A novel single alpha-helix DNA-binding domain in CAF-1 promotes gene silencing and DNA damage survival through tetrasome-length DNA selectivity and spacer function.

The histone chaperone chromatin assembly factor 1 (CAF-1) deposits two nascent histone H3/H4 dimers onto newly replicated DNA forming the central core of the nucleosome known as the tetrasome. How CAF-1 ensures there is sufficient space for the assembly of tetrasomes remains unknown. Structural and biophysical characterization of the lysine/glutamic acid/arginine-rich (KER) region of CAF-1 revealed a 128-Å single alpha-helix (SAH) motif with unprecedented DNA-binding properties. Distinct KER sequence features and length of the SAH drive the selectivity of CAF-1 for tetrasome-length DNA and facilitate function in budding yeast. In vivo, the KER cooperates with the DNA-binding winged helix domain in CAF-1 to overcome DNA damage sensitivity and maintain silencing of gene expression. We propose that the KER SAH links functional domains within CAF-1 with structural precision, acting as a DNA-binding spacer element during chromatin assembly.

Reprogramming the Cleavage Specificity of Botulinum Neurotoxin Serotype B1.

Proteases with reprogrammed specificity for nonnative substrates are highly desired in synthetic biology and biomedicine. However, generating reprogrammed proteases that are orthogonal and highly specific for a new target has been a major challenge. In this work, we sought to expand the versatility of protease systems by engineering an orthogonal botulinum neurotoxin serotype B (BoNT/B) protease that recognizes an orthogonal substrate. We designed and validated an orthogonal BoNT/B protease system in mammalian cells, combining mutations in the protease with compensatory mutations in the protease substrate and incorporating a truncated target sequence and then demonstrated use of this orthogonal BoNT/B protease-substrate combination to regulate complex transcriptional circuitry in mammalian cells. Transposing this platform into yeast, we demonstrated utility of this approach for in vivo protease evolution. We tested this platform with the newly designed orthogonal protease and then used it in a high-throughput screen to identify novel orthogonal protease/protease substrate combinations. While carrying out this work, we also generated new cleavage reporters that could be used to report botulinum toxin protease activity in mammalian cells using simple fluorescent readouts. We envision that these approaches will expand the applications of botulinum protease in new directions and aid in the development of new reprogrammed proteases.

Characterization of a Primordial Major Capsid-Scaffolding Protein Complex in Icosahedral Virus Shell Assembly.

Capsid assembly pathways are strongly conserved in the complex dsDNA viruses, where major capsid proteins (MCP) self-assemble into icosahedral procapsid shells, chaperoned by a scaffolding protein. Without a scaffold, the capsid proteins aggregate and form aberrant structures. This, coupled with the rapid co-polymerization of MCP and scaffolding proteins, has thwarted characterization of the earliest steps in shell assembly. Here we interrogate the structure and biophysical properties of a soluble, assembly-deficient phage lambda major capsid protein, MCP(W308A). The mutant protein is folded, soluble to high concentrations and binds to the scaffolding protein in an apparent SP2:MCP(W308A)1 stoichiometry but does not assemble beyond this initiating complex. The MCP(W308A) crystal structure was solved to 2.7 Å revealing the canonical HK97 fold in a "pre-assembly" conformation featuring the conserved N-arm and E-loops folded into the body of the protein. Structural, biophysical and computational analyses suggest that MCP(W308A) is thermodynamically trapped in this pre-assembly conformation precluding self-association interactions required for shell assembly. A model is described wherein dynamic interactions between MCP proteins play an essential role in high fidelity viral shell assembly. Scaffold-chaperoned MCP polymerization is a strongly conserved process in all the large dsDNA viruses and our results provide insight into this primordial complex in solution and have broad biological significance in our understanding of virus assembly mechanisms.

Cytosine methylation of mitochondrial DNA at CpG sequences impacts transcription factor A DNA binding and transcription.

In eukaryotes, cytosine methylation of nuclear DNA at CpG sequences (5mCpG) regulates epigenetic inheritance through alterations in chromatin structure. However, mitochondria lack nucleosomal chromatin, therefore the molecular mechanisms by which 5mCpG influences mitochondria must be different and are as yet unknown. Mitochondrial Transcription Factor A (TFAM) is both the primary DNA-compacting protein in the mitochondrial DNA (mtDNA) nucleoid and a transcription-initiation factor. TFAM must encounter hundreds of CpGs in mtDNA, so the occurrence of 5mCpG has the potential to impact TFAM-DNA recognition. We used biophysical approaches to determine whether 5mCpG alters any TFAM-dependent activities. 5mCpG in the heavy strand promoter (HSP1) increased the binding affinity of TFAM and induced TFAM multimerization with increased cooperativity compared to nonmethylated DNA. However, 5mCpG had no apparent effect on TFAM-dependent DNA compaction. Additionally, 5mCpG had a clear and context-dependent effect on transcription initiating from the three mitochondrial promoters. Taken together, our findings demonstrate that 5mCpG in the mitochondrial promoter region does impact TFAM-dependent activities in vitro.

