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Alu retrotransposons, which form the largest family of mobile DNA elements in the human genome, have recently come to attention as a potential source of regulatory novelties, most notably by participating in enhancer function. Even though Alu transcription by RNA polymerase III is subjected to tight epigenetic silencing, their expression has long been known to increase in response to various types of stress, including viral infection. Here we show that, in primary human fibroblasts, adenovirus small e1a triggered derepression of hundreds of individual Alus by promoting TFIIIB recruitment by Alu-bound TFIIIC. Epigenome profiling revealed an e1a-induced decrease of H3K27 acetylation and increase of H3K4 monomethylation at derepressed Alus, making them resemble poised enhancers. The enhancer nature of e1a-targeted Alus was confirmed by the enrichment, in their upstream regions, of the EP300/CBP acetyltransferase, EP400 chromatin remodeler and YAP1 and FOS transcription factors. The physical interaction of e1a with EP400 was critical for Alu derepression, which was abrogated upon EP400 ablation. Our data suggest that e1a targets a subset of enhancer Alus whose transcriptional activation, which requires EP400 and is mediated by the e1a-EP400 interaction, may participate in the manipulation of enhancer activity by adenoviruses.

Centromeric localization of evolutionarily conserved CENP-A (Cse4 in Saccharomyces cerevisiae) is essential for chromosomal stability. Mislocalization of overexpressed CENP-A to noncentromeric regions contributes to chromosomal instability in yeasts, flies, and humans. Overexpression and mislocalization of CENP-A observed in many cancers are associated with poor prognosis. Previous studies have shown that F-box proteins, Cdc4 and Met30 of the Skp, Cullin, F-box ubiquitin ligase cooperatively regulate proteolysis of Cse4 to prevent Cse4 mislocalization and chromosomal instability under normal physiological conditions. Mck1-mediated phosphorylation of Skp, Cullin, F-box-Cdc4 substrates such as Cdc6 and Rcn1 enhances the interaction of the substrates with Cdc4. Here, we report that Mck1 interacts with Cse4, and Mck1-mediated proteolysis of Cse4 prevents Cse4 mislocalization for chromosomal stability. Our results showed that mck1Δ strain overexpressing CSE4 (GAL-CSE4) exhibits lethality, defects in ubiquitin-mediated proteolysis of Cse4, mislocalization of Cse4, and reduced Cse4-Cdc4 interaction. Strain expressing GAL-cse4-3A with mutations in three potential Mck1 phosphorylation consensus sites (S10, S16, and T166) also exhibits growth defects, increased stability with mislocalization of Cse4-3A, chromosomal instability, and reduced interaction with Cdc4. Constitutive expression of histone H3 (Δ16H3) suppresses the chromosomal instability phenotype of GAL-cse4-3A strain, suggesting that the chromosomal instability phenotype is linked to Cse4-3A mislocalization. We conclude that Mck1 and its three potential phosphorylation sites on Cse4 promote Cse4-Cdc4 interaction and this contributes to ubiquitin-mediated proteolysis of Cse4 preventing its mislocalization and chromosomal instability. These studies advance our understanding of pathways that regulate cellular levels of CENP-A to prevent mislocalization of CENP-A in human cancers.

In frontotemporal lobar degeneration (FTLD), pathological protein aggregation in specific brain regions is associated with declines in human-specialized social-emotional and language functions. In most patients, disease protein aggregates contain either TDP-43 (FTLD-TDP) or tau (FTLD-tau). Here, we explored whether FTLD-associated regional degeneration patterns relate to regional gene expression of human accelerated regions (HARs), conserved sequences that have undergone positive selection during recent human evolution. To this end, we used structural neuroimaging from patients with FTLD and human brain regional transcriptomic data from controls to identify genes expressed in FTLD-targeted brain regions. We then integrated primate comparative genomic data to test our hypothesis that FTLD targets brain regions linked to expression levels of recently evolved genes. In addition, we asked whether genes whose expression correlates with FTLD atrophy are enriched for genes that undergo cryptic splicing when TDP-43 function is impaired. We found that FTLD-TDP and FTLD-tau subtypes target brain regions with overlapping and distinct gene expression correlates, highlighting many genes linked to neuromodulatory functions. FTLD atrophy-correlated genes were strongly enriched for HARs. Atrophy-correlated genes in FTLD-TDP showed greater overlap with TDP-43 cryptic splicing genes and genes with more numerous TDP-43 binding sites compared with atrophy-correlated genes in FTLD-tau. Cryptic splicing genes were enriched for HAR genes, and vice versa, but this effect was due to the confounding influence of gene length. Analyses performed at the individual-patient level revealed that the expression of HAR genes and cryptically spliced genes within putative regions of disease onset differed across FTLD-TDP subtypes. Overall, our findings suggest that FTLD targets brain regions that have undergone recent evolutionary specialization and provide intriguing potential leads regarding the transcriptomic basis for selective vulnerability in distinct FTLD molecular-anatomical subtypes.

