Insights Into the Chromatin Structure of Thermoplasma volcanium: Archaeal HU Regulates Alba‐Mediated DNA Compaction

The fatty membrane that surrounds cells is an essential feature of all living things. It is a selective barrier, only allowing certain substances to enter and exit the cell, and it contains the proteins and carbohydrates that the cell uses to interact with its environment. In bacteria, the carbohydrates on the outer side of the membrane can become ‘tagged’ or modified with small chemical entities which often prove useful for the cell. Acyl groups, for example, allow disease-causing bacteria to evade the immune system and contribute to infections persisting in the body. As a rule, activated acyl groups are only found inside the cell, so they need to move across the membrane before they can be attached onto the carbohydrates at the surface. This transfer is performed by a group of proteins that sit within the membrane called the acyltransferase-3 (AT3) family. The structure of these proteins and the mechanism by which they facilitate membrane crossing have remained unclear. Newman, Tindall et al. combined computational and structural modelling techniques with existing experimental data to establish how this family of proteins moves acyl groups across the membrane. They focused on OafB, an AT3 protein from the foodborne bacterial pathogen Salmonella typhimurium. The experimental data used by the team included information about which parts of OafB are necessary for this protein to acylate carbohydrates molecules. In their experiments, Newman, Tindall et al. studied how different parts of OafB move, how they interact with the molecules that carry an acyl group to the membrane, and how the acyl group is then transferred to the carbohydrate acceptor. Their results suggest that AT3 family proteins have a central pore or hole, plugged by a loop. This loop moves and therefore ‘unplug’ the pore, resulting in the emergence of a channel across the membrane. This channel can accommodate the acyl-donating molecule, presenting the acyl group to the outer surface of the membrane where it can be transferred to the acceptor carbohydrate. The AT3 family of proteins participates in many cellular processes involving the membrane, and a range of bacterial pathogens rely on these proteins to successfully infect human hosts. The results of Newman Tindall et al. could therefore be used across the biological sciences to provide more detailed understanding of the membrane, and to inform the design of drugs to fight bacterial diseases.

Deinococcus radiodurans contains a highly condensed nucleoid that remains to be unaltered following the exposure to high doses of gamma-irradiation. Proteins belonging to the structural maintenance of chromosome protein (SMC) family are present in all organisms and were shown to be involved in chromosome condensation, pairing, and/or segregation. Here, we have inactivated the smc gene in the radioresistant bacterium D. radiodurans, and, unexpectedly, found that smc null mutants showed no discernible phenotype except an increased sensitivity to gyrase inhibitors suggesting a role of SMC in DNA folding. A defect in the SMC-like SbcC protein exacerbated the sensitivity to gyrase inhibitors of cells devoid of SMC. We also showed that the D. radiodurans SMC protein forms discrete foci at the periphery of the nucleoid suggesting that SMC could locally condense DNA. The phenotype of smc null mutant leads us to speculate that other, not yet identified, proteins drive the compact organization of the D. radiodurans nucleoid.

According to the research, obesity is associated with hyperlipidemia, hypertension, and type 2 diabetes mellitus, which are grouped as metabolic syndrome. Notably, under the obese status, the adipocyte could accumulate excessive lipid as lipid droplets (LDs), leading the dysfunctional fat mass. Recently, emerging evidence has shown that the cell death-inducing DNA fragmentation factor 45-like effector protein (CIDE) family played an important role in regulating lipid metabolism. In addition, diverse CIDE proteins were also confirmed to influence the intracellular lipid metabolism, such as within adipocyte, hepatocyte, and macrophage. Nevertheless, the results which showed the regulatory influence of CIDE proteins are significantly contradictory from in vitro experiments and in vivo clinical studies. Similarly, recent studies have changed the perception of these proteins, redefining them as regulators of lipid droplet dynamics and fat metabolism, which contribute to a healthy metabolic phenotype in humans. However, the underlying mechanisms by which the diverse CIDE proteins alter lipid metabolism are not elucidated. In the current review, the understandings of CIDE proteins in lipid catabolism were well-summarized. On the other hand, the relatively mechanisms were also proposed for the further understandings of the CIDE protein family.