Mechanism of agonism and antagonism of the Pseudomonas aeruginosa quorum sensing regulator QscR with non-native ligands.

Pseudomonas aeruginosa is an opportunistic pathogen that uses the process of quorum sensing (QS) to coordinate the expression of many virulence genes. During quorum sensing, N-acyl-homoserine lactone (AHL) signaling molecules regulate the activity of three LuxR-type transcription factors, LasR, RhlR and QscR. To better understand P. aeruginosa QS signal reception, we examined the mechanism underlying the response of QscR to synthetic agonists and antagonists using biophysical and structural approaches. The structure of QscR bound to a synthetic agonist reveals a novel mode of ligand binding supporting a general mechanism for agonist activity. In turn, antagonists of QscR with partial agonist activity were found to destabilize and greatly impair QscR dimerization and DNA binding. These results highlight the diversity of LuxR-type receptor responses to small molecule agonists and antagonists and demonstrate the potential for chemical strategies for the selective targeting of individual QS systems.

Functional Impact of Chromatin Remodeling Gene Mutations and Predictive Signature for Therapeutic Response in Bladder Cancer.

Urothelial carcinoma accounts for most of the bladder cancer cases. Using next-generation sequencing (NGS) technology, we found that a significant percentage (83%) of tumors had mutations in chromatin-remodeling genes. Here, we examined the functional relevance of mutations in two chromatin-remodeling genes, EP300 and its paralog, CREBBP, which are mutated in almost one-third of patients. Interestingly, almost half of missense mutations cluster in the histone-acetyltransferase (HAT) domain of EP300/CREBBP. This domain catalyzes the transfer of an acetyl group to target molecules such as histones, thereby regulating chromatin dynamics. Thus, patients with EP300 or CREBBP mutations may have alterations in the ability of the corresponding proteins to modify histone proteins and control transcriptional profiles. In fact, it was determined that many of the missense HAT mutations in EP300 (64%) and CREBBP (78%) were HAT-inactivating. These inactivating mutations also correlated with invasive disease in patients. Strikingly, the prediction software Mutation Assessor accurately predicted the functional consequences of each HAT missense mutation. Finally, a gene expression signature was developed that associated with loss of HAT activity and that this signature was associated with more aggressive cancer in four patient datasets. Further supporting the notion that this score accurately reflects HAT activity, we found it is responsive to treatment of cancer cells to mocetinostat, a histone deacetylase (HDAC) inhibitor.Implication: This study provides a rationale for targeted sequencing of EP300 and CREBBP and use of a gene profiling signature for predicting therapeutic response in patients. Mol Cancer Res; 16(1); 69-77. ©2017 AACR.

De Novo Mutations in EBF3 Cause a Neurodevelopmental Syndrome.

Early B cell factor 3 (EBF3) is an atypical transcription factor that is thought to influence the laminar formation of the cerebral cortex. Here, we report that de novo mutations in EBF3 cause a complex neurodevelopmental syndrome. The mutations were identified in two large-scale sequencing projects: the UK Deciphering Developmental Disorders (DDD) study and the Canadian Clinical Assessment of the Utility of Sequencing and Evaluation as a Service (CAUSES) study. The core phenotype includes moderate to severe intellectual disability, and many individuals exhibit cerebellar ataxia, subtle facial dysmorphism, strabismus, and vesicoureteric reflux, suggesting that EBF3 has a widespread developmental role. Pathogenic de novo variants identified in EBF3 include multiple loss-of-function and missense mutations. Structural modeling suggested that the missense mutations affect DNA binding. Functional analysis of mutant proteins with missense substitutions revealed reduced transcriptional activities and abilities to form heterodimers with wild-type EBF3. We conclude that EBF3, a transcription factor previously unknown to be associated with human disease, is important for brain and other organ development and warrants further investigation.

Mechanism of Mitochondrial Transcription Factor A Attenuation of CpG-Induced Antibody Production.

