The Gene-Silencing Protein MORC-1 Topologically Entraps DNA and Forms Multimeric Assemblies to Cause DNA Compaction.

Microrchidia (MORC) ATPases are critical for gene silencing and chromatin compaction in multiple eukaryotic systems, but the mechanisms by which MORC proteins act are poorly understood. Here, we apply a series of biochemical, single-molecule, and cell-based imaging approaches to better understand the function of the Caenorhabditis elegans MORC-1 protein. We find that MORC-1 binds to DNA in a length-dependent but sequence non-specific manner and compacts DNA by forming DNA loops. MORC-1 molecules diffuse along DNA but become static as they grow into foci that are topologically entrapped on DNA. Consistent with the observed MORC-1 multimeric assemblies, MORC-1 forms nuclear puncta in cells and can also form phase-separated droplets in vitro. We also demonstrate that MORC-1 compacts nucleosome templates. These results suggest that MORCs affect genome structure and gene silencing by forming multimeric assemblages to topologically entrap and progressively loop and compact chromatin.

Post-translational modifications on RNA-binding proteins: accelerators, brakes, or passengers in neurodegeneration?

RNA-binding proteins (RBPs) are critical players in RNA expression and metabolism, thus, the proper regulation of this class of proteins is critical for cellular health. Regulation of RBPs often occurs through post-translational modifications (PTMs), which allow the cell to quickly and efficiently respond to cellular and environmental stimuli. PTMs have recently emerged as important regulators of RBPs implicated in neurodegenerative disorders, in particular amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here, we summarize how disease-associated PTMs influence the biophysical properties, molecular interactions, subcellular localization, and function of ALS/FTD-linked RBPs, such as FUS and TDP-43. We will discuss how PTMs are believed to play pathological, protective, or ambiguous roles in these neurodegenerative disorders.

A conserved signaling pathway activates bacterial CBASS immune signaling in response to DNA damage.

To protect themselves from the constant threat of bacteriophage (phage) infection, bacteria have evolved diverse immune systems including restriction-modification, CRISPR-Cas, and many others. Here, we describe the discovery of a two-protein transcriptional regulator module associated with hundreds of CBASS immune systems and demonstrate that this module drives the expression of its associated CBASS system in response to DNA damage. We show that the helix-turn-helix transcriptional repressor CapH binds the promoter region of its associated CBASS system to repress transcription until it is cleaved by the metallopeptidase CapP. CapP is activated in vitro by single-stranded DNA, and in cells by DNA-damaging drugs. Together, CapH and CapP drive increased expression of their associated CBASS system in response to DNA damage. We identify CapH- and CapP-related proteins associated with diverse known and putative bacterial immune systems including DISARM and Pycsar antiphage operons. Overall, our data highlight a mechanism by which bacterial immune systems can sense and respond to a universal signal of cell stress, potentially enabling multiple immune systems to mount a coordinated defensive response against an invading pathogen.

DNAJA2 and Hero11 mediate similar conformational extension and aggregation suppression of TDP-43.

Many RNA binding proteins (RBPs) contain low-complexity domains (LCDs) with prion-like compositions. These long intrinsically disordered regions regulate their solubility, contributing to their physiological roles in RNA processing and organization. However, this also makes these RBPs prone to pathological misfolding and aggregation that are characteristic of neurodegenerative diseases. For example, TAR DNA-binding protein 43 (TDP-43) forms pathological aggregates associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). While molecular chaperones are well-known suppressors of these aberrant events, we recently reported that highly disordered, hydrophilic and charged heat-resistant obscure (Hero) proteins may have similar effects. Specifically, Hero proteins can maintain the activity of other proteins from denaturing conditions in vitro, while their overexpression can suppress cellular aggregation and toxicity associated with aggregation-prone proteins. However, it is unclear how these protective effects are achieved. Here, we utilized single-molecule FRET to monitor the conformations of the aggregation-prone prion-like LCD of TDP-43. While we observed high conformational heterogeneity in wild-type LCD, the ALS-associated mutation A315T promoted collapsed conformations. In contrast, an Hsp40 chaperone, DNAJA2, and a Hero protein, Hero11 stabilized extended states of the LCD, consistent with their ability to suppress the aggregation of TDP-43. Our results link single-molecule effects on conformation to macro effects on bulk aggregation, where a Hero protein, like a chaperone, can maintain the conformational integrity of a client protein to prevent its aggregation.

