Aberrant activation of Wnt/β-catenin signaling is a major driving force in colon cancer. Wnt/β-catenin signaling induces the expression of the transcription factor c-Myc, leading to cell proliferation and tumorigenesis. c-Myc regulates multiple biological processes through its ability to directly modulate gene expression. Here, we identify a direct target of c-Myc, termed MYU, and show that MYU is upregulated in most colon cancers and required for the tumorigenicity of colon cancer cells. Furthermore, we demonstrate that MYU associates with the RNA binding protein hnRNP-K to stabilize CDK6 expression and thereby promotes the G1-S transition of the cell cycle. These results suggest that the MYU/hnRNP-K/CDK6 pathway functions downstream of Wnt/c-Myc signaling and plays a critical role in the proliferation and tumorigenicity of colon cancer cells.

In meiosis, telomeres attach to the inner nuclear membrane (INM) and drive the chromosome movement required for homolog pairing and recombination. Here, we address the question of how telomeres are structurally adapted for the meiotic task. We identify a multi-subunit meiotic telomere-complex, TERB1/2-MAJIN, which takes over telomeric DNA from the shelterin complex in mouse germ cells. TERB1/2-MAJIN initially assembles on the INM sequestered by its putative transmembrane subunit MAJIN. In early meiosis, telomere attachment is achieved by the formation of a chimeric complex of TERB1/2-MAJIN and shelterin. The chimeric complex matures during prophase into DNA-bound TERB1/2-MAJIN by releasing shelterin, forming a direct link between telomeric DNA and the INM. These hierarchical processes, termed "telomere cap exchange," are regulated by CDK-dependent phosphorylation and the DNA-binding activity of MAJIN. Further, we uncover a positive feedback between telomere attachment and chromosome movement, revealing a comprehensive regulatory network underlying meiosis-specific telomere function in mammals.

Cornelia de Lange syndrome (CdLS) is a dominantly inherited congenital malformation disorder, caused by mutations in the cohesin-loading protein NIPBL for nearly 60% of individuals with classical CdLS, and by mutations in the core cohesin components SMC1A (~5%) and SMC3 (<1%) for a smaller fraction of probands. In humans, the multisubunit complex cohesin is made up of SMC1, SMC3, RAD21 and a STAG protein. These form a ring structure that is proposed to encircle sister chromatids to mediate sister chromatid cohesion and also has key roles in gene regulation. SMC3 is acetylated during S-phase to establish cohesiveness of chromatin-loaded cohesin, and in yeast, the class I histone deacetylase Hos1 deacetylates SMC3 during anaphase. Here we identify HDAC8 as the vertebrate SMC3 deacetylase, as well as loss-of-function HDAC8 mutations in six CdLS probands. Loss of HDAC8 activity results in increased SMC3 acetylation and inefficient dissolution of the ‘used’ cohesin complex released from chromatin in both prophase and anaphase. SMC3 with retained acetylation is loaded onto chromatin, and chromatin immunoprecipitation sequencing analysis demonstrates decreased occupancy of cohesin localization sites that results in a consistent pattern of altered transcription seen in CdLS cell lines with either NIPBL or HDAC8 mutations.

Sister chromatid cohesion is mediated by cohesin and is essential for accurate chromosome segregation. The cohesin subunits SMC1, SMC3, and Rad21 form a tripartite ring within which sister chromatids are thought to be entrapped. This event requires the acetylation of SMC3 and the association of sororin with cohesin by the acetyltransferases Esco1 and Esco2 in humans, but the functional mechanisms of these acetyltransferases remain elusive. Here, we showed that Esco1 requires Pds5, a cohesin regulatory subunit bound to Rad21, to form cohesion via SMC3 acetylation and the stabilization of the chromatin association of sororin, whereas Esco2 function was not affected by Pds5 depletion. Consistent with the functional link between Esco1 and Pds5, Pds5 interacted exclusively with Esco1, and this interaction was dependent on a unique and conserved Esco1 domain. Crucially, this interaction was essential for SMC3 acetylation and sister chromatid cohesion. Esco1 localized to cohesin localization sites on chromosomes throughout interphase in a manner that required the Esco1-Pds5 interaction, and it could acetylate SMC3 before and after DNA replication. These results indicate that Esco1 acetylates SMC3 via a mechanism different from that of Esco2. We propose that, by interacting with a unique domain of Esco1, Pds5 recruits Esco1 to chromatin-bound cohesin complexes to form cohesion. Furthermore, Esco1 acetylates SMC3 independently of DNA replication.

