Topography of histone H3-H4 interaction with the Hat1-Hat2 acetyltransferase complex.

Chaperones influence histone conformation and intermolecular interaction in multiprotein complexes, and the structures obtained with full-length histones often provide more accurate and comprehensive views. Here, our structure of the Hat1-Hat2 acetyltransferase complex bound to Asf1-H3-H4 shows that the core domains of H3 and H4 are involved in binding Hat1 and Hat2, and the N-terminal tail of H3 makes extensive interaction with Hat2. These findings expand the knowledge about histone-protein interaction and implicate a function of Hat2/RbAp46/48, which is a versatile histone chaperone found in many chromatin-associated complexes, in the passing of histones between chaperones.

The RING E3 ligase CLG1 targets GS3 for degradation via the endosome pathway to determine grain size in rice.

G-protein signaling and ubiquitin-dependent degradation are both involved in grain development in rice, but how these pathways are coordinated in regulating this process is unknown. Here, we show that Chang Li Geng 1 (CLG1), which encodes an E3 ligase, regulates grain size by targeting the Gγ protein GS3, a negative regulator of grain length, for degradation. Overexpression of CLG1 led to increased grain length, while overexpression of mutated CLG1 with changes in three conserved amino acids decreased grain length. We found that CLG1 physically interacts with and ubiquitinats GS3which is subsequently degraded through the endosome degradation pathway, leading to increased grain size. Furthermore, we identified a critical SNP in the exon 3 of CLG1 that is significantly associated with grain size variation in a core collection of cultivated rice. This SNP results in an amino acid substitution from Arg to Ser at position 163 of CLG1 that enhances the E3 ligase activity of CLG1 and thus increases rice grain size. Both the expression level of CLG1 and the SNP CLG1163S may be useful variations for manipulating grain size in rice.

Understanding the mechanism of asymmetric gene regulation determined by the VqmA of vibriophage.

The quorum-sensing (QS) system between the phages and their hosts is important for the phage lysis-lysogeny decision. In Vibrio cholerae, the QS system consists of a LuxR-type receptor VqmA (VqmAVc) and an autoinducer molecule 3,5-dimethylpyrazin-2-ol (DPO). A VqmA homolog encoded by vibriophage VP882 (VqmAPhage) can intervene the host QS system via binding to both the host-produced DPO and its cognate promoter (Pqtip) to induce the phage lysogeny-to-lysis transition, whereas VqmAVc cannot influence the VqmAPhage-induced pathway, suggesting an asymmetry regulation. In this study, we report the crystal structure of VqmAPhage-DPO complex at 2.65 Å and reveal that the mechanism of DPO recognition is conserved in VqmA homologs. Besides, we identify a non-classical palindrome sequence in Pqtip, which can be effectively recognized by VqmAPhage but not VqmAVc. The sequence contains an interval longer than that in the vqmR promoter recognized by VqmAVc. In addition, the two DBD regions in the VqmAPhage dimer exhibit more relaxed architecture than that of the reported VqmAVc, which is likely to be in the conformation that may easily bind to target promoter containing a longer interval. In summary, our findings provide a structural and biochemical basis for the DBD-dependent DNA recognition in different promoter regions in the phage lysogeny-to-lysis decision communication system, and provide clues for developing phage therapies against Vibrio cholerae infection.

DNA polymerase α interacts with H3-H4 and facilitates the transfer of parental histones to lagging strands.

How parental histones, the carriers of epigenetic modifications, are deposited onto replicating DNA remains poorly understood. Here, we describe the eSPAN method (enrichment and sequencing of protein-associated nascent DNA) in mouse embryonic stem (ES) cells and use it to detect histone deposition onto replicating DNA strands with a relatively small number of cells. We show that DNA polymerase α (Pol α), which synthesizes short primers for DNA synthesis, binds histone H3-H4 preferentially. A Pol α mutant defective in histone binding in vitro impairs the transfer of parental H3-H4 to lagging strands in both yeast and mouse ES cells. Last, dysregulation of both coding genes and noncoding endogenous retroviruses is detected in mutant ES cells defective in parental histone transfer. Together, we report an efficient eSPAN method for analysis of DNA replication-linked processes in mouse ES cells and reveal the mechanism of Pol α in parental histone transfer.

Acetylation of histone H3K27 signals the transcriptional elongation for estrogen receptor alpha.

As approximately 70% of human breast tumors are estrogen receptor α (ERα)-positive, estrogen and ERα play essential roles in breast cancer development. By interrupting the ERα signaling pathway, endocrine therapy has been proven to be an effective therapeutic strategy. In this study, we identified a mechanism by which Transcription Start Site (TSS)-associated histone H3K27 acetylation signals the Super Elongation Complex (SEC) to regulate transcriptional elongation of the ESR1 (ERα) gene. SEC interacts with H3K27ac on ESR1 TSS through its scaffold protein AFF4. Depletion of AFF4 by siRNA or CRISPR/Cas9 dramatically reduces expression of ESR1 and its target genes, consequently inhibiting breast cancer cell growth. More importantly, a AFF4 mutant which lacks H3K27ac interaction failed to rescue ESR1 gene expression, suggesting H3K27 acetylation at TSS region is a key mark bridging the transition from transcriptional initiation to elongation, and perturbing SEC function can be an alternative strategy for targeting ERα signaling pathway at chromatin level.