Somatic hypermutation in cancer has gained momentum with the increased use of tumour mutation burden as a biomarker for immune checkpoint inhibitors. Spontaneous deamination of 5-methylcytosine to thymine at CpG dinucleotides is one of the most ubiquitous endogenous mutational processes in normal and cancer cells. Here, we performed a systematic investigation of somatic CpG hypermutation at a pan-cancer level. We studied 30,191 cancer patients and 103 cancer types and developed an algorithm to identify somatic CpG hypermutation. Across cancer types, we observed the highest prevalence in paediatric leukaemia (3.5%), paediatric high-grade glioma (1.7%), and colorectal cancer (1%). We discovered germline variants and somatic mutations in the mismatch repair complex MutSα (MSH2-MSH6) as genetic drivers of somatic CpG hypermutation in cancer, which frequently converged on CpG sites and TP53 driver mutations. We further observe an association between somatic CpG hypermutation and response to immune checkpoint inhibitors. Overall, our study identified novel cancer types that display somatic CpG hypermutation, strong association with MutSα-deficiency, and potential utility in cancer immunotherapy.

Chordoma is a rare bone cancer with variable clinical outcomes. Here, we recruited 184 sporadic chordoma patients from the US and Canada and collected their clinical and treatment data. The average age at diagnosis was 45.5 years (Range 5-78) and the chordoma site distribution was 49.2% clivus, 26.2% spinal, and 24.0% sacral. Most patients (97.5%) received surgery as the primary treatment, among whom 85.3% also received additional treatment. Except for the most prevalent cancers like prostate, lung, breast, and skin cancer, there was no discernible enrichment for any specific cancer type among patients or their family members. Among a subset of patients (N = 70) with tumor materials, we conducted omics analyses and obtained targeted panel sequencing and SNP array genotyping data for 51 and 49 patients, respectively. The most recurrent somatic driver mutations included PIK3CA (12%), followed by chromatin remodeling genes PBRM1 and SETD2. Amplification of the 6q27 region, containing the chordoma susceptibility gene TBXT, was detected in eight patients (16.3%). Clival patients appeared to be less likely to carry driver gene mutations, chromosome arm level deletion events (e.g., 5p, 5p, and 9p), or 6q27 amplification compared to sacral patients. After adjusting for age, sex, tumor site, and additional treatment, patients with somatic deletions of 14q (OR = 13.73, 95% CI 1.96-96.02, P = 0.008) and 18p (OR = 13.68, 95% CI 1.77-105.89, P = 0.012) were more likely to have persistent chordoma. The study highlights genomic heterogeneity in chordoma, potentially linked to location and clinical progression.

IntroductionVariants of uncertain significance (VUS) surged with affordable genetic testing, posing challenges for determining pathogenicity. We examine the pathogenicity of a novel VUS P93S in Annexin A11 (ANXA11) - an amyotrophic lateral sclerosis/frontotemporal dementia-associated gene - in a corticobasal syndrome kindred. Established ANXA11 mutations cause ANXA11 aggregation, altered lysosomal-RNA granule co-trafficking, and transactive response DNA binding protein of 43 kDa (TDP-43) mis-localization.MethodsWe described the clinical presentation and explored the phenotypic diversity of ANXA11 variants. P93S's effect on ANXA11 function and TDP-43 biology was characterized in induced pluripotent stem cell-derived neurons alongside multiomic neuronal and microglial profiling.ResultsANXA11 mutations were linked to corticobasal syndrome cases. P93S led to decreased lysosome colocalization, neuritic RNA, and nuclear TDP-43 with cryptic exon expression. Multiomic microglial signatures implicated immune dysregulation and interferon signaling pathways.DiscussionThis study establishes ANXA11 P93S pathogenicity, broadens the phenotypic spectrum of ANXA11 mutations, underscores neuronal and microglial dysfunction in ANXA11 pathophysiology, and demonstrates the potential of cellular models to determine variant pathogenicity.HighlightsANXA11 P93S is a pathogenic variant. Corticobasal syndrome is part of the ANXA11 phenotypic spectrum. Hybridization chain reaction fluorescence in situ hybridization (HCR FISH) is a new tool for the detection of cryptic exons due to TDP-43-related loss of splicing regulation. Microglial ANXA11 and related immune pathways are important drivers of disease. Cellular models are powerful tools for adjudicating variants of uncertain significance.