Mitochondrial transcription factor A (TFAM) had previously been shown to act as a damage associated molecular pattern with the ability to enhance CpG-A phosphorothioate oligodeoxynucleotide (ODN)-mediated stimulation of IFNα production from human plasmacytoid dendritic cells. Examination of the mechanism by which TFAM might influence CpG ODN mediated innate immune responses revealed that TFAM binds directly, tightly and selectively to the structurally related CpG-A, -B, and -C ODN. TFAM also modulated the ability of the CpG-B or -C to stimulate the production of antibodies from human B cells. TFAM showed a dose-dependent modulation of CpG-B, and -C -induced antibody production from human B cells in vitro, with enhancement of high dose and inhibition of low doses of CpG stimulation. This effect was linked to the ability of TFAM to directly inhibit the binding of CpG ODNs to B cells, in a manner consistent with the relative binding affinities of TFAM for the ODNs. These data suggest that TFAM alters the free concentration of the CpG available to stimulate B cells by sequestering this ODN in a TFAM-CpG complex. Thus, TFAM has the potential to decrease the pathogenic consequences of exposure to natural CpG-like hypomethylated DNA in vivo, as well as such as that found in traumatic injury, infection, autoimmune disease and during pregnancy.

The sea urchin mitochondrial transcription factor A binds and bends DNA efficiently despite its unusually short C-terminal tail.

Mitochondrial transcription factor A (TFAM) is a key component for the protection and transcription of the mitochondrial genome. TFAM belongs to the high mobility group (HMG) box family of DNA binding proteins that are able to bind to and bend DNA. Human TFAM (huTFAM) contains two HMG box domains separated by a linker region, and a 26 amino acid C-terminal tail distal to the second HMG box. Previous studies on huTFAM have shown that requisites for proper DNA bending and specific binding to the mitochondrial genome are specific intercalating residues and the C-terminal tail. We have characterized TFAM from the sea urchin Paracentrotus lividus (suTFAM). Differently from human, suTFAM contains a short 9 amino acid C-terminal tail, yet it still has the ability to specifically bind to mtDNA. To provide information on the mode of binding of the protein we used fluorescence resonance energy transfer (FRET) assays and found that, in spite of the absence of a canonical C-terminal tail, suTFAM distorts DNA at a great extent and recognizes specific target with high affinity. Site directed mutagenesis showed that the two Phe residues placed in corresponding position of the two intercalating Leu of huTFAM are responsible for the strong bending and the great binding affinity of suTFAM.

Nuclear PTEN functions as an essential regulator of SRF-dependent transcription to control smooth muscle differentiation.

Vascular disease progression is associated with marked changes in vascular smooth muscle cell (SMC) phenotype and function. SMC contractile gene expression and, thus differentiation, is under direct transcriptional control by the transcription factor, serum response factor (SRF); however, the mechanisms dynamically regulating SMC phenotype are not fully defined. Here we report that the lipid and protein phosphatase, PTEN, has a novel role in the nucleus by functioning as an indispensible regulator with SRF to maintain the differentiated SM phenotype. PTEN interacts with the N-terminal domain of SRF and PTEN-SRF interaction promotes SRF binding to essential promoter elements in SM-specific genes. Factors inducing phenotypic switching promote loss of nuclear PTEN through nucleo-cytoplasmic translocation resulting in reduced myogenically active SRF, but enhanced SRF activity on target genes involved in proliferation. Overall decreased expression of PTEN was observed in intimal SMCs of human atherosclerotic lesions underlying the potential clinical importance of these findings.

Development of novel Asf1-H3/H4 inhibitors.

The histone chaperone anti-silencing function 1 (Asf1) has emerged as a promising target for therapeutic intervention for multiple cancers (Cell2006, 127, 458). Asf1 is involved in the packaging of the eukaryotic genome into chromatin, which is essential for normal growth, development, and differentiation, as this regulates all nuclear processes that use DNA as a substrate. Starting from a collection of HTS leads, we identified a series of N-acyl hydrazones as novel inhibitors of the Asf-histone H3/H4 interaction. These compounds represent the first example of inhibitors capable of disrupting the Asf1-H3/H4 complex.

A solo luxI-type gene directs acylhomoserine lactone synthesis and contributes to motility control in the marine sponge symbiont Ruegeria sp. KLH11.

Marine sponges harbour abundant and diverse bacterial communities, providing an ideal environment for bacterial cell-density-dependent cell-cell signalling, termed quorum sensing. The marine sponge symbiont Ruegeria sp. KLH11 produces mainly long chain acylhomoserine lactones (AHLs) and has been developed as a quorum sensing model for roseobacterial sponge symbionts. Two pairs of luxR/I homologues were identified by genetic screening and were designated ssaRI and ssbRI (sponge-associated symbiont locus A or B, luxR/luxI homologue). In this study, we identified a third luxI-type gene, named sscI. The sscI gene does not have a cognate luxR homologue present at an adjacent locus and thus sscI is an AHL synthase solo. The sscI gene is required for production of long-chain hydroxylated AHLs, contributes to AHL pools and modestly influences flagellar motility in KLH11. A triple mutant for all luxI-type genes cannot produce AHLs, but still synthesizes para-coumaroyl-homoserine lactone.

