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Background Glioblastoma multiforme and other solid malignancies are heterogeneous, containing subpopulations of tumor cells that exhibit stem characteristics. Oct4, also known as POU5F1, is a key transcription factor in the self-renewal, proliferation, and differentiation of stem cells. Although it has been detected in advanced gliomas, the biological function of Oct4, and transcriptional machinery maintained by the stemness of Oct4 protein-mediated glioma stem cells (GSC), has not been fully determined. Methods The expression of Oct4 variants was evaluated in brain cancer cell lines, and in brain tumor tissues, by quantitative real-time PCR, western blotting, and immunohistochemical analysis. The palmitoylation level of Oct4A was determined by the acyl-biotin exchange method, and the effects of palmitoylation Oct4A on GSCs were investigated by a series of in vitro (neuro-sphere formation assay, double immunofluorescence, pharmacological treatment, luciferase assay, and coimmunoprecipitation) and in vivo (xenograft model) experiments. Results Here, we report that all three variants of Oct4 are expressed in different types of cerebral cancer, while Oct4A is important for maintaining tumorigenicity in GSCs. Palmitoylation mediated by ZDHHC17 was indispensable for preserving Oct4A from lysosome degradation to maintain its protein stability. Oct4A palmitoylation also helped to integrate Sox4 and Oct4A in the SOX2 enhancement subregion to maintain the stem performance of GSCs. We also designed Oct4A palmitoylation competitive inhibitors, inhibiting the self-renewal ability and tumorigenicity of GSCs. Conclusions These findings indicate that Oct4A acts on the tumorigenic activity of glioblastoma, and Oct4A palmitoylation is a candidate therapeutic target.

Purpose: This study aims to provide a better set of DNA methylation markers in urine sediments for sensitive and specific detection of bladder cancer.Experimental Design: Fifty-nine tumor-associated genes were profiled in three bladder cancer cell lines, a small cohort of cancer biopsies and urine sediments by methylation-specific PCR. Twenty-one candidate genes were then profiled in urine sediments from 132 bladder cancer patients (8 cases for stage 0a; 68 cases for stage I; 50 cases for stage II; 4 cases for stages III; and 2 cases for stage IV), 23 age-matched patients with noncancerous urinary lesions, 6 neurologic diseases, and 7 healthy volunteers.Results: Despite six incidences of four genes reported in 3 of 23 noncancerous urinary lesion patients analyzed, cancer-specific hypermethylation in urine sediments were reported for 15 genes (P < 0.05). Methylation assessment of an 11-gene set (SALL3, CFTR, ABCC6, HPR1, RASSF1A, MT1A, RUNX3, ITGA4, BCL2, ALX4, MYOD1, DRM, CDH13, BMP3B, CCNA1, RPRM, MINT1, and BRCA1) confirmed the existing diagnosis of 121 among 132 bladder cancer cases (sensitivity, 91.7%) with 87% accuracy. Significantly, more than 75% of stage 0a and 88% of stage I disease were detected, indicating its value in the early diagnosis of bladder cancer. Interestingly, the cluster of reported methylation markers used in the U.S. bladder cancers is distinctly different from that identified in this study, suggesting a possible epigenetic disparity between the American and Chinese cases.Conclusions: Methylation profiling of an 11-gene set in urine sediments provides a sensitive and specific detection of bladder cancer.

Supplementary Methods, Figures 1-4 from Rho Kinase Phosphorylation Promotes Ezrin-Mediated Metastasis in Hepatocellular Carcinoma

Enhancer–promoter communication is known to regulate spatiotemporal dynamics of gene expression. Several methods are available to capture enhancer–promoter interactions, but they either require large amounts of starting materials and are costly, or provide a relative low resolution in chromatin contact maps. Here, we present nicking enzyme-assisted open chromatin interaction capture (NicE-C), a method that leverages nicking enzyme–mediated open chromatin profiling and chromosome conformation capture to enable robust and cost-effective detection of open chromatin interactions at high resolution, especially enhancer–promoter interactions. Using TNF stimulation and mouse kidney aging as models, we applied NicE-C to reveal characteristics of dynamic enhancer–promoter interactions.