Z-nucleic acid structures play vital roles in cellular processes and have implications in innate immunity due to their recognition by Zα domains containing proteins (Z-DNA/Z-RNA binding proteins, ZBPs). Although Zα domains have been identified in six proteins, including viral E3L, ORF112, and I73R, as well as, cellular ADAR1, ZBP1, and PKZ, their prevalence across living organisms remains largely unexplored. In this study, we introduce a computational approach to predict Zα domains, leading to the revelation of previously unidentified Zα domain-containing proteins in eukaryotic organisms, including non-metazoan species. Our findings encompass the discovery of new ZBPs in previously unexplored giant viruses, members of the Nucleocytoviricota phylum. Through experimental validation, we confirm the Zα functionality of select proteins, establishing their capability to induce the B-to-Z conversion. Additionally, we identify Zα-like domains within bacterial proteins. While these domains share certain features with Zα domains, they lack the ability to bind to Z-nucleic acids or facilitate the B-to-Z DNA conversion. Our findings significantly expand the ZBP family across a wide spectrum of organisms and raise intriguing questions about the evolutionary origins of Zα-containing proteins. Moreover, our study offers fresh perspectives on the functional significance of Zα domains in virus sensing and innate immunity and opens avenues for exploring hitherto undiscovered functions of ZBPs.

IGHMBP2 is a nonessential, superfamily 1 DNA/RNA helicase that is mutated in patients with rare neuromuscular diseases SMARD1 and CMT2S. IGHMBP2 is implicated in translational and transcriptional regulation via biochemical association with ribosomal proteins, pre-rRNA processing factors, and tRNA-related species. To uncover the cellular consequences of perturbing IGHMBP2, we generated full and partial IGHMBP2 deletion K562 cell lines. Using polysome profiling and a nascent protein synthesis assay, we found that IGHMBP2 deletion modestly reduces global translation. We performed Ribo-seq and RNA-seq and identified diverse gene expression changes due to IGHMBP2 deletion, including ATF4 up-regulation. With recent studies showing the integrated stress response (ISR) can contribute to tRNA metabolism-linked neuropathies, we asked whether perturbing IGHMBP2 promotes ISR activation. We generated ATF4 reporter cell lines and found IGHMBP2 knockout cells demonstrate basal, chronic ISR activation. Our work expands upon the impact of IGHMBP2 in translation and elucidates molecular mechanisms that may link mutant IGHMBP2 to severe clinical phenotypes.

Active loop extrusion—the process of formation of dynamically growing chromatin loops due to the motor activity of DNA–binding protein complexes—is a firmly established mechanism responsible for chromatin spatial organization at different stages of a cell cycle in eukaryotes and bacteria. The theoretical insight into the effect of loop extrusion on the experimentally measured statistics of chromatin conformation can be gained with an appropriately chosen polymer model. Here, we consider the simplest analytically solvable model of an interphase chromosome, which is treated as an ideal chain with disorder of sufficiently sparse random loops whose conformations are sampled from the equilibrium ensemble. This framework allows us to arrive at the closed-form analytical expression for the mean-squared distance between pairs of genomic loci, which is valid beyond the one-loop approximation in diagrammatic representation. In addition, we analyze the loop-induced deviation of chain conformations from the Gaussian statistics by calculating kurtosis of probability density of the pairwise separation vector. The presented results suggest the possible ways of estimating the characteristics of the loop extrusion process based on the experimental data on the scale-dependent statistics of intra-chromosomal pair-wise distances. [ABSTRACT FROM AUTHOR]

UNLABELLED: Vibrio cholerae, the causative agent of the diarrheal disease cholera, poses an ongoing health threat due to its wide repertoire of horizontally acquired elements (HAEs) and virulence factors. New clinical isolates of the bacterium with improved fitness abilities, often associated with HAEs, frequently emerge. The appropriate control and expression of such genetic elements is critical for the bacteria to thrive in the different environmental niches they occupy. H-NS, the histone-like nucleoid structuring protein, is the best-studied xenogeneic silencer of HAEs in gamma-proteobacteria. Although H-NS and other highly abundant nucleoid-associated proteins (NAPs) have been shown to play important roles in regulating HAEs and virulence in model bacteria, we still lack a comprehensive understanding of how different NAPs modulate transcription in V. cholerae. By obtaining genome-wide measurements of protein occupancy and active transcription in a clinical isolate of V. cholerae, harboring recently discovered HAEs encoding for phage defense systems, we show that a lack of H-NS causes a robust increase in the expression of genes found in many HAEs. We further found that TsrA, a protein with partial homology to H-NS, regulates virulence genes primarily through modulation of H-NS activity. We also identified few sites that are affected by TsrA independently of H-NS, suggesting TsrA may act with diverse regulatory mechanisms. Our results demonstrate how the combinatorial activity of NAPs is employed by a clinical isolate of an important pathogen to regulate recently discovered HAEs. IMPORTANCE: New strains of the bacterial pathogen Vibrio cholerae, bearing novel horizontally acquired elements (HAEs), frequently emerge. HAEs provide beneficial traits to the bacterium, such as antibiotic resistance and defense against invading bacteriophages. Xenogeneic silencers are proteins that help bacteria harness new HAEs and silence those HAEs until they are needed. H-NS is the best-studied xenogeneic silencer; it is one of the nucleoid-associated proteins (NAPs) in gamma-proteobacteria and is responsible for the proper regulation of HAEs within the bacterial transcriptional network. We studied the effects of H-NS and other NAPs on the HAEs of a clinical isolate of V. cholerae. Importantly, we found that H-NS partners with a small and poorly characterized protein, TsrA, to help domesticate new HAEs involved in bacterial survival and in causing disease. A proper understanding of the regulatory state in emerging isolates of V. cholerae will provide improved therapies against new isolates of the pathogen.

