Role of CCNB1, CENPF, and neutrophils in lung cancer diagnosis and prognosis.

This study aimed to investigate CCNB1, CENPF, and Neutrophils as diagnostic predictors of lung cancer and to explore their association with clinical prognosis. Clinical data were obtained for a total of 52 patients. In addition, we downloaded 555 lung cancer-related samples from the cancer genome atlas (TCGA) database. Differentially expressed genes were further screened. Immune cell infiltration and survival analysis were performed. Immunohistochemistry was used to confirm gene expression. Peripheral blood analysis showed that neutrophil percentages were significantly reduced in patients with lung cancer. The least absolute shrinkage and selection operator and multivariate regression analysis revealed that CCNB1 and CENPF were lung cancer risk factors. Both CCNB1 and CENPF are overexpressed in lung cancer. The clinical diagnostic model constructed using CCNB1, CENPF, and neutrophils had a C-index of 0.994. This model area under the curve (AUC) and internal validation C-index values were 0.994 and 0.993, respectively. The elevated expression of CCNB1 and CENPF showed that the survival rate of lung cancer patients was reduced. CCNB1 and CENPF expression was positively correlated with the clinical stage of lung cancer. Further studies confirmed that CCNB1 and CENPF are overexpressed in lung cancer tissues. The clinically constructed model with high accuracy based on CCNB1, CENPF, and neutrophils demonstrated that these are crucial indicators for lung cancer diagnosis. High expression of CCNB1 and CENPF indicates a poor prognosis in patients with lung cancer.

Synthetic Antibodies Detect Distinct Cellular States of Chromosome Passenger Complex Proteins.

High performance affinity reagents are essential tools to enable biologists to profile the cellular location and composition of macromolecular complexes undergoing dynamic reorganization. To support further development of such tools, we have assembled a high-throughput phage display pipeline to generate Fab-based affinity reagents that target different dynamic forms of a large macromolecular complex, using the Chromosomal Passenger Complex (CPC), as an example. The CPC is critical for the maintenance of chromosomal and cytoskeleton processes during cell division. The complex contains 4 protein components: Aurora B kinase, survivin, borealin and INCENP. The CPC acts as a node to dynamically organize other partnering subcomplexes to build multiple functional structures during mitotic progression. Using phage display mutagenesis, a cohort of synthetic antibodies (sABs) were generated against different domains of survivin, borealin and INCENP. Immunofluorescence established that a set of these sABs can discriminate between the form of the CPC complex in the midbody versus the spindle. Others localize to targets, which appear to be less organized, in the nucleus or cytoplasm. This differentiation suggests that different CPC epitopes have dynamic accessibility depending upon the mitotic state of the cell. An Immunoprecipitation/Mass Spectrometry analysis was performed using sABs that bound specifically to the CPC in either the midbody or MT spindle macromolecular assemblies. Thus, sABs can be exploited as high performance reagents to profile the accessibility of different components of the CPC within macromolecular assemblies during different stages of mitosis suggesting this high throughput approach will be applicable to other complex macromolecular systems.

Characterization of Opa interacting protein 5 as a new biomarker and therapeutic target for oral cancer.

Oral cancer is a leading cause of cancer­related death worldwide. Current treatment for oral cancer includes surgery, radiotherapy, and chemotherapy; however, their effectiveness is still limited. To identify a new prognostic biomarker and therapeutic target for oral cancer, the Opa interacting protein 5 (OIP5), which plays an essential role in the proper segregation of chromosomes, was examined. Immunohistochemical staining using tissue microarrays indicated that OIP5 was expressed in 120 of 164 (73.2%) oral cancers but was minimally expressed in normal oral tissues. OIP5 expression was significantly associated with poor prognosis in patients with oral cancer. Overexpression of OIP5 enhanced the growth of oral cancer cells, whereas OIP5 knockdown using small interfering RNAs (siRNAs) significantly inhibited cell growth through cell cycle arrest at the G2/M phase. Suppression of OIP5 expression also induced senescence of oral cancer cells. Overall, the findings of the present study suggest that OIP5 may be a candidate prognostic biomarker and therapeutic target in oral cancer.