The outer kinetochore serves as a platform for the initiation of the spindle assembly checkpoint (SAC) and for mediating kinetochore–microtubule attachments. How the inner kinetochore subcomplex CENP-S–CENP-X is involved in regulating the SAC and kinetochore–microtubule attachments has not been well characterized. Using live-cell microscopy and yeast genetics, we found that Mhf1–Mhf2, the CENP-S–CENP-X counterpart in the fission yeast Schizosaccharomyces pombe, plays crucial roles in promoting the SAC and regulating chromosome segregation. The absence of Mhf2 attenuates the SAC, impairs the kinetochore localization of most of the components in the constitutive centromere-associated network (CCAN), and alters the localization of the kinase Ark1 (yeast homolog of Aurora B) to the kinetochore. Hence, our findings constitute a model in which Mhf1–Mhf2 ensures faithful chromosome segregation by regulating the accurate organization of the CCAN complex, which is required for promoting SAC signaling and for regulating kinetochore–microtubule attachments. This article has an associated First Person interview with the first author of the paper.

Chromosome segregation in mitosis is orchestrated by the dynamic interactions between the kinetochore and spindle microtubules. Our recent studies show that mitotic motor CENP-E cooperates with SKAP and forms a link between kinetochore core MIS13 complex and spindle microtubule plus-ends to achieve accurate chromosome alignment in mitosis. However, it remains elusive how SKAP regulates kinetochore attachment from lateral association to end-on attachment during metaphase alignment. Here, we identify a novel interaction between Aurora B and SKAP that orchestrates accurate interaction between the kinetochore and dynamic spindle microtubules. Interestingly, SKAP spontaneously phase-separates in vitro via weak, multivalent interactions into droplets with fast internal dynamics. SKAP and Aurora B form heterogeneous coacervates in vitro, which recapitulate the dynamics and behavior of SKAP comets in vivo. Importantly, SKAP interaction with Aurora B via phase separation is essential for accurate chromosome segregation and alignment. Based on those findings, we reason that SKAP–Aurora B interaction via phase separation constitutes a dynamic pool of Aurora B activity during the lateral to end-on conversion of kinetochore–microtubule attachments to achieve faithful cell division.

The molecular mechanisms underlying the establishment of the monopolar growth of fission yeast spores have been less characterized. Here, we report that the Cdc42 GTPase-activating protein (GAP) Rga6 is required for promoting monopolar growth during spore germination. The absence of Rga6 increases the number of spores that grow in a bipolar fashion. Rga6 decorates the non-growing cortical region, binds phosphatidylinositol 4,5-bisphosphate, and colocalizes with the phosphatidylinositol 4,5-bisphosphate-binding protein Opy1. Overexpression of Opy1 diminishes the cortical localization of Rga6. The characteristic localization of Rga6 on the cell cortex depends on the C-terminal PBR region of Rga6. Moreover, engineered chimera composed of the Rga6 C-terminal PBR region fused to the GAP domain of Rga3 or Rga4 are sufficient to rescue the spore growth phenotype caused by the absence of Rga6. Hence, our work establishes a paradigm in which the lipid composition of the plasma membrane directs polarized cell growth by specifying the cortical localization of a GAP protein.

Stable transmission of genetic information during cell division requires faithful mitotic spindle assembly and chromosome segregation. In eukaryotic cells, nuclear envelope breakdown (NEBD) is required for proper chromosome segregation. Although a list of mitotic kinases has been implicated in NEBD, how they coordinate their activity to dissolve the nuclear envelope and protein machinery such as nuclear pore complexes was unclear. Here, we identified a regulatory mechanism in which Nup62 is acetylated by TIP60 in human cell division. Nup62 is a novel substrate of TIP60, and the acetylation of Lys432 by TIP60 dissolves nucleoporin Nup62–Nup58–Nup54 complex during entry into mitosis. Importantly, this acetylation-elicited remodeling of nucleoporin complex promotes the distribution of Nup62 to the mitotic spindle, which is indispensable for orchestrating correct spindle orientation. Moreover, suppression of Nup62 perturbs accurate chromosome segregation during mitosis. These results establish a previously uncharacterized regulatory mechanism in which TIP60-elicited nucleoporin dynamics promotes chromosome segregation in mitosis.