Eukaryotic genomes are highly complex structures that must be efficiently packaged into relatively small nuclei in order to accommodate multiple DNA-dependent processes, from transcription and DNA replication to DNA repair and recombination. The compaction of genomes is hierarchically achieved at two distinct levels: (1) the compaction of DNA into nucleosome arrays and (2) the three-dimensional (3D) folding of nucleosome arrays within the nucleus. The compaction of genomes is required for the proper regulation of DNA-dependent processes, and disruption of either is associated with complex human diseases.1,2 The 3D folding of nucleosome arrays within the nucleus is highly dynamic, with discrete chromosomes occupying distinct non-random “territories”3. Within each chromosome territory, specific DNA “loops” are formed that uniquely juxtapose distally located DNA loci, bringing them into close proximity.3 DNA loops have been implicated in transcriptional regulation and transcriptional memory, although the molecular mechanisms for these phenomena remain to be determined. The compaction of DNA into nucleosome arrays is accomplished by wrapping DNA around an octamer of histone proteins. Eukaryotic organisms regulate DNA-dependent process through nucleosome arrays using highly conserved ATP-dependent chromatin remodeling enzymes that utilizing the energy released from ATP hydrolysis to slide, evict or replace histones within nucleosomes.4 ATP-dependent chromatin remodeling enzymes are highly abundant, yet function only at very specific loci. How such abundant enzymes are targeted to specific loci genome-wide remains a very important unanswered question. In our recent article,5 we established that the primary mechanism for the targeting of Isw2, a highly conserved ATP-dependent chromatin remodeling enzyme in S. cerevisiae, is through sequence-specific transcription factor (TF)-dependent recruitment. Using chromatin immunoprecipitation on whole genome tiled microarrays (ChIP-chip), we showed that the TFs Ume6, Cin5, Sok2 and Nrg1 target Isw2 to their binding sites genome-wide. These “canonical” Isw2 targets represent the classical model of protein targeting. Unexpectedly, we found that more than half of the TF-dependent Isw2 targets do not have the corresponding TF binding site. This suggested that TFs target Isw2 to specific loci via a previously unknown mechanism(s). A hint for this mechanism(s) came from the observation that Isw2 is targeted to both the 5′- and 3′-ends of the same gene at a highly statistically significant frequency. Because the 5′- and 3′-ends of yeast genes have been shown to form DNA loops,6 we hypothesized that DNA looping may mediate Isw2 targeting to loci that do not have TF binding sites (Fig. 1). Using Ume6-dependent Isw2 targets as a model, we demonstrated by chromosome conformation capture (3C) that DNA looping does indeed take place between an Isw2 target with a Ume6 binding site (canonical targets) and one lacking a Ume6 binding site (ectopic targets). We further discovered that DNA looping-dependent ectopic Isw2 targets require both the general TF TFIIB and the sequence-specific DNA binding repressor Ume6. Finally, we provided evidence suggesting that Ume6-dependent DNA looping is associated with both chromatin remodeling and transcriptional repression. Therefore, our results reveal two distinct mechanisms for TF-dependent targeting of a chromatin remodeling factor (Fig. 1): (1) targeting directly to its binding sites (canonical targets) and (2) via DNA looping (ectopic targets). Significantly, our finding that DNA looping-dependent Isw2 targeting likely takes place very widely across the budding yeast genome, suggests a model where the 3D folding of nucleosome arrays within the nucleus is intimately linked to both the regulation of chromatin structure and DNA-dependent processes. In addition, our results identified a molecular mechanism by which DNA looping affects DNA-dependent processes and a novel biological function of DNA looping. Our results have raised several interesting questions. For example, are there different biological consequences associated with recruitment of Isw2 to canonical vs. ectopic targets? Bioinformatics analysis does in fact suggest this might be the case: canonical targets are associated with genes involved in meiosis and DNA recombination, while ectopic targets are associated with housekeeping genes involved in translation and glucose metabolism. Isw2 is also known to repress non-coding RNA (ncRNA),7,8but the role for DNA looping-dependent targeting of Isw2 in the repression of ncRNA remains unknown. It is possible that canonical and ectopic targets have different specificities for coding and ncRNA. Finally, it is unknown how dynamic DNA looping-dependent Isw2 targeting is. It is likely that DNA looping is a far more dynamic and transient process than TF binding to its recognition sites. If this were the case, DNA looping-dependent Isw2 targeting may lead to more variable chromatin remodeling at ectopic targets within a cell population, which would lead to variable transcriptional repression. Investigating biological functions of DNA looping-dependent Isw2 targeting will likely reveal novel aspects of chromatin regulation. Figure 1. Two distinct mechanisms of TF-dependent Isw2 targeting. Transcription factor Ume6 can target Isw2 to the vicinity of its binding sites via physical interactions (canonical targets) or by TFIIB- and Ume6-dependent DNA looping (ectopic targets).