Overexpression of CENPF is associated with progression and poor prognosis of lung adenocarcinoma.

Background and aim: The molecular signatures of lung adenocarcinoma (LUAD) are not well understood. Centromere protein F (CENPF) has been shown to promote oncogenesis in many cancers; however, its role in LUAD has not been illustrated. We explored the role of CENPF in LUAD. Methods: CENPF expression level was investigated in public online database firstly, the prognosis of CENPF in LUAD were also assessed by Kaplan-Meier analysis. Then quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed using 13 matched pairs of clinical LUAD tissue samples. Subsequently, the impact of CENPF expression on cell proliferation, cell cycle, apoptosis, colony formation was investigated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), flow cytometric analysis and colony formation assay, respectively. Finally, experimental xenograft lung cancer model of nude mice armpit of right forelimb to determine the effect of CENPF on LUAD tumorigenesis. Results: CENPF mRNA expression was significantly elevated in LUAD tissues compared with adjacent non-tumor lung tissues in Gene Expression Profiling Interactive Analysis (GEPIA) (P < 0.001). Up-regulated CENPF was remarkably positively associated with pathological stage, relapse free survival (RFS) as well as overall survival (OS) of LUAD patients. Besides, CENPF knockdown greatly suppressed A549 cell proliferation, induced S phase arrest, promoted apoptosis and decreased colony numbers of LUAD cells. Furthermore, knockdown of CENPF significantly inhibited the tumor growth of the LUAD cells in an experimental xenograft lung cancer model of nude mice armpit of right forelimb. Conclusion: Taken together, these results demonstrated that CENPF may serve as a potential biomarker of prognostic relevance and a potential therapeutic target for LUAD.

The Nucleus of Intestinal Cells of the Bacterivore Nematode Caenorhabditis elegans as a Sensitive Sensor of Environmental Pollutants.

Prevalent environmental challenges are climate change, the biodiversity crisis, and the global scale of environmental pollution. We identified the cell nucleus as a sensitive sensor for bio-effects of pollutants such as mercury and nanoparticles. As a major route of pollutant uptake into organisms is ingestion, we have developed a test system that uses single intestinal cells of the nematode roundworm Caenorhabditis elegans. Microscopic observation of the cell nucleus in reporter worms for the methyltransferase fibrillarin (FIB-1::GFP) revealed nuclear staining patterns that are specific for pollutants such as silica nanoparticles, BULK silica particles, silver nanoparticles, ionic AgNO3, and inorganic mercury (HgCl2). While the underlying molecular mechanisms need further investigation, cultivation of the reporter worms in liquid culture on microtiter plates now enables cost-effective screening of more pollutants and samples from the environment, e.g., mesocosm analyses. As C. elegans leads a dual life in the lab and in ecosystems, alteration of nuclear structure and function may likewise explain how environmental pollutants reduce the fitness of wild worms and thus may negatively affect biodiversity.

SKA1 regulates actin cytoskeleton remodelling via activating Cdc42 and influences the migration of pancreatic ductal adenocarcinoma cells.