Improved prediction of DNA and RNA binding proteins with deep learning models.

Nucleic acid-binding proteins (NABPs), including DNA-binding proteins (DBPs) and RNA-binding proteins (RBPs), play important roles in essential biological processes. To facilitate functional annotation and accurate prediction of different types of NABPs, many machine learning-based computational approaches have been developed. However, the datasets used for training and testing as well as the prediction scopes in these studies have limited their applications. In this paper, we developed new strategies to overcome these limitations by generating more accurate and robust datasets and developing deep learning-based methods including both hierarchical and multi-class approaches to predict the types of NABPs for any given protein. The deep learning models employ two layers of convolutional neural network and one layer of long short-term memory. Our approaches outperform existing DBP and RBP predictors with a balanced prediction between DBPs and RBPs, and are more practically useful in identifying novel NABPs. The multi-class approach greatly improves the prediction accuracy of DBPs and RBPs, especially for the DBPs with ~12% improvement. Moreover, we explored the prediction accuracy of single-stranded DNA binding proteins and their effect on the overall prediction accuracy of NABP predictions.

DNA-binding and protein structure of nuclear factors likely acting in genetic information processing in the Paulinella chromatophore.

The chromatophores in Paulinella are evolutionary-early-stage photosynthetic organelles. Biological processes in chromatophores depend on a combination of chromatophore and nucleus-encoded proteins. Interestingly, besides proteins carrying chromatophore-targeting signals, a large arsenal of short chromatophore-targeted proteins (sCTPs; <90 amino acids) without recognizable targeting signals were found in chromatophores. This situation resembles endosymbionts in plants and insects that are manipulated by host-derived antimicrobial peptides. Previously, we identified an expanded family of sCTPs of unknown function, named here "DNA-binding (DB)-sCTPs". DB-sCTPs contain a ~45 amino acid motif that is conserved in some bacterial proteins with predicted functions in DNA processing. Here, we explored antimicrobial activity, DNA-binding capacity, and structures of three purified recombinant DB-sCTPs. All three proteins exhibited antimicrobial activity against bacteria involving membrane permeabilization, and bound to bacterial lipids in vitro. A combination of in vitro assays demonstrated binding of recombinant DB-sCTPs to chromatophore-derived genomic DNA sequences with an affinity in the low nM range. Additionally, we report the 1.2 Å crystal structure of one DB-sCTP. In silico docking studies suggest that helix α2 inserts into the DNA major grove and the exposed residues, that are highly variable between different DB-sCTPs, confer interaction with the DNA bases. Identification of photosystem II subunit CP43 as a potential interaction partner of one DB-sCTP, suggests DB-sCTPs to be involved in more complex regulatory mechanisms. We hypothesize that membrane binding of DB-sCTPs is related to their import into chromatophores. Once inside, they interact with the chromatophore genome potentially providing nuclear control over genetic information processing.

Arid5a uses disordered extensions of its core ARID domain for distinct DNA- and RNA-recognition and gene regulation.

AT-rich interacting domain (ARID)-containing proteins, Arids, are a heterogeneous DNA-binding protein family involved in transcription regulation and chromatin processing. For the member Arid5a, no exact DNA-binding preference has been experimentally defined so far. Additionally, the protein binds to mRNA motifs for transcript stabilization, supposedly through the DNA-binding ARID domain. To date, however, no unbiased RNA motif definition and clear dissection of nucleic acid-binding through the ARID domain have been undertaken. Using NMR-centered biochemistry, we here define the Arid5a DNA preference. Further, high-throughput in vitro binding reveals a consensus RNA-binding motif engaged by the core ARID domain. Finally, transcriptome-wide binding (iCLIP2) reveals that Arid5a has a weak preference for (A)U-rich regions in pre-mRNA transcripts of factors related to RNA processing. We find that the intrinsically disordered regions flanking the ARID domain modulate the specificity and affinity of DNA binding, while they appear crucial for RNA interactions. Ultimately, our data suggest that Arid5a uses its extended ARID domain for bifunctional gene regulation and that the involvement of IDR extensions is a more general feature of Arids in interacting with different nucleic acids at the chromatin-mRNA interface.

