alterations in cell cycle-related genes (p&amp;lt;.01), with 70% of cases carrying at least one of those mutations (vs. 35% of E-AML, p&amp;lt;.01). The mutated genes played different functions across cell cycle phases, including DNA replication, centrosome dynamics, chromatid cohesion, chromosome segregation and spindle-assembly checkpoint. Moreover, 29% of A-AML displayed mutations of TP53 or a TP53-related gene (DDIAS, USP10), compared with 5.9% of E-AML cases (p = .01), with enrichment for a transcriptional signature of p53 downregulation in the aneuploid cohort (p&amp;lt;.05). Among the differentially expressed genes, we identified a cell cycle-related signature characterized by increased CDC20 and UBE2C and reduced RAD50, ATR and CCNA1 in A-AML (p&amp;lt;.001), confirmed at protein level, which was able to separate A-AML and E-AML. A-AML also showed upregulation of a KRAS transcriptional signature, irrespective of KRAS mutational status (p&amp;lt;.05). Our data show a link between aneuploidy and genomic instability in AML and highlight novel molecular mechanisms for the acquisition and/or maintenance of the aneuploid phenotype. Deregulation of the cell cycle machinery and DNA damage/repair checkpoints, either through mutations, copy number and transcriptomic alterations, cooperate with leukemogenic pathways, as KRAS signaling, to develop A-AML and overcome the unfitness barrier. This evidence suggests that a number of A-AML patients may benefit from pharmacological reactivation of TP53 and inhibition of KRAS pathway. Supported by: FP7 NGS-PTL project, ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi). Citation Format: Giorgia Simonetti, Antonella Padella, Marco Manfrini, ĺtalo Faria do Valle, Cristina Papayannidis, Carmen Baldazzi, Maria Chiara Fontana, Viviana Guadagnuolo, Anna Ferrari, Elisa Zago, Marianna Garonzi, Simona Bernardi, Annalisa Astolfi, Maria Chiara Abbenante, Giovanni Marconi, Elisa Zuffa, Eugenia Franchini, Ilaria Iacobucci, Michele Cavo, Emanuela Ottaviani, Nicoletta Testoni, Alberto Ferrarini, Massimo Delledonne, Torsten Haferlach, Daniel Remondini, Giovanni Martinelli. A cell cycle-related genomic and transcriptomic signature distinguish aneuploid and euploid acute myeloid leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 90.

Epigenetic Histone Marks of Extended Meta-Polycentric Centromeres of Lathyrus and Pisum Chromosomes.

Species of the legume genera Lathyrus and Pisum possess chromosomes that exhibit a unique structure of their centromeric regions, which is clearly apparent during metaphase by the formation of extended primary constrictions which span up to a third of the length of the chromosome. In addition, these species express two different variants of the CenH3 protein which are co-localized in multiple domains along the poleward surface of the primary constrictions. Here, we show that the constrictions represent a distinct type of chromatin differing from the chromosome arms. In metaphase, histone phosphorylation patterns including H3S10ph, H3S28ph, and H3T3ph were observed along the entire constriction, in a way similar to holocentric chromosomes. On the other hand, distribution of phosphorylated H2AT120 was different from that previously reported from either, holocentric and monocentric chromosomes, occurring at chromatin surrounding but not overlapping CenH3 domains. Since some of these phosphorylations play a role in chromatid cohesion, it can be assumed that they facilitate correct chromosome segregation by ensuring that multiple separate CenH3 domains present on the same chromatid are oriented toward the same pole. The constrictions also displayed distinct patterns of histone methylation marks, being enriched in H3K9me2 and depleted in H3K4me3 and H3K27me2 compared to the chromosome arms. Super-resolution fluorescence microscopy revealed that although both CenH3 protein variants are present in all CenH3 domains detected on metaphase chromosomes, they are only partially co-localized while there are chromatin subdomains which are mostly made of only one CenH3 variant. Taken together, these data revealed specific features of extended primary constrictions of Lathyrus and Pisum and support the idea that they may represent an intermediate stage between monocentric and holocentric chromosomes.

Reversing chromatin accessibility differences that distinguish homologous mitotic metaphase chromosomes.