OBJECTIVES: Spindle and kinetochore-associated protein 1(SKA1), originally identified as a protein essential for proper chromosome segregation, has been recently linked to multiple malignancies. This study aimed to explore the biological, clinical role and molecular mechanism of SKA1 in pancreatic carcinogenesis. MATERIALS AND METHODS: SKA1 expression was detected in 145 pancreatic ductal adenocarcinoma (PDAC) specimens by immunohistochemistry. Biological behaviour assays were used to determine the role of SKA1 in PDAC progression in vitro and in vivo. Using isobaric tags for relative and absolute quantitation (iTRAQ), SKA1's downstream proteins were examined. Moreover, cytochalasin B and ZCL278 were used to explore the changes of SKA1-induced signalling and cell morphology, with further confirmation by immunoblotting and immunofluorescence assays. RESULTS: Increased SKA1 expression was significantly correlated with tumour size and cellular differentiation degree in PDAC tissues. Furthermore, elevated levels of SKA1 reflected shorter overall survival (P = .019). As for biological behaviour, SKA1 acted as a tumour promotor in PDAC, overexpression of SKA1 facilitates cell proliferation, migration and invasion in vitro and in vivo. Mechanistically, we demonstrated that SKA1 enhanced pancreatic cancer aggressiveness by inhibiting G2/M arrest and regulating actin cytoskeleton organization via activating Cdc42. CONCLUSIONS: This study revealed novel roles for SKA1 as an important regulator of actin cytoskeleton organization and an oncogene in PDAC cells, which may provide insights into developing novel therapeutics.

A multi-layered structure of the interphase chromocenter revealed by proximity-based biotinylation.

During interphase centromeres often coalesce into a small number of chromocenters, which can be visualized as distinct, DAPI dense nuclear domains. Intact chromocenters play a major role in maintaining genome stability as they stabilize the transcriptionally silent state of repetitive DNA while ensuring centromere function. Despite its biological importance, relatively little is known about the molecular composition of the chromocenter or the processes that mediate chromocenter formation and maintenance. To provide a deeper molecular insight into the composition of the chromocenter and to demonstrate the usefulness of proximity-based biotinylation as a tool to investigate those questions, we performed super resolution microscopy and proximity-based biotinylation experiments of three distinct proteins associated with the chromocenter in Drosophila. Our work revealed an intricate internal architecture of the chromocenter suggesting a complex multilayered structure of this intranuclear domain.

DEK Is a Potential Biomarker Associated with Malignant Phenotype in Gastric Cancer Tissues and Plasma.

Gastric cancer (GC) is the second most widespread cause of cancer-related mortality worldwide. The discovery of novel biomarkers of oncoproteins can facilitate the development of therapeutic strategies for GC treatment. In this study, we identified novel biomarkers by integrating isobaric tags for relative and absolute quantitation (iTRAQ), a human plasma proteome database, and public Oncomine datasets to search for aberrantly expressed oncogene-associated proteins in GC tissues and plasma. One of the most significantly upregulated biomarkers, DEK, was selected and its expression validated. Our immunohistochemistry (IHC) (n = 92) and quantitative real-time polymerase chain reaction (qRT-PCR) (n = 72) analyses disclosed a marked increase in DEK expression in tumor tissue, compared with paired nontumor mucosa. Importantly, significantly higher preoperative plasma DEK levels were detected in GC patients than in healthy controls via enzyme-linked immunosorbent assay (ELISA). In clinicopathological analysis, higher expression of DEK in both tissue and plasma was significantly associated with advanced stage and poorer survival outcomes of GC patients. Data from receiver operating characteristic (ROC) curve analysis disclosed a better diagnostic accuracy of plasma DEK than carcinoembryonic antigen (CEA), carbohydrate antigen 19.9 (CA 19.9), and C-reactive protein (CRP), highlighting its potential as an effective plasma biomarker for GC. Plasma DEK is also more sensitive in tumor detection than the other three biomarkers. Knockdown of DEK resulted in inhibition of GC cell migration via a mechanism involving modulation of matrix metalloproteinase MMP-2/MMP-9 level and vice versa. Our results collectively support plasma DEK as a useful biomarker for making diagnosis and prognosis of GC patients.

Epigenetic impairments in development of parthenogenetic preimplantation mouse embryos.