Borrelia burgdorferi PlzA is a cyclic-di-GMP dependent DNA and RNA binding protein.

The PilZ domain-containing protein, PlzA, is the only known cyclic di-GMP binding protein encoded by all Lyme disease spirochetes. PlzA has been implicated in the regulation of many borrelial processes, but the effector mechanism of PlzA was not previously known. Here, we report that PlzA can bind DNA and RNA and that nucleic acid binding requires c-di-GMP, with the affinity of PlzA for nucleic acids increasing as concentrations of c-di-GMP were increased. A mutant PlzA that is incapable of binding c-di-GMP did not bind to any tested nucleic acids. We also determined that PlzA interacts predominantly with the major groove of DNA and that sequence length and G-C content play a role in DNA binding affinity. PlzA is a dual-domain protein with a PilZ-like N-terminal domain linked to a canonical C-terminal PilZ domain. Dissection of the domains demonstrated that the separated N-terminal domain bound nucleic acids independently of c-di-GMP. The C-terminal domain, which includes the c-di-GMP binding motifs, did not bind nucleic acids under any tested conditions. Our data are supported by computational docking, which predicts that c-di-GMP binding at the C-terminal domain stabilizes the overall protein structure and facilitates PlzA-DNA interactions via residues in the N-terminal domain. Based on our data, we propose that levels of c-di-GMP during the various stages of the enzootic life cycle direct PlzA binding to regulatory targets.

StackDPP: a stacking ensemble based DNA-binding protein prediction model.

BACKGROUND: DNA-binding proteins (DNA-BPs) are the proteins that bind and interact with DNA. DNA-BPs regulate and affect numerous biological processes, such as, transcription and DNA replication, repair, and organization of the chromosomal DNA. Very few proteins, however, are DNA-binding in nature. Therefore, it is necessary to develop an efficient predictor for identifying DNA-BPs. RESULT: In this work, we have proposed new benchmark datasets for the DNA-binding protein prediction problem. We discovered several quality concerns with the widely used benchmark datasets, PDB1075 (for training) and PDB186 (for independent testing), which necessitated the preparation of new benchmark datasets. Our proposed datasets UNIPROT1424 and UNIPROT356 can be used for model training and independent testing respectively. We have retrained selected state-of-the-art DNA-BP predictors in the new dataset and reported their performance results. We also trained a novel predictor using the new benchmark dataset. We extracted features from various feature categories, then used a Random Forest classifier and Recursive Feature Elimination with Cross-validation (RFECV) to select the optimal set of 452 features. We then proposed a stacking ensemble architecture as our final prediction model. Named Stacking Ensemble Model for DNA-binding Protein Prediction, or StackDPP in short, our model achieved 0.92, 0.92 and 0.93 accuracy in 10-fold cross-validation, jackknife and independent testing respectively. CONCLUSION: StackDPP has performed very well in cross-validation testing and has outperformed all the state-of-the-art prediction models in independent testing. Its performance scores in cross-validation testing generalized very well in the independent test set. The source code of the model is publicly available at https://github.com/HasibAhmed1624/StackDPP . Therefore, we expect this generalized model can be adopted by researchers and practitioners to identify novel DNA-binding proteins.

A CsoR family transcriptional regulator, TTHA1953, controls the sulfur oxidation pathway in Thermus thermophilus HB8.

Transcription regulation is a critical means by which microorganisms sense and adapt to their environments. Bacteria contain a wide range of highly conserved families of transcription factors that have evolved to regulate diverse sets of genes. It is increasingly apparent that structural similarities between transcription factors do not always equate to analogous transcription regulatory networks. For example, transcription factors within the copper-sensing operon repressor (CsoR)-resistance to cobalt and nickel repressor family have been found to repress a wide range of gene targets, including various metal efflux genes, as well as genes involved in sulfide and formaldehyde detoxification machinery. In this study, we identify the preferred DNA-binding sequence for the CsoR-like protein, TTHA1953, from the model extremophile Thermus thermophilus HB8 using the iterative selection approach, restriction endonuclease, protection, selection, and amplification. By mapping significant DNA motifs to the T. thermophilus HB8 genome, we identify potentially regulated genes that we validate with in vitro and in vivo methodologies. We establish TTHA1953 as a master regulator of the sulfur oxidation pathway, providing the first link between CsoR-like proteins and Sox regulation.