Error-free mitosis depends on accurate chromosome attachment to spindle microtubules via a fine structure called the centromere that is epigenetically specified by the enrichment of CENP-A nucleosomes. Centromere maintenance during mitosis requires CENP-A-mediated deposition of constitutive centromere-associated network that establishes the inner kinetochore and connects centromeric chromatin to spindle microtubules during mitosis. Although previously proposed to be an adaptor of retinoic acid receptor, here, we show that CENP-R synergizes with CENP-OPQU to regulate kinetochore–microtubule attachment stability and ensure accurate chromosome segregation in mitosis. We found that a phospho-mimicking mutation of CENP-R weakened its localization to the kinetochore, suggesting that phosphorylation may regulate its localization. Perturbation of CENP-R phosphorylation is shown to prevent proper kinetochore–microtubule attachment at metaphase. Mechanistically, CENP-R phosphorylation disrupts its binding with CENP-U. Thus, we speculate that Aurora B-mediated CENP-R phosphorylation promotes the correction of improper kinetochore–microtubule attachment in mitosis. As CENP-R is absent from yeast, we reasoned that metazoan evolved an elaborate chromosome stability control machinery to ensure faithful chromosome segregation in mitosis.

Cell polarity is essential for spatially regulating of physiological processes in metazoans by which hormonal stimulation‒secretion coupling is precisely coupled for tissue homeostasis and organ communications. However, the molecular mechanisms underlying epithelial cell polarity establishment remain elusive. Here, we show that septin cytoskeleton interacts with catenin complex to organize a functional domain to separate apical from basal membranes in polarized epithelial cells. Using polarized epithelial cell monolayer as a model system with transepithelial electrical resistance as functional readout, our studies show that septins are essential for epithelial cell polarization. Our proteomic analyses discovered a novel septin‒catenin complex during epithelial cell polarization. The functional relevance of septin‒catenin complex was then examined in three-dimensional (3D) culture in which suppression of septins resulted in deformation of apical lumen in cysts, a hallmark seen in polarity-deficient 3D cultures and animals. Mechanistically, septin cytoskeleton stabilizes the association of adherens catenin complex with actin cytoskeleton, and depletion or disruption of septin cytoskeleton liberates adherens junction and polarity complexes into the cytoplasm. Together, these findings reveal a previously unrecognized role for septin cytoskeleton in the polarization of the apical‒basal axis and lumen formation in polarized epithelial cells.

The Spalt-like 4 transcription factor (SALL4) plays an essential role in controlling the pluripotent property of embryonic stem cells via binding to AT-rich regions of genomic DNA, but structural details on this binding interaction have not been fully characterized. Here, we present crystal structures of the zinc finger cluster 4 (ZFC4) domain of SALL4 (SALL4

Spalt-like 4 (SALL4) plays an essential role in controlling the pluripotent property of embryonic stem cells (ESCs) via binding to AT-rich regions of genomic DNA. Here we present crystal structures of the the zinc finger cluster 4 (ZFC4) domain of SALL4 (SALL4ZFC4) bound with different double stranded DNAs containing a conserved AT-rich motif. In the structures, two zinc fingers of SALL4ZFC4 coordinatively recognize an AATA tetranucleotide. We also solved the DNA-bound structures of SALL3ZFC4 and SALL4ZFC1. These structures illuminate a common recognition mode for AT-rich DNA by the SALL family proteins. The DNA binding activity is essential for SALL4 function as DNA-binding defective mutants of mouse Sall4 failed to repress aberrant gene expression in Sall4-/- mESCs. Thus, these analyses provide new insights into the mechanisms of action underlying SALL family in controlling cell fate via preferentially targeted to AT-rich sites within genomic DNAs during cell differentiation.