Intrinsically disordered regions (IDRs) of proteins remain understudied with enigmatic sequence features relevant to their functions. Members of the myotubularin-related protein (MTMR) family contain uncharacterized IDRs. After decades of research on their phosphatase activity, recent work on the C-terminal IDRs of MTMR7 revealed new interactions and important new functions beyond the phosphatase function. Here we take a broader look at the C-terminal domains (CTDs) of 14 human MTMRs and use bioinformatic tools and biophysical methods to ask which other functions may be probable in this protein family. The predictions show that the CTDs are disordered and carry short linear motifs (SLiMs) important for targeting of MTMRs to defined subcellular compartments and implicating them in signaling, phase separation, interaction with diverse proteins, including transcription factors and are of relevance for cancer research and neuroscience. We also present experimental methods to study the CTDs and use them to characterize the coiled coil (CC) domains of MTMR7 and MTMR9. We show homo- and hetero-oligomerization with preference for MTMR7-CC to form dimers, while MTMR9-CC forms trimers. We relate the results to sequence features and make predictions for the structural landscape of other MTMRs. Our work gives a broad insight into the so far unrecognized features and SLiMs in MTMR-CTDs, and provides the basis for more in-depth experimental research on this diverse protein family and understudied IDRs in proteins in general.

The Statistical Inference of Function Through Evolutionary Relationships (SIFTER) framework uses a statistical graphical model that applies phylogenetic principles to automate precise protein function prediction. Here we present a revised approach (SIFTER version 2.0) that enables annotations on a genomic scale. SIFTER 2.0 produces equivalently precise predictions compared to the earlier version on a carefully studied family and on a collection of 100 protein families. We have added an approximation method to SIFTER 2.0 and show a 500-fold improvement in speed with minimal impact on prediction results in the functionally diverse sulfotransferase protein family. On the Nudix protein family, previously inaccessible to the SIFTER framework because of the 66 possible molecular functions, SIFTER achieved 47.4% accuracy on experimental data (where BLAST achieved 34.0%). Finally, we used SIFTER to annotate all of the Schizosaccharomyces pombe proteins with experimental functional characterizations, based on annotations from proteins in 46 fungal genomes. SIFTER precisely predicted molecular function for 45.5% of the characterized proteins in this genome, as compared with four current function prediction methods that precisely predicted function for 62.6%, 30.6%, 6.0%, and 5.7% of these proteins. We use both precision-recall curves and ROC analyses to compare these genome-scale predictions across the different methods and to assess performance on different types of applications. SIFTER 2.0 is capable of predicting protein molecular function for large and functionally diverse protein families using an approximate statistical model, enabling phylogenetics-based protein function prediction for genome-wide analyses. The code for SIFTER and protein family data are available at http://sifter.berkeley.edu.

DNA-damage formation and repair are coupled to the structure and accessibility of DNA in chromatin. DNA damage may compromise protein binding, thereby affecting function. We have studied the effect of TATA-binding protein (TBP) on damage formation by ultraviolet light and on DNA repair by photolyase and nucleotide excision repair in yeast and in vitro. In vivo, selective and enhanced formation of (6-4)-photoproducts (6-4PPs) was found within the TATA boxes of the active SNR6 and GAL10 genes, engaged in transcription initiation by RNA polymerase III and RNA polymerase II, respectively. Cyclobutane pyrimidine dimers (CPDs) were generated at the edge and outside of the TATA boxes, and in the inactive promoters. The same selective and enhanced 6-4PP formation was observed in a TBP-TATA complex in vitro at sites where crystal structures revealed bent DNA. We conclude that similar DNA distortions occur in vivo when TBP is part of the initiation complexes. Repair analysis by photolyase revealed inhibition of CPD repair at the edge of the TATA box in the active SNR6 promoter in vitro, but not in the GAL10 TATA box or in the inactive SNR6 promoter. Nucleotide excision repair was not inhibited, but preferentially repaired the 6-4PPs. We conclude that TBP can remain bound to damaged promoters and that nucleotide excision repair is the predominant pathway to remove UV damage in active TATA boxes.

DNA folding is not desirable for solid-state nanopore techniques when analyzing the interaction of a biomolecule with its specific binding sites on DNA since the signal derived from the binding site could be buried by a large signal from the folding of DNA nearby. To resolve the problems associated with DNA folding, ionic liquids (ILs), which are known to interact with DNA through charge-charge and hydrophobic interactions are employed. 1-n-butyl-3-methylimidazolium chloride (C4 mim) is found to be the most effective in lowering the incident of DNA folding during its translocation through solid-state nanopores (4-5 nm diameter). The rate of folding signals from the translocation of DNA-C4 mim is decreased by half in comparison to that from the control bare DNA. The conformational changes of DNA upon complexation with C4 mim are further examined using atomic force microscopy, showing that the entanglement of DNA which is common in bare DNA is not observed when treated with C4 mim. The stretching effect of C4 mim on DNA strands improves the detection accuracy of nanopore for identifying the location of zinc finger protein bound to its specific binding site in DNA by lowering the incident of DNA folding.