Aflatoxin contamination of crops is a serious problem worldwide. Utilization of aflatoxin production inhibitors is attractive, as the elucidation of their modes of action contributes to clarifying the mechanism of aflatoxin production. Here, we identified mitochondrial protease ClpP as the target of dioctatin, an inhibitor of aflatoxin production of Aspergillus flavus. Dioctatin conferred uncontrolled caseinolytic capacity on ClpP of A. flavus and Escherichia coli. Dioctatin-bound ClpP selectively degraded mitochondrial energy-related proteins in vitro, including a subunit of respiratory chain complex V, which was also reduced by dioctatin in a ClpP-dependent manner in vivo. Dioctatin enhanced glycolysis and alcohol fermentation while reducing tricarboxylic acid cycle metabolites. These disturbances were accompanied by reduced histone acetylation and reduced expression of aflatoxin biosynthetic genes. Our results suggest that dioctatin inhibits aflatoxin production by inducing ClpP-mediated degradation of mitochondrial energy-related components, and that mitochondrial energy metabolism functions as a key determinant of aflatoxin production.

The molluscan shell is a biomineral that comprises calcium carbonate and organic matrices controlling the crystal growth of calcium carbonate. The main components of organic matrices are insoluble chitin and proteins. Various kinds of proteins have been identified by solubilizing them with reagents, such as acid or detergent. However, insoluble proteins remained due to the formation of a solid complex with chitin. Herein, we identified these proteins from the nacreous layer, prismatic layer, and hinge ligament of Pinctada fucata using mercaptoethanol and trypsin. Most identified proteins contained a methionine-rich region in common. We focused on one of these proteins, NU-5, to examine the function in shell formation. Gene expression analysis of NU-5 showed that NU-5 was highly expressed in the mantle, and a knockdown of NU-5 prevented the formation of aragonite tablets in the nacre, which suggested that NU-5 was required for nacre formation. Dynamic light scattering and circular dichroism revealed that recombinant NU-5 had aggregation activity and changed its secondary structure in the presence of calcium ions. These findings suggest that insoluble proteins containing methionine-rich regions may be important for scaffold formation, which is an initial stage of biomineral formation.

Drosophila Piwi associates with PIWI-interacting RNAs (piRNAs) and represses transposons transcriptionally through heterochromatinization; however, this process is poorly understood. Here, we identify Brahma (Brm), the core adenosine triphosphatase of the SWI/SNF chromatin remodeling complex, as a new Piwi interactor, and show Brm involvement in activating transcription of Piwi-targeted transposons before silencing. Bioinformatic analyses indicated that Piwi, once bound to target RNAs, reduced the occupancies of SWI/SNF and RNA polymerase II (Pol II) on target loci, abrogating transcription. Artificial piRNA-driven targeting of Piwi to RNA transcripts enhanced repression of Brm-dependent reporters compared with Brm-independent reporters. This was dependent on Piwi cofactors, Gtsf1/Asterix (Gtsf1), Panoramix/Silencio (Panx), and Maelstrom (Mael), but not Eggless/dSetdb (Egg)-mediated H3K9me3 deposition. The λN-box B-mediated tethering of Mael to reporters repressed Brm-dependent genes in the absence of Piwi, Panx, and Gtsf1. We propose that Piwi, via Mael, can rapidly suppress transcription of Brm-dependent genes to facilitate heterochromatin formation.

Transcription factor binding to genomic DNA is generally prevented by nucleosome formation, in which the DNA is tightly wrapped around the histone octamer. In contrast, pioneer transcription factors efficiently bind their target DNA sequences within the nucleosome. OCT4 has been identified as a pioneer transcription factor required for stem cell pluripotency. To study the nucleosome binding by OCT4, we prepared human OCT4 as a recombinant protein, and biochemically analyzed its interactions with the nucleosome containing a natural OCT4 target, the LIN28B distal enhancer DNA sequence, which contains three potential OCT4 target sequences. By a combination of chemical mapping and cryo-electron microscopy single-particle analysis, we mapped the positions of the three target sequences within the nucleosome. A mutational analysis revealed that OCT4 preferentially binds its target DNA sequence located near the entry/exit site of the nucleosome. Crosslinking mass spectrometry consistently showed that OCT4 binds the nucleosome in the proximity of the histone H3 N-terminal region, which is close to the entry/exit site of the nucleosome. We also found that the linker histone H1 competes with OCT4 for the nucleosome binding. These findings provide important information for understanding the molecular mechanism by which OCT4 binds its target DNA in chromatin.