Class I HDAC inhibition stimulates cardiac protein SUMOylation through a post-translational mechanism.

Lysine residues are subject to a multitude of reversible post-translational modifications, including acetylation and SUMOylation. In the heart, enhancement of lysine acetylation or SUMOylation using histone deacetylase (HDAC) inhibitors or SUMO-1 gene transfer, respectively, has been shown to be cardioprotective. Here, we addressed whether there is crosstalk between lysine acetylation and SUMOylation in the heart. Treatment of cardiomyocytes and cardiac fibroblasts with pharmacological inhibitors of HDAC catalytic activity robustly increased conjugation of SUMO-1, but not SUMO-2/3, to several high molecular weight proteins in both cell types. The use of a battery of selective HDAC inhibitors and short hairpin RNAs demonstrated that HDAC2, which is a class I HDAC, is the primary HDAC isoform that controls cardiac protein SUMOylation. HDAC inhibitors stimulated protein SUMOylation in the absence of de novo gene transcription or protein synthesis, revealing a post-translational mechanism of HDAC inhibitor action. HDAC inhibition did not suppress the activity of de-SUMOylating enzymes, suggesting that increased protein SUMOylation in HDAC inhibitor-treated cells is due to stimulation of SUMO-1 conjugation rather than blockade of SUMO-1 cleavage. Consistent with this, multiple components of the SUMO conjugation machinery were capable of being acetylated in vitro. These findings reveal a novel role for reversible lysine acetylation in the control of SUMOylation in the heart, and suggest that cardioprotective actions of HDAC inhibitors are in part due to stimulation of protein SUMO-1-ylation in myocytes and fibroblasts.

Binding of the histone chaperone ASF1 to the CBP bromodomain promotes histone acetylation.

The multifunctional Creb-binding protein (CBP) protein plays a pivotal role in many critical cellular processes. Here we demonstrate that the bromodomain of CBP binds to histone H3 acetylated on lysine 56 (K56Ac) with higher affinity than to its other monoacetylated binding partners. We show that autoacetylation of CBP is critical for the bromodomain-H3 K56Ac interaction, and we propose that this interaction occurs via autoacetylation-induced conformation changes in CBP. Unexpectedly, the bromodomain promotes acetylation of H3 K56 on free histones. The CBP bromodomain also interacts with the histone chaperone anti-silencing function 1 (ASF1) via a nearby but distinct interface. This interaction is necessary for ASF1 to promote acetylation of H3 K56 by CBP, indicating that the ASF1-bromodomain interaction physically delivers the histones to the histone acetyl transferase domain of CBP. A CBP bromodomain mutation manifested in Rubinstein-Taybi syndrome has compromised binding to both H3 K56Ac and ASF1, suggesting that these interactions are important for the normal function of CBP.

Reversed-phase ion-pair liquid chromatography method for purification of duplex DNA with single base pair resolution.

Obtaining quantities of highly pure duplex DNA is a bottleneck in the biophysical analysis of protein-DNA complexes. In traditional DNA purification methods, the individual cognate DNA strands are purified separately before annealing to form DNA duplexes. This approach works well for palindromic sequences, in which top and bottom strands are identical and duplex formation is typically complete. However, in cases where the DNA is non-palindromic, excess of single-stranded DNA must be removed through additional purification steps to prevent it from interfering in further experiments. Here we describe and apply a novel reversed-phase ion-pair liquid chromatography purification method for double-stranded DNA ranging in lengths from 17 to 51 bp. Both palindromic and non-palindromic DNA can be readily purified. This method has the unique ability to separate blunt double-stranded DNA from pre-attenuated (n-1, n-2, etc) synthesis products, and from DNA duplexes with single base pair overhangs. Additionally, palindromic DNA sequences with only minor differences in the central spacer sequence of the DNA can be separated, and the purified DNA is suitable for co-crystallization of protein-DNA complexes. Thus, double-stranded ion-pair liquid chromatography is a useful approach for duplex DNA purification for many applications.

Genetic and structure-function studies of missense mutations in human endothelial lipase.