DNA replication stress (RS) is a widespread phenomenon in carcinogenesis, causing genomic instability and extensive chromatin alterations. DNA damage leads to activation of innate immune signaling, but little is known about transcriptional regulators mediating such signaling upon RS. Using a chemical screen, we identified protein arginine methyltransferase 5 (PRMT5) as a key mediator of RS-dependent induction of interferon-stimulated genes (ISGs). This response is also associated with reactivation of endogenous retroviruses (ERVs). Using quantitative mass spectrometry, we identify proteins with PRMT5-dependent symmetric dimethylarginine (SDMA) modification induced upon RS. Among these, we show that PRMT5 targets and modulates the activity of ZNF326, a zinc finger protein essential for ISG response. Our data demonstrate a role for PRMT5-mediated SDMA in the context of RS-induced transcriptional induction, affecting physiological homeostasis and cancer therapy.

TDP-43 proteinopathy is a salient neuropathologic feature in a subset of frontotemporal lobar degeneration (FTLD-TDP), in amyotrophic lateral sclerosis (ALS-TDP), and in limbic-predominant age-related TDP-43 encephalopathy neuropathologic change (LATE-NC), and is associated with hippocampal sclerosis of aging (HS-A). We examined TDP-43-related pathology data in the National Alzheimer's Coordinating Center (NACC) in two parts: (I) availability of assessments, and (II) associations with clinical diagnoses and other neuropathologies in those with all TDP-43 measures available. Part I: Of 4326 participants with neuropathology data collected using forms that included TDP-43 assessments, data availability was highest for HS-A (97%) and ALS (94%), followed by FTLD-TDP (83%). Regional TDP-43 pathologic assessment was available for 77% of participants, with hippocampus the most common region. Availability for the TDP-43-related measures increased over time, and was higher in centers with high proportions of participants with clinical FTLD. Part II: In 2142 participants with all TDP-43-related assessments available, 27% of participants had LATE-NC, whereas ALS-TDP or FTLD-TDP (ALS/FTLD-TDP) was present in 9% of participants, and 2% of participants had TDP-43 related to other pathologies ("Other TDP-43"). HS-A was present in 14% of participants, of whom 55% had LATE-NC, 20% ASL/FTLD-TDP, 3% Other TDP-43, and 23% no TDP-43. LATE-NC, ALS/FTLD-TDP, and Other TDP-43, were each associated with higher odds of dementia, HS-A, and hippocampal atrophy, compared to those without TDP-43 pathology. LATE-NC was associated with higher odds for Alzheimer's disease (AD) clinical diagnosis, AD neuropathologic change (ADNC), Lewy bodies, arteriolosclerosis, and cortical atrophy. ALS/FTLD-TDP was associated with higher odds of clinical diagnoses of primary progressive aphasia and behavioral-variant frontotemporal dementia, and cortical/frontotemporal lobar atrophy. When using NACC data for TDP-43-related analyses, researchers should carefully consider the incomplete availability of the different regional TDP-43 assessments, the high frequency of participants with ALS/FTLD-TDP, and the presence of other forms of TDP-43 pathology.