Prioritizing cardiovascular disease-associated variants altering NKX2-5 and TBX5 binding through an integrative computational approach.

Cardiovascular diseases (CVDs) are the leading cause of death worldwide and are heavily influenced by genetic factors. Genome-wide association studies have mapped >90% of CVD-associated variants within the noncoding genome, which can alter the function of regulatory proteins, such as transcription factors (TFs). However, due to the overwhelming number of single-nucleotide polymorphisms (SNPs) (>500,000) in genome-wide association studies, prioritizing variants for in vitro analysis remains challenging. In this work, we implemented a computational approach that considers support vector machine (SVM)-based TF binding site classification and cardiac expression quantitative trait loci (eQTL) analysis to identify and prioritize potential CVD-causing SNPs. We identified 1535 CVD-associated SNPs within TF footprints and putative cardiac enhancers plus 14,218 variants in linkage disequilibrium with genotype-dependent gene expression in cardiac tissues. Using ChIP-seq data from two cardiac TFs (NKX2-5 and TBX5) in human-induced pluripotent stem cell-derived cardiomyocytes, we trained a large-scale gapped k-mer SVM model to identify CVD-associated SNPs that altered NKX2-5 and TBX5 binding. The model was tested by scoring human heart TF genomic footprints within putative enhancers and measuring in vitro binding through electrophoretic mobility shift assay. Five variants predicted to alter NKX2-5 (rs59310144, rs6715570, and rs61872084) and TBX5 (rs7612445 and rs7790964) binding were prioritized for in vitro validation based on the magnitude of the predicted change in binding and are in cardiac tissue eQTLs. All five variants altered NKX2-5 and TBX5 DNA binding. We present a bioinformatic approach that considers tissue-specific eQTL analysis and SVM-based TF binding site classification to prioritize CVD-associated variants for in vitro analysis.

Dysregulation of the progranulin-driven autophagy-lysosomal pathway mediates secretion of the nuclear protein TDP-43.

The cytoplasmic accumulation of the nuclear protein transactive response DNA-binding protein 43 kDa (TDP-43) has been linked to the progression of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. TDP-43 secreted into the extracellular space has been suggested to contribute to the cell-to-cell spread of the cytoplasmic accumulation of TDP-43 throughout the brain; however, the underlying mechanisms remain unknown. We herein demonstrated that the secretion of TDP-43 was stimulated by the inhibition of the autophagy-lysosomal pathway driven by progranulin (PGRN), a causal protein of frontotemporal lobar degeneration. Among modulators of autophagy, only vacuolar-ATPase inhibitors, such as bafilomycin A1 (Baf), increased the levels of the full-length and cleaved forms of TDP-43 and the autophagosome marker LC3-II (microtubule-associated proteins 1A/1B light chain 3B) in extracellular vesicle fractions prepared from the culture media of HeLa, SH-SY5Y, or NSC-34 cells, whereas vacuolin-1, MG132, chloroquine, rapamycin, and serum starvation did not. The C-terminal fragment of TDP-43 was required for Baf-induced TDP-43 secretion. The Baf treatment induced the translocation of the aggregate-prone GFP-tagged C-terminal fragment of TDP-43 and mCherry-tagged LC3 to the plasma membrane. The Baf-induced secretion of TDP-43 was attenuated in autophagy-deficient ATG16L1 knockout HeLa cells. The knockdown of PGRN induced the secretion of cleaved TDP-43 in an autophagy-dependent manner in HeLa cells. The KO of PGRN in mouse embryonic fibroblasts increased the secretion of the cleaved forms of TDP-43 and LC3-II. The treatment inducing TDP-43 secretion increased the nuclear translocation of GFP-tagged transcription factor EB, a master regulator of the autophagy-lysosomal pathway in SH-SY5Y cells. These results suggest that the secretion of TDP-43 is promoted by dysregulation of the PGRN-driven autophagy-lysosomal pathway.