Parthenogenesis is an activation process of oocytes that occur without the participation of sperm. Evidence suggests that normal development of embryos requires proper expression of several imprinted genes inherited from both the paternal and maternal genomes. Compared to gene expression, histone modifications and chromatin remodeling are not well-documented. In this research, by using immunofluorescence staining for several developmental-associated histone modifications, we investigated whether epigenetic impairments in parthenogenetic embryos act as constraints for proper development. At early stages, fertilized embryos exhibited high methylation of histone H3 at lysine 9 (Me-H3-K9) and Heterochromatin Protein 1 (HP1) present in the maternal chromatin, while paternal chromatin showed weaker HP1 signals. We found that at the two-cell stage in fertilized embryos, HP1, initially detected around the nucleolus, colocalized with chromocenters at one pole of the blastomere, while parthenotes showed a diffused distribution pattern of HP1 throughout the entire nucleoplasm. At the four-cell stage, methylation of histone H3 at arginine 26 (Me-H3-R26) increased at nascent RNA repression sites in fertilized embryos, while parthenotes recorded weaker signals throughout the nucleoplasm, suggesting differences in pluripotency of the ICM cells between the two types of embryos. Moreover, at the blastocyst stage, we observed that the acetylation level of histone H4 at lysine 12 (Ac-H4-K12) was significantly decreased in parthenogenetic ICM compared to that in its fertilized counterpart. To summarize, differences in epigenetic modifications correlating with paternal chromatin's capacity to regulate nascent RNA repression may contribute to aberrant development and lineage allocation in mouse parthenogenetic embryos.

Dynamic interactions of the homologous pairing 2 (Hop2)-meiotic nuclear divisions 1 (Mnd1) protein complex with meiotic presynaptic filaments in budding yeast.

Homologous recombination (HR) is a universally conserved DNA repair pathway that can result in the exchange of genetic material. In eukaryotes, HR has evolved into an essential step in meiosis. During meiosis many eukaryotes utilize a two-recombinase pathway. This system consists of Rad51 and the meiosis-specific recombinase Dmc1. Both recombinases have distinct activities during meiotic HR, despite being highly similar in sequence and having closely related biochemical activities, raising the question of how these two proteins can perform separate functions. A likely explanation for their differential regulation involves the meiosis-specific recombination proteins Hop2 and Mnd1, which are part of a highly conserved eukaryotic protein complex that participates in HR, albeit through poorly understood mechanisms. To better understand how Hop2-Mnd1 functions during HR, here we used DNA curtains in conjunction with single-molecule imaging to measure and quantify the binding of the Hop2-Mnd1 complex from Saccharomyces cerevisiae to recombination intermediates comprising Rad51- and Dmc1-ssDNA in real time. We found that yeast Hop2-Mnd1 bound rapidly to Dmc1-ssDNA filaments with high affinity and remained bound for ∼1.3 min before dissociating. We also observed that this binding interaction was highly specific for Dmc1 and found no evidence for an association of Hop2-Mnd1 with Rad51-ssDNA or RPA-ssDNA. Our findings provide new quantitative insights into the binding dynamics of Hop2-Mnd1 with the meiotic presynaptic complex. On the basis of these findings, we propose a model in which recombinase specificities for meiotic accessory proteins enhance separation of the recombinases' functions during meiotic HR.

Modular Organization and Assembly of SWI/SNF Family Chromatin Remodeling Complexes.

Mammalian SWI/SNF (mSWI/SNF) ATP-dependent chromatin remodeling complexes are multi-subunit molecular machines that play vital roles in regulating genomic architecture and are frequently disrupted in human cancer and developmental disorders. To date, the modular organization and pathways of assembly of these chromatin regulators remain unknown, presenting a major barrier to structural and functional determination. Here, we elucidate the architecture and assembly pathway across three classes of mSWI/SNF complexes-canonical BRG1/BRM-associated factor (BAF), polybromo-associated BAF (PBAF), and newly defined ncBAF complexes-and define the requirement of each subunit for complex formation and stability. Using affinity purification of endogenous complexes from mammalian and Drosophila cells coupled with cross-linking mass spectrometry (CX-MS) and mutagenesis, we uncover three distinct and evolutionarily conserved modules, their organization, and the temporal incorporation of these modules into each complete mSWI/SNF complex class. Finally, we map human disease-associated mutations within subunits and modules, defining specific topological regions that are affected upon subunit perturbation.