Structural insights into the mechanism of c-di-GMP-bound YcgR regulating flagellar motility in Escherichia coli.

The motile-sessile transition is critical for bacterial survival and growth. Cyclic-di-GMP (c-di-GMP) plays a central role in controlling this transition and regulating biofilm formation via various effectors. As an effector of c-di-GMP in Escherichia coli and related species, the PilZ domain-containing protein YcgR responds to elevated c-di-GMP concentrations and acts on the flagellar motor to suppress bacterial motility in a brakelike fashion, which promotes bacterial surface attachment. To date, several target proteins within the motor, MotA, FliG, and FliM, along with different regulatory mechanisms have been reported. However, how YcgR acts on these components remains unclear. Here, we report that activated YcgR stably binds to MotA at the MotA-FliG interface and thereby regulates bacterial swimming. Biochemical and structural analyses revealed that c-di-GMP rearranges the PilZ domain configuration, resulting in the formation of a MotA-binding patch consisting of an RXXXR motif and the C-tail helix α3. Moreover, we noted that a conserved region in the YcgR-N domain, which is independent of MotA interaction, is necessary for motility regulation. On the basis of these findings, we infer that the YcgR-N domain is required for activity on other motor proteins. We propose that activated YcgR appends to MotA via its PilZ domain and thereby interrupts the MotA-FliG interaction and simultaneously interacts with other motor proteins via its YcgR-N domain to inhibit flagellar motility. Our findings suggest that the mode of interaction between YcgR and motor proteins may be shared by other PilZ family proteins.

The ligand-binding domain of a chemoreceptor from Comamonas testosteroni has a previously unknown homotrimeric structure.

Transmembrane chemoreceptors are widely present in Bacteria and Archaea. They play a critical role in sensing various signals outside and transmitting to the cell interior. Here, we report the structure of the periplasmic ligand-binding domain (LBD) of the transmembrane chemoreceptor MCP2201, which governs chemotaxis to citrate and other organic compounds in Comamonas testosteroni. The apo-form LBD crystal revealed a typical four-helix bundle homodimer, similar to previously well-studied chemoreceptors such as Tar and Tsr of Escherichia coli. However, the citrate-bound LBD revealed a four-helix bundle homotrimer that had not been observed in bacterial chemoreceptor LBDs. This homotrimer was further confirmed with size-exclusion chromatography, analytical ultracentrifugation and cross-linking experiments. The physiological importance of the homotrimer for chemotaxis was demonstrated with site-directed mutations of key amino acid residues in C. testosteroni mutants.

A potential substrate binding pocket of BdcA plays a critical role in NADPH recognition and biofilm dispersal.

Biofilm dispersal is characterized by the cell detachment from biofilms and expected to provide novel "anti-biofilm" approaches of prevention and treatment of biofilms in clinical and industrial settings. The E.coli protein BdcA has been identified as a biofilm dispersal factor and designed to be an important component in engineered applications to control biofilm formation. It belongs to short-chain dehydrogenase/reductase (SDR) family with the specific affinity to NADPH. Here, we show the structure of BdcA in complex with NADPH and confirm that NADPH binding is requisite for BdcA facilitating cell motility and increasing biofilm dispersal. Especially, we observe a potential substrate binding pocket surrounded by hydrophobic residues upon NADPH binding and present evidences that this pocket is essential for BdcA binding NADPH and exerting its biological functions. Our study provides the clues for illuminating the molecular mechanism of BdcA regulating biofilm dispersal and better utilizing BdcA to eliminate the biofilms.

OsMAPK3 Phosphorylates OsbHLH002/OsICE1 and Inhibits Its Ubiquitination to Activate OsTPP1 and Enhances Rice Chilling Tolerance.

Improvement of chilling tolerance is a major target in rice breeding. The signaling pathways regulating chilling consist of complex networks, including key transcription factors and their targets. However, it remains largely unknown how transcription factors are activated by chilling stress. Here, we report that the transcription factor OsbHLH002/OsICE1 is phosphorylated by OsMAPK3 under chilling stress. The osbhlh002-1 knockout mutant and antisense transgenic plants showed chilling hypersensitivity, whereas OsbHLH002-overexpressing plants exhibited enhanced chilling tolerance. OsbHLH002 can directly target OsTPP1, which encodes a key enzyme for trehalose biosynthesis. OsMAPK3 interacts with OsbHLH002 to prevent its ubiquitination by the E3 ligase OsHOS1. Under chilling stress, active OsMAPK3 phosphorylates OsbHLH002, leading to accumulation of phospho-OsbHLH002, which promotes OsTPP1 expression and increases trehalose content and resistance to chilling damage. Taken together, these results indicate that OsbHLH002 is phosphorylated by OsMAPK3, which enhances OsbHLH002 activation to its target OsTPP1 during chilling stress.