Endothelial lipase (EL) plays a pivotal role in HDL metabolism. We sought to characterize EL and its interaction with HDL as well as its natural variants genetically, functionally and structurally. We screened our biethnic population sample (n = 802) for selected missense mutations (n = 5) and identified T111I as the only common variant. Multiple linear regression analyses in Hispanic subjects revealed an unexpected association between T111I and elevated LDL-C (p-value = 0.012) and total cholesterol (p-value = 0.004). We examined lipase activity of selected missense mutants (n = 10) and found different impacts on EL function, ranging from normal to complete loss of activity. EL-HDL lipidomic analyses indicated that EL has a defined remodeling of HDL without exhaustion of the substrate and a distinct and preference for several fatty acids that are lipid mediators and known for their potent pro- and anti-inflammatory properties. Structural studies using homology modeling revealed a novel α/ß motif in the C-domain, unique to EL. The EL dimer was found to have the flexibility to expand and to bind various sizes of HDL particles. The likely impact of the all known missense mutations (n = 18) on the structure of EL was examined using molecular modeling and the impact they may have on EL lipase activity using a novel structure-function slope based on their structural free energy differences. The results of this multidisciplinary approach delineated the impact of EL and its variants on HDL. Moreover, the results suggested EL to have the capacity to modulate vascular health through its role in fatty acid-based signaling pathways.

The C terminus of the histone chaperone Asf1 cross-links to histone H3 in yeast and promotes interaction with histones H3 and H4.

The central histone H3/H4 chaperone Asf1 comprises a highly conserved globular core and a divergent C-terminal tail. While the function and structure of the Asf1 core are well known, the function of the tail is less well understood. Here, we have explored the role of the yeast (yAsf1) and human (hAsf1a and hAsf1b) Asf1 tails in Saccharomyces cerevisiae. We show, using a photoreactive, unnatural amino acid, that Asf1 tail residue 210 cross-links to histone H3 in vivo and, further, that loss of C-terminal tail residues 211 to 279 weakens yAsf1-histone binding affinity in vitro nearly 200-fold. Via several yAsf1 C-terminal truncations and yeast-human chimeric proteins, we found that truncations at residue 210 increase transcriptional silencing and that the hAsf1a tail partially substitutes for full-length yAsf1 with respect to silencing but that full-length hAsf1b is a better overall substitute for full-length yAsf1. In addition, we show that the C-terminal tail of Asf1 is phosphorylated at T270 in yeast. Loss of this phosphorylation site does not prevent coimmunoprecipitation of yAsf1 and Rad53 from yeast extracts, whereas amino acid residue substitutions at the Asf1-histone H3/H4 interface do. Finally, we show that residue substitutions in yAsf1 near the CAF-1/HIRA interface also influence yAsf1's function in silencing.

The high mobility group box: the ultimate utility player of a cell.

High mobility group (HMG) box proteins are abundant and ubiquitous DNA binding proteins with a remarkable array of functions throughout the cell. The structure of the HMG box DNA binding domain and general mechanisms of DNA binding and bending have been known for more than a decade. However, new mechanisms that regulate HMG box protein intracellular translocation, and by which HMG box proteins recognize DNA with and without sequence specificity, have only recently been uncovered. This review focuses primarily on the Sry-like HMG box family, HMGB1, and mitochondrial transcription factor A. For these proteins, structural and biochemical studies have shown that HMG box protein modularity, interactions with other DNA binding proteins and cellular receptors, and post-translational modifications are key regulators of their diverse functions.

CAF-1-induced oligomerization of histones H3/H4 and mutually exclusive interactions with Asf1 guide H3/H4 transitions among histone chaperones and DNA.

Anti-silencing function 1 (Asf1) and Chromatin Assembly Factor 1 (CAF-1) chaperone histones H3/H4 during the assembly of nucleosomes on newly replicated DNA. To understand the mechanism of histone H3/H4 transfer among Asf1, CAF-1 and DNA from a thermodynamic perspective, we developed and employed biophysical approaches using full-length proteins in the budding yeast system. We find that the C-terminal tail of Asf1 enhances the interaction of Asf1 with CAF-1. Surprisingly, although H3/H4 also enhances the interaction of Asf1 with the CAF-1 subunit Cac2, H3/H4 forms a tight complex with CAF-1 exclusive of Asf1, with an affinity weaker than Asf1-H3/H4 or H3/H4-DNA interactions. Unlike Asf1, monomeric CAF-1 binds to multiple H3/H4 dimers, which ultimately promotes the formation of (H3/H4)(2) tetramers on DNA. Thus, transition of H3/H4 from the Asf1-associated dimer to the DNA-associated tetramer is promoted by CAF-1-induced H3/H4 oligomerization.