Mechanistic insights into the nickel-dependent allosteric response of the Helicobacter pylori NikR transcription factor.

In Helicobacter pylori, the nickel-responsive NikR transcription factor plays a key role in regulating intracellular nickel concentrations, which is an essential process for survival of this pathogen in the acidic human stomach. Nickel binding to H. pylori NikR (HpNikR) allosterically activates DNA binding to target promoters encoding genes involved in nickel homeostasis and acid adaptation, to either activate or repress their transcription. We previously showed that HpNikR adopts an equilibrium between an open conformation and DNA-binding competent cis and trans states. Nickel binding slows down conformational exchange between these states and shifts the equilibrium toward the binding-competent states. The protein then becomes stabilized in a cis conformation upon binding the ureA promoter. Here, we investigate how nickel binding creates this response and how it is transmitted to the DNA-binding domains. Through mutagenesis, DNA-binding studies, and computational methods, the allosteric response to nickel was found to be propagated from the nickel-binding sites to the DNA-binding domains via the ß-sheets of the metal-binding domain and a network of residues at the inter-domain interface. Our computational results suggest that nickel binding increases protein rigidity to slow down the conformational exchange. A thymine base in the ureA promoter sequence, known to be critical for high affinity DNA binding by HpNikR, was also found to be important for the allosteric response, while a modified version of this promoter further highlighted the importance of the DNA sequence in modulating the response. Collectively, our results provide insights into regulation of a key protein for H. pylori survival.

Development of in-line anoxic small-angle X-ray scattering and structural characterization of an oxygen-sensing transcriptional regulator.

Oxygen-sensitive metalloenzymes are responsible for many of the most fundamental biochemical processes in nature, from the reduction of dinitrogen in nitrogenase to the biosynthesis of photosynthetic pigments. However, biophysical characterization of such proteins under anoxic conditions can be challenging, especially at noncryogenic temperatures. In this study, we introduce the first in-line anoxic small-angle X-ray scattering (anSAXS) system at a major national synchrotron source, featuring both batch-mode and chromatography-mode capabilities. To demonstrate chromatography-coupled anSAXS, we investigated the oligomeric interconversions of the fumarate and nitrate reduction (FNR) transcription factor, which is responsible for the transcriptional response to changing oxygen conditions in the facultative anaerobe Escherichia coli. Previous work has shown that FNR contains a labile [4Fe-4S] cluster that is degraded when oxygen is present and that this change in cluster composition leads to the dissociation of the DNA-binding dimeric form. Using anSAXS, we provide the first direct structural evidence for the oxygen-induced dissociation of the E. coli FNR dimer and its correlation with cluster composition. We further demonstrate how complex FNR-DNA interactions can be studied by investigating the promoter region of the anaerobic ribonucleotide reductase genes, nrdDG, which contains tandem FNR-binding sites. By coupling size-exclusion chromatography-anSAXS with full-spectrum UV-Vis analysis, we show that the [4Fe-4S] cluster-containing dimeric form of FNR can bind to both sites in the nrdDG promoter region. The development of in-line anSAXS greatly expands the toolbox available for the study of complex metalloproteins and provides a foundation for future expansions.

Interaction with single-stranded DNA-binding protein modulates Escherichia coli RadD DNA repair activities.

The bacterial RadD enzyme is important for multiple genome maintenance pathways, including RecA DNA strand exchange and RecA-independent suppression of DNA crossover template switching. However, much remains unknown about the precise roles of RadD. One potential clue into RadD mechanisms is its direct interaction with the single-stranded DNA binding protein (SSB), which coats single-stranded DNA exposed during genome maintenance reactions in cells. Interaction with SSB stimulates the ATPase activity of RadD. To probe the mechanism and importance of RadD-SSB complex formation, we identified a pocket on RadD that is essential for binding SSB. In a mechanism shared with many other SSB-interacting proteins, RadD uses a hydrophobic pocket framed by basic residues to bind the C-terminal end of SSB. We found that RadD variants that substitute acidic residues for basic residues in the SSB binding site impair RadD:SSB complex formation and eliminate SSB stimulation of RadD ATPase activity in vitro. Additionally, mutant Escherichia coli strains carrying charge reversal radD changes display increased sensitivity to DNA damaging agents synergistically with deletions of radA and recG, although the phenotypes of the SSB-binding radD mutants are not as severe as a full radD deletion. This suggests that cellular RadD requires an intact interaction with SSB for full RadD function.