The mechanisms of hepatic stellate cell (HSC) activation and the development of liver fibrosis are not fully understood. Here, we show that deletion of a nuclear seven transmembrane protein, TM7SF3, accelerates HSC activation in liver organoids, primary human HSCs, and in vivo in metabolic-dysfunction-associated steatohepatitis (MASH) mice, leading to activation of the fibrogenic program and HSC proliferation. Thus, TM7SF3 knockdown promotes alternative splicing of the Hippo pathway transcription factor, TEAD1, by inhibiting the splicing factor heterogeneous nuclear ribonucleoprotein U (hnRNPU). This results in the exclusion of the inhibitory exon 5, generating a more active form of TEAD1 and triggering HSC activation. Furthermore, inhibiting TEAD1 alternative splicing with a specific antisense oligomer (ASO) deactivates HSCs in vitro and reduces MASH diet-induced liver fibrosis. In conclusion, by inhibiting TEAD1 alternative splicing, TM7SF3 plays a pivotal role in mitigating HSC activation and the progression of MASH-related fibrosis.

We construct and analyze monomeric and multimeric models of the stochastic disassembly of a single nucleosome. Our monomeric model predicts the time needed for a number of histone–DNA contacts to spontaneously break, leading to dissociation of a non-fragmented histone from DNA. The dissociation process can be facilitated by DNA binding proteins or processing molecular motors that compete with histones for histone–DNA contact sites. Eigenvalue analysis of the corresponding master equation allows us to evaluate histone detachment times under both spontaneous detachment and protein-facilitated processes. We find that competitive DNA binding of remodeling proteins can significantly reduce the typical detachment time but only if these remodelers have DNA-binding affinities comparable to those of histone–DNA contact sites. In the presence of processive motors, the histone detachment rate is shown to be proportional to the product of the histone single-bond dissociation constant and the speed of motor protein procession. Our simple intact-histone model is then extended to allow for multimeric nucleosome kinetics that reveal additional pathways of disassembly. In addition to a dependence of complete disassembly times on subunit–DNA contact energies, we show how histone subunit concentrations in bulk solutions can mediate the disassembly process by rescuing partially disassembled nucleosomes. Moreover, our kinetic model predicts that remodeler binding can also bias certain pathways of nucleosome disassembly, with higher remodeler binding rates favoring intact-histone detachment. [ABSTRACT FROM AUTHOR]

IntroductionAlthough dementia-related proteinopathy has a strong negative impact on public health, and is highly heritable, understanding of the related genetic architecture is incomplete.MethodsWe applied multidimensional generalized partial credit modeling (GPCM) to test genetic associations with dementia-related proteinopathies. Data were analyzed to identify candidate single nucleotide variants for the following proteinopathies: Aβ, tau, α-synuclein, and TDP-43.ResultsFinal included data comprised 966 participants with neuropathologic and WGS data. Three continuous latent outcomes were constructed, corresponding to TDP-43-, Aβ/Tau-, and α-synuclein-related neuropathology endophenotype scores. This approach helped validate known genotype/phenotype associations: for example, TMEM106B and GRN were risk alleles for TDP-43 pathology; and GBA for α-synuclein/Lewy bodies. Novel suggestive proteinopathy-linked alleles were also discovered, including several (SDHAF1, TMEM68, and ARHGEF28) with colocalization analyses and/or high degrees of biologic credibility.DiscussionA novel methodology using GPCM enabled insights into gene candidates for driving misfolded proteinopathies.HighlightsLatent factor scores for proteinopathies were estimated using a generalized partial credit model. The three latent continuous scores corresponded well with proteinopathy severity. Novel genes associated with proteinopathies were identified. Several genes had high degrees of biologic credibility for dementia risk factors.

XIST noncoding RNA promotes the initiation of X chromosome silencing by recruiting the protein SPEN to one X chromosome in female mammals. The SPEN protein is also called SHARP (SMRT and HDAC-associated repressor protein) and MINT (Msx-2 interacting nuclear target) in humans. SPEN recruits N-CoR2 and HDAC3 to initiate histone deacetylation on the X chromosome, leading to the formation of repressive chromatin marks and silencing gene expression. We dissected the contributions of different RNA and protein regions to the formation of a human XIST-SPEN complex in vitro and identified novel sequence and structure determinants that may contribute to X chromosome silencing initiation. Binding of SPEN to XIST RNA requires RRM 4 of the protein, in contrast to the requirement of RRM 3 and RRM 4 for specific binding to SRA RNA. Measurements of SPEN binding to full-length, dimeric, trimeric, or other truncated versions of the A-repeat region revealed that high-affinity binding of XIST to SPEN in vitro requires a minimum of four A-repeat segments. SPEN binding to XIST A-repeat RNA changes the accessibility of the RNA at specific nucleotide sequences, as indicated by changes in RNA reactivity through chemical structure probing. Based on computational modeling, we found that inter-repeat duplexes formed by multiple A-repeats can present an unpaired adenosine in the context of a double-stranded region of RNA. The presence of this specific combination of sequence and structural motifs correlates with high-affinity SPEN binding in vitro. These data provide new information on the molecular basis of the XIST and SPEN interaction.