BackgroundChromatin-modifying reagents that alter histone associating proteins, DNA conformation or its sequence are well established strategies for studying chromatin structure in interphase (G1, S, G2). Little is known about how these compounds act during metaphase. We assessed the effects of these reagents at genomic loci that show reproducible, non-random differences in accessibility to chromatin that distinguish homologous targets by single copy DNA probe fluorescence in situ hybridization (scFISH). By super-resolution 3-D structured illumination microscopy (3D-SIM) and other criteria, the differences correspond to ‘differential accessibility’ (DA) to these chromosomal regions. At these chromosomal loci, DA of the same homologous chromosome is stable and epigenetic hallmarks of less accessible interphase chromatin are present.ResultsTo understand the basis for DA, we investigate the impact of epigenetic modifiers on these allelic differences in chromatin accessibility between metaphase homologs in lymphoblastoid cell lines. Allelic differences in metaphase chromosome accessibility represent a stable chromatin mark on mitotic metaphase chromosomes. Inhibition of the topoisomerase IIα-DNA cleavage complex reversed DA. Inter-homolog probe fluorescence intensity ratios between chromosomes treated with ICRF-193 were significantly lower than untreated controls. 3D-SIM demonstrated that differences in hybridized probe volume and depth between allelic targets were equalized by this treatment. By contrast, DA was impervious to chromosome decondensation treatments targeting histone modifying enzymes, cytosine methylation, as well as in cells with regulatory defects in chromatid cohesion. These data altogether suggest that DA is a reflection of allelic differences in metaphase chromosome compaction, dictated by the localized catenation state of the chromosome, rather than by other epigenetic marks.ConclusionsInhibition of the topoisomerase IIα-DNA cleavage complex mitigated DA by decreasing DNA superhelicity and axial metaphase chromosome condensation. This has potential implications for the mechanism of preservation of cellular phenotypes that enables the same chromatin structure to be correctly reestablished in progeny cells of the same tissue or individual.Electronic supplementary materialThe online version of this article (doi:10.1186/s13039-015-0159-y) contains supplementary material, which is available to authorized users.

Loss of kinesin-14 results in aneuploidy via kinesin-5-dependent microtubule protrusions leading to chromosome cut.

Aneuploidy – chromosome instability leading to incorrect chromosome number in dividing cells – can arise from defects in centrosome duplication, bipolar spindle formation, kinetochore-microtubule attachment, chromatid cohesion, mitotic checkpoint monitoring, or cytokinesis. As most tumors show some degree of aneuploidy, mechanistic understanding of these pathways has been an intense area of research to provide potential therapeutics. Here, we present a mechanism for aneuploidy in fission yeast based on spindle pole microtubule defocusing by loss of kinesin-14 Pkl1, leading to kinesin-5 Cut7-dependent aberrant long spindle microtubule minus end protrusions that push the properly segregated chromosomes to the site of cell division, resulting in chromosome cut at cytokinesis. Pkl1 localization and function at the spindle pole is mutually dependent on spindle pole-associated protein Msd1. This mechanism of aneuploidy bypasses the known spindle assembly checkpoint that monitors chromosome segregation.

Causes of Chromosomal Instability.

The majority of human cancer cells are highly aneuploid harboring chromosome numbers deviating from the modal number of 46. In cancer, aneuploidy is a consequence of an increased rate of whole chromosome missegregation during mitosis, a process known as chromosomal instability (CIN). In fact, CIN is a hallmark of human cancer and is thought to contribute to tumorigenesis, tumor progression, and the development of therapy resistance by providing a high genetic variability that might foster rapid adaptation processes. However, the molecular mechanisms that cause chromosome missegregation in cancer cells are still poorly understood. So far, several mechanisms underlying CIN have been proposed and some of them are indeed detectable in human cancer cells exhibiting CIN. Examples include, for instance, weakened spindle checkpoint signaling, supernumerary centrosomes, defects in chromatid cohesion, abnormal kinetochore-microtubule attachments and increased spindle microtubule dynamics. Here, the mechanisms leading to CIN in human cancer cells are summarized.

The AtRAD21.1 and AtRAD21.3 Arabidopsis cohesins play a synergistic role in somatic DNA double strand break damage repair.

BackgroundThe RAD21 cohesin plays, besides its well-recognised role in chromatid cohesion, a role in DNA double strand break (dsb) repair. In Arabidopsis there are three RAD21 paralog genes (AtRAD21.1, AtRAD21.2 and AtRAD21.3), yet only AtRAD21.1 has been shown to be required for DNA dsb damage repair. Further investigation of the role of cohesins in DNA dsb repair was carried out and is here reported.ResultsWe show for the first time that not only AtRAD21.1 but also AtRAD21.3 play a role in somatic DNA dsb repair. Comet data shows that the lack of either cohesins induces a similar high basal level of DNA dsb in the nuclei and a slower DNA dsb repair kinetics in both cohesin mutants. The observed AtRAD21.3 transcriptional response to DNA dsb induction reinforces further the role of this cohesin in DNA dsb repair. The importance of AtRAD21.3 in DNA dsb damage repair, after exposure to DNA dsb damage inducing agents, is notorious and recognisably evident at the phenotypical level, particularly when the AtRAD21.1 gene is also disrupted.Data on the kinetics of DNA dsb damage repair and DNA damage sensitivity assays, of single and double atrad21 mutants, as well as the transcription dynamics of the AtRAD21 cohesins over a period of 48 hours after the induction of DNA dsb damage is also shown.ConclusionsOur data demonstrates that both Arabidopsis cohesin (AtRAD21.1 and AtRAD21.3) play a role in somatic DNA dsb repair. Furthermore, the phenotypical data from the atrad21.1 atrad21.3 double mutant indicates that these two cohesins function synergistically in DNA dsb repair. The implications of this data are discussed.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0353-9) contains supplementary material, which is available to authorized users.