Fanconi anemia-associated mutation in RAD51 compromises the coordinated action of DNA-binding and ATPase activities.

Fanconi anemia (FA) is a rare genetic disease caused by a defect in DNA repair pathway for DNA interstrand crosslinks. These crosslinks can potentially impede the progression of the DNA replication fork, consequently leading to DNA double-strand breaks. Heterozygous RAD51-Q242R mutation has been reported to cause FA-like symptoms. However, the molecular defect of RAD51 underlying the disease is largely unknown. In this study, we conducted a biochemical analysis of RAD51-Q242R protein, revealing notable deficiencies in its DNA-dependent ATPase activity and its ATP-dependent regulation of DNA-binding activity. Interestingly, although RAD51-Q242R exhibited the filament instability and lacked the ability to form displacement loop, it efficiently stimulated the formation of displacement loops mediated by wild-type RAD51. These findings facilitate understanding of the biochemical properties of the mutant protein and how RAD51 works in the FA patient cells.

The bacterial nucleoid-associated proteins, HU and Dps, condense DNA into context-dependent biphasic or multiphasic complex coacervates.

The bacterial chromosome, known as its nucleoid, is an amorphous assemblage of globular nucleoprotein domains. It exists in a state of phase separation from the cell's cytoplasm, as an irregularly-shaped, membrane-less, intracellular compartment. This state (the nature of which remains largely unknown) is maintained through bacterial generations ad infinitum. Here, we show that HU and Dps, two of the most abundant nucleoid-associated proteins (NAPs) of Escherichia coli, undergo spontaneous complex coacervation with different forms of DNA/RNA, both individually and in each other's presence, to cause accretion and compaction of DNA/RNA into liquid-liquid phase separated condensates in vitro. Upon mixing with nucleic acids, HU-A and HU-B form (a) biphasic heterotypic mixed condensates in which HU-B helps to lower the Csat of HU-A and also (b) multiphasic heterotypic condensates, with Dps, in which demixed domains display different contents of HU and Dps. We believe that these modes of complex coacervation that are seen in vitro can serve as models for the in vivo relationships among NAPs in nucleoids, involving local and global variations in the relative abundances of the different NAPs, especially in demixed subdomains that are characterized by differing grades of phase separation. Our results clearly demonstrate some quantitative, and some qualitative, differences in the coacervating abilities of different NAPs with DNA, potentially explaining (i) why E. coli has two isoforms of HU, and (ii) why changes in the abundances of HU and Dps facilitate the lag, logarithmic, and stationary phases of E. coli growth.

Transcription factor binding specificities of the oomycete Phytophthora infestans reflect conserved and divergent evolutionary patterns and predict function.

BACKGROUND: Identifying the DNA-binding specificities of transcription factors (TF) is central to understanding gene networks that regulate growth and development. Such knowledge is lacking in oomycetes, a microbial eukaryotic lineage within the stramenopile group. Oomycetes include many important plant and animal pathogens such as the potato and tomato blight agent Phytophthora infestans, which is a tractable model for studying life-stage differentiation within the group. RESULTS: Mining of the P. infestans genome identified 197 genes encoding proteins belonging to 22 TF families. Their chromosomal distribution was consistent with family expansions through unequal crossing-over, which were likely ancient since each family had similar sizes in most oomycetes. Most TFs exhibited dynamic changes in RNA levels through the P. infestans life cycle. The DNA-binding preferences of 123 proteins were assayed using protein-binding oligonucleotide microarrays, which succeeded with 73 proteins from 14 families. Binding sites predicted for representatives of the families were validated by electrophoretic mobility shift or chromatin immunoprecipitation assays. Consistent with the substantial evolutionary distance of oomycetes from traditional model organisms, only a subset of the DNA-binding preferences resembled those of human or plant orthologs. Phylogenetic analyses of the TF families within P. infestans often discriminated clades with canonical and novel DNA targets. Paralogs with similar binding preferences frequently had distinct patterns of expression suggestive of functional divergence. TFs were predicted to either drive life stage-specific expression or serve as general activators based on the representation of their binding sites within total or developmentally-regulated promoters. This projection was confirmed for one TF using synthetic and mutated promoters fused to reporter genes in vivo. CONCLUSIONS: We established a large dataset of binding specificities for P. infestans TFs, representing the first in the stramenopile group. This resource provides a basis for understanding transcriptional regulation by linking TFs with their targets, which should help delineate the molecular components of processes such as sporulation and host infection. Our work also yielded insight into TF evolution during the eukaryotic radiation, revealing both functional conservation as well as diversification across kingdoms.