Despite numerous female contraceptive options, nearly half of all pregnancies are unintended. Family planning choices for men are currently limited to unreliable condoms and invasive vasectomies with questionable reversibility. Here, we report the development of an oral contraceptive approach based on transcriptional disruption of cyclical gene expression patterns during spermatogenesis. Spermatogenesis involves a continuous series of self-renewal and differentiation programs of spermatogonial stem cells (SSCs) that is regulated by retinoic acid (RA)-dependent activation of receptors (RARs), which control target gene expression through association with corepressor proteins. We have found that the interaction between RAR and the corepressor silencing mediator of retinoid and thyroid hormone receptors (SMRT) is essential for spermatogenesis. In a genetically engineered mouse model that negates SMRT-RAR binding (SMRTmRID mice), the synchronized, cyclic expression of RAR-dependent genes along the seminiferous tubules is disrupted. Notably, the presence of an RA-resistant SSC population that survives RAR de-repression suggests that the infertility attributed to the loss of SMRT-mediated repression is reversible. Supporting this notion, we show that inhibiting the action of the SMRT complex with chronic, low-dose oral administration of a histone deacetylase inhibitor reversibly blocks spermatogenesis and fertility without affecting libido. This demonstration validates pharmacologic targeting of the SMRT repressor complex for non-hormonal male contraception.

Mislocalization of overexpressed CENP-A (Cse4 in budding yeast, Cnp1 in fission yeast, CID in flies) contributes to chromosomal instability (CIN) in yeasts, flies, and human cells. Mislocalization of CENP-A is observed in many cancers and this correlates with poor prognosis. Structural mechanisms that contribute to mislocalization of CENP-A are poorly defined. Here, we show that interaction of histone H4 with Cse4 facilitates an in vivo conformational change in Cse4 promoting its mislocalization in budding yeast. We determined that Cse4 Y193A mutant exhibits reduced sumoylation, mislocalization, interaction with histone H4, and lethality in psh1Δ and cdc48-3 strains; all these phenotypes are suppressed by increased gene dosage of histone H4. We developed a new in vivo approach, antibody accessibility (AA) assay, to examine the conformation of Cse4. AA assay showed that wild-type Cse4 with histone H4 is in an open state, while Cse4 Y193A predominantly exhibits a closed state. Increased gene dosage of histone H4 contributes to a shift of Cse4 Y193A to an open state with enhanced sumoylation and mislocalization. We provide molecular insights into how Cse4-H4 interaction changes the conformational state of Cse4 in vivo. These studies advance our understanding for mechanisms that promote mislocalization of CENP-A in human cancers.

Nuclear clearance and cytoplasmic accumulations of the RNA-binding protein TDP-43 are pathological hallmarks in almost all patients with amyotrophic lateral sclerosis (ALS) and up to 50% of patients with frontotemporal dementia (FTD) and Alzheimers disease. In Alzheimers disease, TDP-43 pathology is predominantly observed in the limbic system and correlates with cognitive decline and reduced hippocampal volume. Disruption of nuclear TDP-43 function leads to abnormal RNA splicing and incorporation of erroneous cryptic exons in numerous transcripts including Stathmin-2 (STMN2, also known as SCG10) and UNC13A, recently reported in tissues from patients with ALS and FTD. Here, we identify both STMN2 and UNC13A cryptic exons in Alzheimers disease patients, that correlate with TDP-43 pathology burden, but not with amyloid-β or tau deposits. We also demonstrate that processing of the STMN2 pre-mRNA is more sensitive to TDP-43 loss of function than UNC13A. In addition, full-length RNAs encoding STMN2 and UNC13A are suppressed in large RNA-seq datasets generated from Alzheimers disease post-mortem brain tissue. Collectively, these results open exciting new avenues to use STMN2 and UNC13A as potential therapeutic targets in a broad range of neurodegenerative conditions with TDP-43 proteinopathy including Alzheimers disease.

RNA-binding proteins (RBPs) have pivotal functions in RNA metabolism, but current methods are limited in retrieving RBP-RNA interactions within endogenous biological contexts. Here, we develop INSCRIBE (IN situ Sensitive Capture of RNA-protein Interactions in Biological Environments), circumventing the challenges through in situ RNA labeling by precisely directing a purified APOBEC1-nanobody fusion to the RBP of interest. This method enables highly specific RNA-binding site identification across a diverse range of fixed biological samples such as HEK293T cells and mouse brain tissue and accurately identifies the canonical binding motifs of RBFOX2 (UGCAUG) and TDP-43 (UGUGUG) in native cellular environments. Applicable to any RBP with available primary antibodies, INSCRIBE enables sensitive capture of RBP-RNA interactions from ultra-low input equivalent to ~5 cells. The robust, versatile, and sensitive INSCRIBE workflow is particularly beneficial for precious tissues such as clinical samples, empowering the exploration of genuine RBP-RNA interactions in RNA-related disease contexts.