Elevated DSN1 expression is associated with poor survival in patients with hepatocellular carcinoma.

Dosage suppressor of Nnf1 (DSN1) is a component of the kinetochore protein complex that is required for proper chromosome segregation. Some studies have explored that DSN1 is related to colorectal cancer progression. However, the role of DSN1 in hepatocellular carcinoma (HCC) remains unknown. This study aimed to explore DSN1 expression in HCC tissues. We obtained data from The Cancer Genome Atlas and Gene Expression Omnibus to analyze DSN1 expression in HCC. DSN1 mRNA expression was assessed in 30 pairs of HCC samples via reverse-transcription quantitative polymerase chain reaction. Immunohistochemical analysis of 95 HCC tissue specimens was performed to assess DSN1 expression and examine the clinicopathological characteristics of DSN1 in HCC. Results showed that DSN1 was upregulated in HCC tissues and was strongly associated with sex (P = .031), α-fetoprotein (P < .001), tumor size (P = .032), tumor nodule number (P = .028), cancer embolus (P = .011), and differentiation grade (P = .001). Moreover, Kaplan-Meier and Cox proportional-hazards analyses indicated that high DSN1 expression was related to poor HCC patient survival and that DSN1 can serve as an independent prognostic factor for overall survival and disease-free survival. In conclusion, our findings indicate that DSN1 could serve as a novel prognostic biomarker for HCC patients.

Chromatin assembly factor-1 (CAF-1) chaperone regulates Cse4 deposition into chromatin in budding yeast.

Correct localization of the centromeric histone variant CenH3/CENP-A/Cse4 is an important part of faithful chromosome segregation. Mislocalization of CenH3 could affect chromosome segregation, DNA replication and transcription. CENP-A is often overexpressed and mislocalized in cancer genomes, but the underlying mechanisms are not understood. One major regulator of Cse4 deposition is Psh1, an E3 ubiquitin ligase that controls levels of Cse4 to prevent deposition into non-centromeric regions. We present evidence that Chromatin assembly factor-1 (CAF-1), an evolutionarily conserved histone H3/H4 chaperone with subunits shown previously to interact with CenH3 in flies and human cells, regulates Cse4 deposition in budding yeast. yCAF-1 interacts with Cse4 and can assemble Cse4 nucleosomes in vitro. Loss of yCAF-1 dramatically reduces the amount of Cse4 deposited into chromatin genome-wide when Cse4 is overexpressed. The incorporation of Cse4 genome-wide may have multifactorial effects on growth and gene expression. Loss of yCAF-1 can rescue growth defects and some changes in gene expression associated with Cse4 deposition that occur in the absence of Psh1-mediated proteolysis. Incorporation of Cse4 into promoter nucleosomes at transcriptionally active genes depends on yCAF-1. Overall our findings suggest CAF-1 can act as a CenH3 chaperone, regulating levels and incorporation of CenH3 in chromatin.

SWI/SNF Chromatin-remodeling Complex Status in SMARCB1/INI1-preserved Epithelioid Sarcoma.