Multiple interactions between SRm160 and SR family proteins in enhancer-dependent splicing and development of C. elegans

Australian elapid snakes are among the most venomous in the world. Their venoms contain multiple components that target blood hemostasis, neuromuscular signaling, and the cardiovascular system. We describe here a comprehensive approach to separation and identification of the venom proteins from 18 of these snake species, representing nine genera. The venom protein components were separated by two-dimensional PAGE and identified using mass spectrometry and de novo peptide sequencing. The venoms are complex mixtures showing up to 200 protein spots varying in size from <7 to over 150 kDa and in pI from 3 to >10. These include many proteins identified previously in Australian snake venoms, homologs identified in other snake species, and some novel proteins. In many cases multiple trains of spots were typically observed in the higher molecular mass range (>20 kDa) (indicative of post-translational modification). Venom proteins and their post-translational modifications were characterized using specific antibodies, phosphoprotein- and glycoprotein-specific stains, enzymatic digestion, lectin binding, and antivenom reactivity. In the lower molecular weight range, several proteins were identified, but the predominant species were phospholipase A2 and alpha-neurotoxins, both represented by different sequence variants. The higher molecular weight range contained proteases, nucleotidases, oxidases, and homologs of mammalian coagulation factors. This information together with the identification of several novel proteins (metalloproteinases, vespryns, phospholipase A2 inhibitors, protein-disulfide isomerase, 5'-nucleotidases, cysteine-rich secreted proteins, C-type lectins, and acetylcholinesterases) aids in understanding the lethal mechanisms of elapid snake venoms and represents a valuable resource for future development of novel human therapeutics.

The Impact of Gene Location in the Nucleus on Transcriptional Regulation

The Shc family of adaptor proteins is a group of proteins that lacks intrinsic enzymatic activity. Instead, Shc proteins possess various domains that allow them to recruit different signalling molecules. Shc proteins help to transduce an extracellular signal into an intracellular signal, which is then translated into a biological response. The Shc family of adaptor proteins share the same structural topography, CH2-PTB-CH1-SH2, which is more than an isoform of Shc family proteins; this structure, which includes multiple domains, allows for the posttranslational modification of Shc proteins and increases the functional diversity of Shc proteins. The deregulation of Shc proteins has been linked to different disease conditions, including cancer and Alzheimer’s, which indicates their key roles in cellular functions. Accordingly, a question might arise as to whether Shc proteins could be targeted therapeutically to correct their disturbance. To answer this question, thorough knowledge must be acquired; herein, we aim to shed light on the Shc family of adaptor proteins to understand their intracellular role in normal and disease states, which later might be applied to connote mechanisms to reverse the disease state.

Chromatin plays a crucial role in genome compaction and is fundamental for regulating multiple nuclear processes. Nucleosomes, the basic building blocks of chromatin, are central in regulating these processes, determining chromatin accessibility by limiting access to DNA for various proteins and acting as important signaling hubs. The association of histones with DNA in nucleosomes and the folding of chromatin into higher-order structures are strongly influenced by a variety of epigenetic marks, including DNA methylation, histone variants, and histone post-translational modifications. Additionally, a wide array of chaperones and ATP-dependent remodelers regulate various aspects of nucleosome biology, including assembly, deposition, and positioning. This review provides an overview of recent advances in our mechanistic understanding of how nucleosomes and chromatin organization are regulated by epigenetic marks and remodelers in plants. Furthermore, we present current technologies for profiling chromatin accessibility and organization.

Genome compaction is one of the important subject areas for understanding the mechanisms regulating genes' expression and DNA replication and repair. The basic unit of DNA compaction in the eukaryotic cell is the nucleosome. The main chromatin proteins responsible for DNA compaction have already been identified, but the regulation of chromatin architecture is still extensively studied. Several authors have shown an interaction of ARTD proteins with nucleosomes and proposed that there are changes in the nucleosomes' structure as a result. In the ARTD family, only PARP1, PARP2, and PARP3 participate in the DNA damage response. Damaged DNA stimulates activation of these PARPs, which use NAD+ as a substrate. DNA repair and chromatin compaction need precise regulation with close coordination between them. In this work, we studied the interactions of these three PARPs with nucleosomes by atomic force microscopy, which is a powerful method allowing for direct measurements of geometric characteristics of single molecules. Using this method, we evaluated perturbations in the structure of single nucleosomes after the binding of a PARP. We demonstrated here that PARP3 significantly alters the geometry of nucleosomes, possibly indicating a new function of PARP3 in chromatin compaction regulation.

Modification of calcite crystal growth by abalone shell proteins: an atomic force microscope study