BackgroundStatins lower circulating low-density lipoprotein cholesterol (LDLC) levels and reduce cardiovascular disease risk. Though highly efficacious in general, there is considerable inter-individual variation in statin efficacy that remains largely unexplained.MethodsTo identify novel genes that may modulate statin-induced LDLC lowering, we used RNA-sequencing data from 426 control- and 2 µM simvastatin-treated lymphoblastoid cell lines (LCLs) derived from European and African American ancestry participants of the Cholesterol and Pharmacogenetics (CAP) 40 mg/day 6-week simvastatin clinical trial (ClinicalTrials.gov Identifier: NCT00451828). We correlated statin-induced changes in LCL gene expression with plasma LDLC statin response in the corresponding CAP participants. For the most correlated gene identified (ZNF335), we followed up in vivo by comparing plasma cholesterol levels, lipoprotein profiles, and lipid statin response between wild-type mice and carriers of a hypomorphic (partial loss of function) missense mutation in Zfp335 (the mouse homolog of ZNF335).ResultsThe statin-induced expression changes of 147 human LCL genes were significantly correlated to the plasma LDLC statin responses of the corresponding CAP participants in vivo (FDR = 5%). The two genes with the strongest correlations were zinc finger protein 335 (ZNF335 aka NIF-1, rho = 0.237, FDR-adj p = 0.0085) and CCR4-NOT transcription complex subunit 3 (CNOT3, rho = 0.233, FDR-adj p = 0.0085). Chow-fed mice carrying a hypomorphic missense (R1092W; aka bloto) mutation in Zfp335 had significantly lower non-HDL cholesterol levels than wild-type C57BL/6J mice in a sex combined model (p = 0.04). Furthermore, male (but not female) mice carrying the Zfp335R1092W allele had significantly lower total and HDL cholesterol levels than wild-type mice. In a separate experiment, wild-type mice fed a control diet for 4 weeks and a matched simvastatin diet for an additional 4 weeks had significant statin-induced reductions in non-HDLC (-43 ± 18% and -23 ± 19% for males and females, respectively). Wild-type male (but not female) mice experienced significant reductions in plasma LDL particle concentrations, while male mice carrying Zfp335R1092W allele(s) exhibited a significantly blunted LDL statin response.ConclusionsOur in vitro and in vivo studies identified ZNF335 as a novel modulator of plasma cholesterol levels and statin response, suggesting that variation in ZNF335 activity could contribute to inter-individual differences in statin clinical efficacy.

Protein quality control pathways play important roles in resistance against pathogen infection. For example, the conserved transcription factor SKN-1/NRF up-regulates proteostasis capacity after blockade of the proteasome and also promotes resistance against bacterial infection in the nematode Caenorhabditis elegans. SKN-1/NRF has 3 isoforms, and the SKN-1A/NRF1 isoform, in particular, regulates proteasomal gene expression upon proteasome dysfunction as part of a conserved bounce-back response. We report here that, in contrast to the previously reported role of SKN-1 in promoting resistance against bacterial infection, loss-of-function mutants in skn-1a and its activating enzymes ddi-1 and png-1 show constitutive expression of immune response programs against natural eukaryotic pathogens of C. elegans. These programs are the oomycete recognition response (ORR), which promotes resistance against oomycetes that infect through the epidermis, and the intracellular pathogen response (IPR), which promotes resistance against intestine-infecting microsporidia. Consequently, skn-1a mutants show increased resistance to both oomycete and microsporidia infections. We also report that almost all ORR/IPR genes induced in common between these programs are regulated by the proteasome and interestingly, specific ORR/IPR genes can be induced in distinct tissues depending on the exact trigger. Furthermore, we show that increasing proteasome function significantly reduces oomycete-mediated induction of multiple ORR markers. Altogether, our findings demonstrate that proteasome regulation keeps innate immune responses in check in a tissue-specific manner against natural eukaryotic pathogens of the C. elegans epidermis and intestine.