Histone H3mm18 is a non-allelic H3 variant expressed in skeletal muscle and brain in mice. However, its function has remained enigmatic. We found that H3mm18 is incorporated into chromatin in cells with low efficiency, as compared to H3.3. We determined the structures of the nucleosome core particle (NCP) containing H3mm18 by cryo-electron microscopy, which revealed that the entry/exit DNA regions are drastically disordered in the H3mm18 NCP. Consistently, the H3mm18 NCP is substantially unstable in vitro. The forced expression of H3mm18 in mouse myoblast C2C12 cells markedly suppressed muscle differentiation. A transcriptome analysis revealed that the forced expression of H3mm18 affected the expression of multiple genes, and suppressed a group of genes involved in muscle development. These results suggest a novel gene expression regulation system in which the chromatin landscape is altered by the formation of unusual nucleosomes with a histone variant, H3mm18, and provide important insight into understanding transcription regulation by chromatin.

The Pacific oyster, Crassostrea gigas, is a traditional food worldwide. The soft body of the oyster can easily accumulate heavy metals such as cadmium (Cd). To clarify the molecular mechanism of Cd accumulation in the viscera of C. gigas, we identified Cd-binding proteins. 5,10,15,20-Tetraphenyl-21H,23H-porphinetetrasulfonic acid, disulfuric acid, tetrahydrate, and Cd-binding competition experiments using immobilized metal ion affinity chromatography revealed the binding of water-soluble high molecular weight proteins to Cd, including C. gigas protein disulfide isomerase (cgPDI). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses revealed two CGHC motifs in cgPDI. The binding between Cd and rcgPDI was confirmed through a Cd-binding experiment using the TPPS method. Isothermal titration calorimetry (ITC) revealed the binding of two Cd ions to one molecule of rcgPDI. Circular dichroism (CD) spectrum and tryptophan fluorescence analyses demonstrated that the rcgPDI bound to Cd. The binding markedly changed the two-dimensional or three-dimensional structures. The activity of rcgPDI measured by a PDI Activity Assay Kit was more affected by the addition of Cd than by human PDI. Immunological analyses indicated that C. gigas contained cgPDI at a concentration of 1.0 nmol/g (viscera wet weight). The combination of ITC and quantification results revealed that Cd-binding to cgPDI accounted for 20% of the total bound Cd in the visceral mass. The findings provide new insights into the defense mechanisms of invertebrates against Cd.

The nuclear pore complex embedded within the nuclear envelope is the essential architecture for trafficking macromolecules, such as proteins and RNAs, between the cytoplasm and nucleus. The nuclear pore complex assembly occurs on chromatin in the post-mitotic phase of the cell cycle. ELYS (MEL-28/AHCTF1) binds to the nucleosome, which is the basic chromatin unit, and promotes assembly of the complex around the chromosomes in cells. Here we show that the Arg-Arg-Lys (RRK) stretch of the C-terminal ELYS region plays an essential role in the nucleosome binding. The cryo-EM structure and the crosslinking mass spectrometry reveal that the ELYS C-terminal region directly binds to the acidic patch of the nucleosome. These results provide mechanistic insight into the ELYS-nucleosome interaction, which promotes the post-mitotic nuclear pore complex formation around chromosomes in cells.

The ring-shaped chromosomal cohesin complex holds sister chromatids together by topological embrace, a prerequisite for accurate chromosome segregation. Cohesin plays additional roles in genome organization, transcriptional regulation and DNA repair. The cohesin ring includes an ABC family ATPase, but the molecular mechanism by which the ATPase contributes to cohesin function is not yet understood. Here we have purified budding yeast cohesin, as well as its Scc2-Scc4 cohesin loader complex, and biochemically reconstituted ATP-dependent topological cohesin loading onto DNA. Our results reproduce previous observations obtained using fission yeast cohesin, thereby establishing conserved aspects of cohesin behavior. Unexpectedly, we find that non-hydrolyzable ATP ground state mimetics ADP·BeF2, ADP·BeF3 - and ADP·AlFx, but not a hydrolysis transition state analog ADP·VO4 3-, support cohesin loading. The energy from nucleotide binding is sufficient to drive the DNA entry reaction into the cohesin ring. ATP hydrolysis, thought to be essential for in vivo cohesin loading, must serve a subsequent reaction step. These results provide molecular insight into cohesin function and open new experimental opportunities that the budding yeast model affords.