The SWI/SNF chromatin-remodeling complex, which is composed of evolutionarily conserved core subunits such as SMARCB1/INI1 (INI1), SMARCA4/BRG1 (BRG1), SMARCC1/BAF155 (BAF155), and SMARCC2/BAF170 (BAF170), can be viewed as the prototype of an epigenetic regulator of gene expression that is involved in tumor suppression. Epithelioid sarcoma, which classified as a tumor of uncertain differentiation, shows an almost complete loss of INI1. However, some cases of epithelioid sarcoma have preserved INI1, and the clinicopathologic features of these cases are uncertain. To date, there has been no investigation focused on the SWI/SNF chromatin-remodeling complex in INI1-preserved epithelioid sarcoma cases. First, an investigation of INI1 immunoexpression statuses in 60 formalin-fixed paraffin-embedded epithelioid sarcoma specimens (proximal type, 29 cases; conventional type, 31 cases) was performed. In the available INI1-preserved epithelioid sarcoma cases, we analyzed the BRG1, BAF155, and BAF170 protein expressions. INI1 preservation was observed in 6 of 29 (21%) proximal-type and 2 of 31 (6%) conventional-type epithelioid sarcoma cases. Six cases of INI1-preserved epithelioid sarcomas of proximal type were available for further immunohistochemical study. One proximal type showed loss of BAF170, and 2 proximal-type cases revealed loss of BRG1 with preservation of the other remaining core subunit proteins. One proximal-type case showed a mosaic pattern of BRG1 and loss of BAF155. However, in the remaining 2 proximal-type cases, all core subunit proteins were preserved. Overall, these results suggest that loss of expression of SWI/SNF chromatin-remodeling complex proteins has an important role in tumorigenesis. The remaining 2 INI1-preserved epithelioid sarcoma cases may have had other abnormalities causing dysfunction of SWI/SNF chromatin remodeling.

Extraocular sebaceous carcinoma accompanied by invasive squamous cell carcinoma: The first case report and consideration of histogenesis.

A 61-year-old man presented with a dome-shaped nodule, 1.2 cm in size, with a central crater covered by keratinous material near the left lateral malleolus. Histological findings demonstrated a basophilic circular cone in the center, surrounded and sharply demarcated by a broad eosinophilic area. The central conical mass was composed mainly of atypical basaloid cells intermingled with scattered atypical sebaceous cells with scalloped nuclei and microvesicular cytoplasms, suggesting sebaceous carcinoma. The peripheral area consisted of atypical keratinizing squamoid cells without sebaceous cells, suggesting invasive squamous cell carcinoma. Atypical sebaceous cells were positive for adipophilin. Atypical basaloid cells were positive for 34ßE12 and CAM5.2. Peripheral squamoid cells were positive for 34ßB4 and 34ßE12 throughout, and were positive for LHP1 in the superficial layer. We herein describe the first case of extraocular sebaceous carcinoma accompanied by invasive squamous cell carcinoma, which might have arisen from biphasic differentiation of cancer stem cells.

Skin-Specific Deletion of Mis18α Impedes Proliferation and Stratification of Epidermal Keratinocytes.

The Mis18 proteins (Mis18α, Mis18ß, and M18BP1) are pivotal to the deposition of CENP-A at the centromere during cell cycle progression and are indispensable for embryonic development. Here, we show that Mis18α is critical for the proliferation of keratinocytes and stratification of the epidermis. Mice lacking Mis18α in the epidermis died shortly after birth, showing skin abnormalities like thin and translucent skin and defective skin barrier functions. The epidermis of newborn Mis18α-deficient mice lacked distinct stratification and mature hair follicles, with a reduction in the number of proliferating cells and increased cell death in the basal layer. Earlier expression of the Cre recombinase from keratin-14 promoter in the ventral region resulted in earlier keratinocyte death in the ventral part compared with the dorsal part in the absence of Mis18α, leading to more severe malformation of the ventral epidermal layers. As observed in Mis18α-deficient mouse keratinocytes, knockdown of Mis18α in HaCaT cells caused marked loss of centromeric CENP-A dots and chromosomal misalignment. Overall, we propose that Mis18α is important for epidermal cell proliferation and stratification, because it is required for the deposition of CENP-A at the centromeric nucleosomes.

The composition and organization of Drosophila heterochromatin are heterogeneous and dynamic.