CK1δ/ε kinases regulate TDP-43 phosphorylation and are therapeutic targets for ALS-related TDP-43 hyperphosphorylation.

Hyperphosphorylated TAR DNA-binding protein 43 (TDP-43) aggregates in the cytoplasm of neurons is the neuropathological hallmark of amyotrophic lateral sclerosis (ALS) and a group of neurodegenerative diseases collectively referred to as TDP-43 proteinopathies that includes frontotemporal dementia, Alzheimer's disease, and limbic onset age-related TDP-43 encephalopathy. The mechanism of TDP-43 phosphorylation is poorly understood. Previously we reported casein kinase 1 epsilon gene (CSNK1E gene encoding CK1ε protein) as being tightly correlated with phosphorylated TDP-43 (pTDP-43) pathology. Here we pursued studies to investigate in cellular models and in vitro how CK1ε and CK1δ (a closely related family sub-member) mediate TDP-43 phosphorylation in disease. We first validated the binding interaction between TDP-43 and either CK1δ and CK1ε using kinase activity assays and predictive bioinformatic database. We utilized novel inducible cellular models that generated translocated phosphorylated TDP-43 (pTDP-43) and cytoplasmic aggregation. Reducing CK1 kinase activity with siRNA or small molecule chemical inhibitors resulted in significant reduction of pTDP-43, in both soluble and insoluble protein fractions. We also established CK1δ and CK1ε are the primary kinases that phosphorylate TDP-43 compared to CK2α, CDC7, ERK1/2, p38α/MAPK14, and TTBK1, other identified kinases that have been implicated in TDP-43 phosphorylation. Throughout our studies, we were careful to examine both the soluble and insoluble TDP-43 protein fractions, the critical protein fractions related to protein aggregation diseases. These results identify CK1s as critical kinases involved in TDP-43 hyperphosphorylation and aggregation in cellular models and in vitro, and in turn are potential therapeutic targets by way of CK1δ/ε inhibitors.

Transcription factor-like 5 is a potential DNA- and RNA-binding protein essential for maintaining male fertility in mice.

Transcription factor-like 5 (TCFL5) is a testis-specific protein that contains the basic helix-loop-helix domain, but the in vivo functions of TCFL5 remain unknown. Herein, we generated CRISPR/Cas9-mediated knockout mice to dissect the function of TCFL5 in mouse testes. Surprisingly, we found that it was difficult to generate homozygous mice with the Tcfl5 deletion as the heterozygous males (Tcfl5+/-) were infertile. However, we did observe markedly abnormal phenotypes of spermatids and spermatozoa in the testes and epididymides of Tcfl5+/- mice. Mechanistically, we demonstrated that TCFL5 transcriptionally and post-transcriptionally regulated a set of genes participating in male germ cell development via TCFL5 ChIP-DNA and eCLIP-RNA high-throughput sequencing. We also identified a known RNA-binding protein, FXR1, as an interacting partner of TCFL5 that may coordinate the transition and localization of TCFL5 in the nucleus. Collectively, we herein report for the first time that Tcfl5 is haploinsufficient in vivo and acts as a dual-function protein that mediates DNA and RNA to regulate spermatogenesis. This article has an associated First Person interview with the first author of the paper.