Sister-chromatid cohesion is established by Eco1-mediated acetylation on two conserved tandem lysines in the cohesin Smc3 subunit. However, the molecular basis of Eco1 substrate recognition and acetylation in cohesion is not fully understood. Here, we discover and rationalize the substrate specificity of Eco1 using mass spectrometry coupled with in-vitro acetylation assays and crystallography. Our structures of the X. laevis Eco2 (xEco2) bound to its primary and secondary Smc3 substrates demonstrate the plasticity of the substrate-binding site, which confers substrate specificity by concerted conformational changes of the central β hairpin and the C-terminal extension.

Sister chromatid cohesion, mediated by cohesin, is required for accurate chromosome segregation [1, 2]. This process requires acetylation of cohesin subunit SMC3 by evolutionarily conserved cohesin acetyltransferases: Eco1 in budding yeast; XEco1 and XEco2 in Xenopus; and ESCO1 and ESCO2 in human [3-10]. Eco1 is recruited to chromatin through physical interaction with PCNA [11] and is degraded by the Skp1/Cul1/F-box protein complex after DNA replication to prevent ectopic cohesion formation [12]. In contrast, XEco2 recruitment to chromatin requires prereplication complex formation [13] and is degraded by the anaphase-promoting complex (APC) [14]. In human, whereas ESCO1 is expressed throughout the cell cycle, ESCO2 is detectable in S phase and is degraded after DNA replication [6, 15]. Although PDS5, a cohesin regulator, preferentially promotes ESCO1-dependent SMC3 acetylation [16], little is known about the molecular basis of the temporal regulation of ESCO2. Here, we show that ESCO2 is recruited to chromatin before PCNA accumulation. Whereas no interaction between PCNA and ESCO proteins is observed, ESCO2, but not ESCO1, interacts with the MCM complex through a unique ESCO2 domain. Interestingly, the interaction is required to protect ESCO2 from proteasomal degradation and is attenuated in late S phase. We also found that ESCO2 physically interacts with the CUL4-DDB1-VPRBP E3 ubiquitin ligase complex in late S phase and that post-replicative ESCO2 degradation requires the complex as well as APC. Thus, we propose that the MCM complex couples ESCO2 with DNA replication and that the CUL4-DDB1-VPRBP complex promotes post-replicative ESCO2 degradation, presumably to suppress cohesion formation during mitosis.

Several types of shell matrix proteins (SMPs) have been identified in molluskan shells. Their diversity is the consequence of various molecular processes, including domain shuffling and gene duplication. However, the evolutionary origin of most SMPs remains unclear. In this study, we investigated the evolutionary process EGF-like and zona pellucida (ZP) domains containing SMPs. Two types of the proteins (EGF-like protein (EGFL) and EGF-like and ZP domains containing protein (EGFZP)) were found in the pearl oyster, Pinctada fucata. In contrast, only EGFZP was identified in the gastropods. Phylogenetic analysis and genomic arrangement studies showed that EGFL and EGFZP formed a clade in bivalves, and their encoding genes were localized in tandem repeats on the same scaffold. In P. fucata, EGFL genes were expressed in the outer part of mantle epithelial cells are related to the calcitic shell formation. However, in both P. fucata and the limpet Nipponacmea fuscoviridis, EGFZP genes were expressed in the inner part of the mantle epithelial cells are related to aragonitic shell formation. Furthermore, our analysis showed that in P. fucata, the ZP domain interacts with eight SMPs that have various functions in the nacreous shell mineralization. The data suggest that the ZP domain can interact with other SMPs, and EGFL evolution in pterimorph bivalves represents an example of neo-functionalization that involves the acquisition of a novel protein through gene duplication.