Evolution has enabled living cells to adopt their structural and functional complexity by organizing intricate cellular compartments, such as membrane-bound and membraneless organelles (MLOs), for spatiotemporal catalysis of physiochemical reactions essential for cell plasticity control. Emerging evidence and view support the notion that MLOs are built by multivalent interactions of biomolecules via phase separation and transition mechanisms. In healthy cells, dynamic chemical modifications regulate MLO plasticity, and reversible phase separation is essential for cell homeostasis. Emerging evidence revealed that aberrant phase separation results in numerous neurodegenerative disorders, cancer, and other diseases. In this review, we provide molecular underpinnings on (i) mechanistic understanding of phase separation, (ii) unifying structural and mechanistic principles that underlie this phenomenon, (iii) various mechanisms that are used by cells for the regulation of phase separation, and (iv) emerging therapeutic and other applications.

Degrons are elements within protein substrates that mediate the interaction with specific degradation machineries to control proteolysis. Recently, a few classes of C-terminal degrons (C-degrons) that are recognized by dedicated cullin-RING ligases (CRLs) have been identified. Specifically, CRL2 using the related substrate adapters FEM1A/B/C was found to recognize C degrons ending with arginine (Arg/C-degron). Here, we uncover the molecular mechanism of Arg/C-degron recognition by solving a subset of structures of FEM1 proteins in complex with Arg/C-degron-bearing substrates. Our structural research, complemented by binding assays and global protein stability (GPS) analyses, demonstrates that FEM1A/C and FEM1B selectively target distinct classes of Arg/C-degrons. Overall, our study not only sheds light on the molecular mechanism underlying Arg/C-degron recognition for precise control of substrate turnover, but also provides valuable information for development of chemical probes for selectively regulating proteostasis.

The outer kinetochore serves as a platform for initiating the spindle assembly checkpoint and for mediating kinetochore-microtubule attachments. How the inner kinetochore subcomplex CENP-S/CENP-X is involved in regulating the spindle assembly checkpoint and kinetochore-microtubule attachments has not been well characterized. Employing live-cell microscopy and yeast genetics, we found that the fission yeast CENP-S/CENP-X counterpart Mhf1/Mhf2 plays crucial roles in promoting the spindle assembly checkpoint and regulating chromosome segregation. The absence of Mhf2 attenuates the spindle assembly checkpoint, impairs the kinetochore localization of most of the components in the constitutive centromere-associated network (CCAN), and alters the localization of the kinase Aurora-B/Ark1 to the kinetochore. Hence, our findings constitute a model in which Mhf1/Mhf2 ensures faithful chromosome segregation by regulating the accurate organization of the CCAN complex that is required for promoting SAC signaling and for regulating kinetochore-microtubule attachments.

Hepatocellular carcinoma (HCC) still presents a high incidence of malignant tumours with poor prognosis. There is an urgent need for new therapeutic agents with high specificity, low toxicity and favourable solubility for the clinical treatment of HCC.The bioactivity of human umbilical cord serum was investigated by proteomics biotechnology and a primitive peptide with certain biological activity was identified. The antitumour effect of RR-171 was detected by cell viability assay in vitro, and determined by subcutaneous xenograft models assay and miniPDX assay in vivo. Pull-down experiments were conducted to identify the potential targeting proteins of RR-171. Immunofluorescence assay and tubulin polymerization assay were conducted to explore the relationship between RR-171 and α-tubulin. Fluorescence imaging in xenograft models was used to explore the biodistribution of RR-171 in vivo. A phosphospecific protein microarray was performed to uncover the underlying signalling pathway by which RR-171 induces tumour cell death.The results indicated that RR-171 could be effective in the treatment of HCC in vivo and in vitro. RR-171 could aggregate significantly in solid tumours and had no obvious systemic toxicity in vivo. RR-171 could interact with α-tubulin and activate the NF-Kappa B pathway in HCC cells.Taken together, RR-171 exhibited significant antitumour activity against HCC in vivo and in vitro and could potentially be used in the clinical application of HCC.