Heterochromatin is enriched for specific epigenetic factors including Heterochromatin Protein 1a (HP1a), and is essential for many organismal functions. To elucidate heterochromatin organization and regulation, we purified Drosophila melanogaster HP1a interactors, and performed a genome-wide RNAi screen to identify genes that impact HP1a levels or localization. The majority of the over four hundred putative HP1a interactors and regulators identified were previously unknown. We found that 13 of 16 tested candidates (83%) are required for gene silencing, providing a substantial increase in the number of identified components that impact heterochromatin properties. Surprisingly, image analysis revealed that although some HP1a interactors and regulators are broadly distributed within the heterochromatin domain, most localize to discrete subdomains that display dynamic localization patterns during the cell cycle. We conclude that heterochromatin composition and architecture is more spatially complex and dynamic than previously suggested, and propose that a network of subdomains regulates diverse heterochromatin functions.

Aspergillus fumigatus spore proteomics and genetics reveal that VeA represses DefA-mediated DNA damage response.

Aspergillus fumigatus reproduces and infects host by forming a high number of small asexual spores (conidia). The velvet proteins are global transcriptional regulators governing the complex process of conidiogenesis in this fungus. Here, to further understand the velvet-mediated regulation, we carried out comparative proteomic analyses of conidia of wild type (WT) and three velvet mutants (ΔveA, ΔvelB and ΔvosA). Cluster analysis of 184 protein spots showing at least 1.5-fold differential accumulation between WT and mutants reveal the clustering of WT- ΔveA and ΔvelB-ΔvosA. Among 43 proteins identified by Nano-LC-ESI-MS/MS, 23 including several heat shock proteins showed more than two-fold reduction in both the ∆velB and ∆vosA conidia. On the contrary, three proteins exhibited more than five-fold increase in ∆veA only, including the putative RNA polymerase II degradation factor DefA. The deletion of defA resulted in a reduced number of conidia and restricted colony growth. In addition, the defA deletion mutant conidia showed hypersensitivity against the DNA damaging agents NQO and MMS, while the ΔveA mutant conidia were more resistant against to NQO. Taken together, we propose that VeA controls protein level of DefA in conidia, which are dormant and equipped with multiple layers of protection against environmental cues.

Identifying Centromeric RNAs Involved in Histone Dynamics In Vivo.

Over the last decade, the long accepted dogma that heterochromatin is silent has been challenged by increasing evidence of active transcription in these apocryphally annotated quiescent regions of the genome. The recent discovery of noncoding RNAs (ncRNAs) originating from, or localizing to, centromeres, pericentromeres, and telomeres (ie, constitutive heterochromatin) suggest a potential role for ncRNAs in genome integrity. This new paradigm suggests that ncRNAs may recruit chromatin-binding factors, stabilize the higher order folded state of the chromatin fiber, and participate in regulation of processes such as transcription-mediated nucleosome assembly. Thus, identifying, purifying, and elucidating the function of ncRNAs has the potential to provide key insights into genome organization and is currently a topic of intense experimental investigation.

Coexisting Liquid Phases Underlie Nucleolar Subcompartments.

The nucleolus and other ribonucleoprotein (RNP) bodies are membrane-less organelles that appear to assemble through phase separation of their molecular components. However, many such RNP bodies contain internal subcompartments, and the mechanism of their formation remains unclear. Here, we combine in vivo and in vitro studies, together with computational modeling, to show that subcompartments within the nucleolus represent distinct, coexisting liquid phases. Consistent with their in vivo immiscibility, purified nucleolar proteins phase separate into droplets containing distinct non-coalescing phases that are remarkably similar to nucleoli in vivo. This layered droplet organization is caused by differences in the biophysical properties of the phases-particularly droplet surface tension-which arises from sequence-encoded features of their macromolecular components. These results suggest that phase separation can give rise to multilayered liquids that may facilitate sequential RNA processing reactions in a variety of RNP bodies. PAPERCLIP.