Bombyx Papi acts as a scaffold for Siwi-piRISC biogenesis on the mitochondrial surface. Papi binds first to Siwi via the Tudor domain and subsequently to piRNA precursors loaded onto Siwi via the K-homology (KH) domains. This second action depends on phosphorylation of Papi. However, the underlying mechanism remains unknown. Here, we show that Siwi targets Par-1 kinase to Papi to phosphorylate Ser547 in the auxiliary domain. This modification enhances the ability of Papi to bind Siwi-bound piRNA precursors via the KH domains. The Papi S547A mutant bound to Siwi, but evaded phosphorylation by Par-1, abrogating Siwi-piRISC biogenesis. A Papi mutant that lacked the Tudor and auxiliary domains escaped coordinated regulation by Siwi and Par-1 and bound RNAs autonomously. Another Papi mutant that lacked the auxiliary domain bound Siwi but did not bind piRNA precursors. A sophisticated mechanism by which Siwi cooperates with Par-1 kinase to promote Siwi-piRISC biogenesis was uncovered.

Concomitant with DNA replication, the chromosomal cohesin complex establishes cohesion between newly replicated sister chromatids. Several replication-fork-associated "cohesion establishment factors," including the multifunctional Ctf18-RFC complex, aid this process in as yet unknown ways. Here, we show that Ctf18-RFC's role in sister chromatid cohesion correlates with PCNA loading but is separable from its role in the replication checkpoint. Ctf18-RFC loads PCNA with a slight preference for the leading strand, which is dispensable for DNA replication. Conversely, the canonical Rfc1-RFC complex preferentially loads PCNA onto the lagging strand, which is crucial for DNA replication but dispensable for sister chromatid cohesion. The downstream effector of Ctf18-RFC is cohesin acetylation, which we place toward a late step during replication maturation. Our results suggest that Ctf18-RFC enriches and balances PCNA levels at the replication fork, beyond the needs of DNA replication, to promote establishment of sister chromatid cohesion and possibly other post-replicative processes.

The cohesin complex participates in many structural and functional aspects of genome organization. Cohesin recruitment onto chromosomes requires nucleosome-free DNA and the Scc2-Scc4 cohesin loader complex that catalyzes topological cohesin loading. Additionally, the cohesin loader facilitates promoter nucleosome clearance in a yet unknown way, and it recognizes chromatin receptors such as the RSC chromatin remodeler. Here, we explore the cohesin loader-RSC interaction. Amongst multi-pronged contacts by Scc2 and Scc4, we find that Scc4 contacts a conserved patch on the RSC ATPase motor module. The cohesin loader directly stimulates in vitro nucleosome sliding by RSC, providing an explanation how it facilitates promoter nucleosome clearance. Furthermore, we observe cohesin loader interactions with a wide range of chromatin remodelers. Our results provide mechanistic insight into how the cohesin loader recognizes, as well as influences, the chromatin landscape, with implications for our understanding of human developmental disorders including Cornelia de Lange and Coffin-Siris syndromes.

Transposable elements (TEs) are genomic parasites that propagate within the host genome and introduce mutations. Long interspersed nuclear element-1 (LINE-1 or L1) is the major TE class, which occupies nearly 20% of the mouse genome. L1 is highly active in mammalian preimplantation embryos, posing a major threat to genome integrity, but the mechanism of stage-specific protection against L1 retrotransposition is unknown. Here, we show that TAR DNA-binding protein 43 (TDP-43), mutations in which constitute a major risk factor for amyotrophic lateral sclerosis, inhibits L1 retrotransposition in mouse embryonic stem cells (mESCs) and preimplantation embryos. Knockdown of TDP-43 resulted in massive genomic L1 expansion and impaired cell growth in preimplantation embryos and ESCs. Functional analysis demonstrated that TDP-43 interacts with L1 open reading frame 1 protein (L1 ORF1p) to mediate genomic protection, and loss of this interaction led to derepression of L1 retrotransposition. Our results identify TDP-43 as a guardian of the embryonic genome.

Reducing sugars can covalently react with proteins to generate a heterogeneous and complex group of compounds called advanced glycation end products (AGEs). AGEs are generally considered as pathogenic molecules, mediating a pro-inflammatory response and contributing to the development of a number of human diseases. However, the intrinsic function of AGEs remains to be elucidated. We now provide multiple lines of evidence showing that AGEs can specifically bind histone localized on the cell surface as an AGE-binding protein, regulate the function of histone as a plasminogen receptor, and result in the regulation of monocytes/macrophage recruitment to the site of inflammation. Our finding of histone as a cell-surface receptor for AGEs suggests that, beside our common concept of AGEs as danger-associated molecular patterns mediating a pro-inflammatory response, they may also be involved in the homeostatic response via binding to histone.