Stress granules (SGs) form through phase separation of biomacromolecules to assist cells in resisting environmental stresses. Numbers of SG proteins contain Arg-Gly-Gly (RGG) motifs, indicating their RNA binding ability, and providing a substrate platform for asymmetric dimethylation of arginine (ADMA), whose roles in SG assembly remain unclear. Here, we demonstrated that Caprin1-mediated recruitment of PRMT1 asymmetrically dimethylates RGGs to provide multiple binding sites for TDRD3, a typical ADMA reader, which in turn bridges the multivalent interactions between RGG motifs and RNA to promote phase separation. This process was suppressed by a bivalent inhibitor of TDRD3, eventually inhibiting proliferation more effectively than arsenite treatment alone. Our work reveals the role of ADMA in SG assembly and the potential of targeting condensates for cancer therapy.

Accurate mitotic progression relies on the dynamic phosphorylation of multiple substrates by key mitotic kinases. Cyclin‐dependent kinase 1 is a master kinase that coordinates mitotic progression and requires its regulatory subunit Cyclin B to ensure full kinase activity and substrate specificity. The function of Cyclin B2, which is a closely related family member of Cyclin B1, remains largely elusive. Here, we show that Mad2 promotes the kinetochore localization of Cyclin B2 and that their interaction at the kinetochores guides accurate chromosome segregation. Our biochemical analyses have characterized the Mad2‐Cyclin B2 interaction and delineated a novel Mad2‐interacting motif (MIM) on Cyclin B2. The functional importance of the Cyclin B2‐Mad2 interaction was demonstrated by real‐time imaging in which MIM‐deficient mutant Cyclin B2 failed to rescue the chromosomal segregation defects. Taken together, we have delineated a previously undefined function of Cyclin B2 at the kinetochore and have established, in human cells, a mechanism of action by which Mad2 contributes to the spindle checkpoint.

Liquid–liquid phase separation (LLPS) of biomolecules drives the formation of subcellular compartments with distinct physicochemical properties. These compartments, free of lipid bilayers and therefore called membraneless organelles, include nucleoli, centrosomes, heterochromatin, and centromeres. These have emerged as a new paradigm to account for subcellular organization and cell fate decisions. Here we summarize recent studies linking LLPS to mitotic spindle, heterochromatin, and centromere assembly and their plasticity controls in the context of the cell division cycle, highlighting a functional role for phase behavior and material properties of proteins assembled onto heterochromatin, centromeres, and central spindles via LLPS. The techniques and tools for visualizing and harnessing membraneless organelle dynamics and plasticity in mitosis are also discussed, as is the potential for these discoveries to promote new research directions for investigating chromosome dynamics, plasticity, and interchromosome interactions in the decision-making process during mitosis.

Error-free mitosis depends on accurate chromosome attachment to spindle microtubules, which is monitored by the spindle assembly checkpoint (SAC) signaling. As an upstream factor of SAC, the precise and dynamic kinetochore localization of Mps1 kinase is critical for initiating and silencing SAC signaling. However, the underlying molecular mechanism remains elusive. Here, we demonstrated that the multisite interactions between Mps1 and Ndc80 complex (Ndc80C) govern Mps1 kinetochore targeting. Importantly, we identified direct interaction between Mps1 tetratricopeptide repeat domain and Ndc80C. We further identified that Mps1 C-terminal fragment, which contains the protein kinase domain and C-tail, enhances Mps1 kinetochore localization. Mechanistically, Mps1 C-terminal fragment mediates its dimerization. Perturbation of C-tail attenuates the kinetochore targeting and activity of Mps1, leading to aberrant mitosis due to compromised SAC function. Taken together, our study highlights the importance of Mps1 dimerization and multisite interactions with Ndc80C in enabling responsive SAC signaling.