NIPBL, a Cohesion Loading Factor, Is Somatically Mutated in Gastric and Colorectal Cancers with High Microsatellite Instability

Timely segregation of sister chromatids during the cell cycle is essential for maintaining euploidy [1]. Aberrant segregation into daughter cells promotes aneuploidy and chromosome instability, which are features of many tumors [1]. Chromatid cohesion, a physical linkage of replicated sister chromatids, is mediated by a multiprotein complex called cohesin [1]. NIPBL is a cohesion loading factor that is required for cohesin to interact with chromosomes [2, 3]. NIPBL mutations (missense, nonsense, frameshift and deletion mutations) account for approximately 60 % of Cornelia de Lange syndrome, a congenital disorder with multisystem developmental abnormalities [3]. In Cornelia de Lange syndrome, mutated NIPBL interferes with its interaction with its partners in the cohesion complex [3]. Components in the cohesin complex and cohesin regulatory proteins are frequently altered at expression and mutation levels in human cancers [1]. For example, cohesin protector SGOL1 down-regulation leads to chromosomal instability in colorectal cancers (CRC) [4]. Somatic mutations of cohesion regulatory factor PDS5B have been identified in prostate cancers [5]. These data suggest that cohesin-related genes could be considered potential tumor suppressor genes (TSG) [1]. Somatic mutations of the NIPBL gene have been found in CRC with stable miscrosatellite instability (MSI) that may underlie chromosomal instability in CRC [6]. Together, these data suggest a possibility that NIPBL is a candidate TSG. In a public genome database (http://genome.cse.ucsc. edu/), we found that the human NIPBL gene had mononucleotide repeats in its coding sequences that could be targets for frameshift mutation in cancers with high MSI (MSI-H) [7]. To data, however, the data on NIPBL somatic mutation in gastric (GC) and CRC with MSI-H are lacking. To see whether the mononucleotide repeats in the NIPBL gene are mutated in GC and CRC, we analyzed two A7 and one A8 repeats in NIPBL exon 10 by polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) assay with three primer pairs. For this, we used 32 GC with MSI-H, 59 GC with MSS, 47 CRC with MSI-H and 53 CRC with MSS in methacarnfixed tissues. All of the patients of the cancers were Koreans. In cancer tissues, malignant cells and normal cells were selectively procured from hematoxylin and eosinstained slides by microdissection [8]. Radioisotope ([P]dCTP) was incorporated into the PCR products for detection by autoradiogram. The PCR products were subsequently displayed in SSCP gels. After SSCP, direct DNA sequencing reactions were performed in the cancers with mobility shifts in the SSCP. On the SSCP, we observed aberrant bands of the NIPBL gene in 11 cancers (Fig. 1). DNA from normal tissue Min Sung Kim and Chang Hyeok An contributed equally to this study.

Appearance and heterochromatin localization of HP1α in early mouse embryos depends on cytoplasmic clock and H3S10 phosphorylation

Pericentric constitutive heterochromatin surrounds centromeric regions and is important for centromere function and chromatid cohesion. HP1 (heterochromatin protein 1), a homolog of yeast Swi6, has been shown to be indispensible for proper heterochromatin structure and function. In mammalian somatic cells, two HP1 isoforms, HP1α and HP1β, are constitutively present in pericentric heterochromatin until late G2, when they dissociate from heterochromatin. Subsequently, they re-associate with heterochromatin at late anaphase. In one-cell mouse embryos, pericentric heterochromatin has a unique configuration and features. It does not form heterochromatin clusters observed in somatic cells and known as chromocenters. Instead, in both pronuclei, it surrounds nucleolar precursor bodies (NBPs), forming ring-like structures. These regions contain HP1β but lack HP1α in both pronuclei. In subsequent interphases, HP1β is constitutively found in heterochromatin until the blastocyst stage. It is not known when HP1α appears and what is its function in early mouse embryos. Here, we show that HP1α appears for the first time at late S phase of two-cell stage, at the time when pericentric heterochromatin is replicated. Its appearance is regulated at the level of translation. In two-cell embryos, the amount of HP1α that can bind to these regions is regulated by phosphorylation of serine 10 of histone H3 (H3S10Ph). Elimination of HP1α by siRNA interfered with centromere relocation from heterochromatin surrounding NPBs to pro-chromocenters at the two-cell stage but did not affect preimplantation develoment to the blastocyst stage.