Muscle wasting is a serious complication in heart failure patients. Oxidative stress and inflammation are implicated in the pathogenesis of muscle wasting. Oxidative stress leads to the formation of toxic lipid peroxidation products, such as 4-hydroxy-2-nonenal (HNE), which covalently bind with proteins and DNA and activate atrophic pathways. Whether the formation of lipid peroxidation products and metabolic pathways that remove these toxic products are affected during heart failure-associated skeletal muscle wasting has never been studied. Male C57BL/6J mice were subjected to sham and transverse aortic constriction (TAC) surgeries for 4, 8 or 14 weeks. Different skeletal muscle beds were weighed, and the total cross-sectional area of the gastrocnemius muscle was measured via immunohistochemistry. Muscle function and muscle stiffness were measured by a grip strength meter and atomic force microscope, respectively. Atrophic and inflammatory marker levels were measured via qRT‒PCR. The levels of acrolein and HNE-protein adducts, aldehyde-removing enzymes, the histidyl dipeptide-synthesizing enzyme carnosine synthase (CARNS), and amino acid transporters in the gastrocnemius muscle were measured via Western blotting and qRT‒PCR. Histidyl dipeptides and histidyl dipeptide aldehyde conjugates in the Gastrocnemius and soleus muscles were analyzed by LC/MS–MS. Body weight, gastrocnemius muscle and soleus muscle weights and the total cross-sectional area of the gastrocnemius muscle were decreased after 14 weeks of TAC. Heart weight, cardiac function, grip strength and muscle stiffness were decreased in the TAC-operated mice. Expression of the atrophic and inflammatory markers Atrogin1 and TNF-α, respectively, was increased ~ 1.5–2fold in the gastrocnemius muscle after 14 weeks of TAC (p < 0.05 and p = 0.004 vs sham). The formation of HNE and acrolein protein adducts was increased, and the expression of the aldehyde-removing enzyme aldehyde dehydrogenase (ALDH2) was decreased in the gastrocnemius muscle of TAC mice. Carnosine (sham: 5.76 ± 1.3 vs TAC: 4.72 ± 0.7 nmol/mg tissue, p = 0.04) and total histidyl dipeptide levels (carnosine and anserine; sham: 11.97 ± 1.5 vs TAC: 10.13 ± 1.4 nmol/mg tissue, p < 0.05) were decreased in the gastrocnemius muscle of TAC mice. Depletion of histidyl dipeptides diminished the aldehyde removal capacity of the atrophic gastrocnemius muscle. Furthermore, CARNS and TAUT protein expression were decreased in the atrophic gastrocnemius muscle. Our data reveals that reduced expression of ALDH2 and depletion of histidyl dipeptides in the gastrocnemius muscle during heart failure leads to the accumulation of toxic aldehydes and might contribute to muscle wasting. [ABSTRACT FROM AUTHOR]

Neurocristopathies such as CHARGE syndrome result from aberrant neural crest development. A large proportion of CHARGE cases are attributed to pathogenic variants in the gene encoding CHD7, chromodomain helicase DNA binding protein 7, which remodels chromatin. While the role for CHD7 in neural crest development is well documented, how this factor is specifically up-regulated in neural crest cells is not understood. Here, we use epigenomic profiling of chick and human neural crest to identify a cohort of enhancers regulating Chd7 expression in neural crest cells and other tissues. We functionally validate upstream transcription factor binding at candidate enhancers, revealing novel epistatic relationships between neural crest master regulators and Chd7, showing tissue-specific regulation of a globally acting chromatin remodeller. Furthermore, we find conserved enhancer features in human embryonic epigenomic data and validate the activity of the human equivalent CHD7 enhancers in the chick embryo. Our findings embed Chd7 in the neural crest gene regulatory network and offer potentially clinically relevant elements for interpreting CHARGE syndrome cases without causative allocation. CHARGE syndrome results from aberrant neural crest development, affecting eye, heart, and facial structures, and has been attributed to pathogenic variants of the chromatin remodeler CHD7. This study characterizes the enhancers and tissue specific transcription factors that regulate Chd7 expression in the developing chick and human neural crest. [ABSTRACT FROM AUTHOR]

Objective: To explore the pharmacodynamic ingredients and pharmacologic mechanism of Haizao Yuhu Decoction (HYD) in treating hyperthyroidism via an analysis integrating network pharmacology, molecular docking, and nontargeted serum metabolomics. Methods: Therapeutic targets of hyperthyroidism were searched through multiarray analyses in the Gene Expression Omnibus (GEO) database. Hub genes were subjected to the construction of a protein-protein interaction (PPI) network, and GO and KEGG enrichment analyses. Targets of active pharmaceutical ingredients (APIs) in HYD and those of hyperthyroidism were intersected to yield hub genes, followed by validations via molecular docking and non-targeted serum metabolomics. Results: 112 hub genes were identified by intersecting APIs of HYD and therapeutic targets of hyperthyroidism. Using ultra-high performance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) in both negative and positive ion polarity modes, 279 compounds of HYD absorbed in the plasma were fingerprinted. Through summarizing data yielded from network pharmacology and non-targeted serum metabolomics, 214 common targets were identified from compounds of HYD absorbed in the plasma and therapeutic targets of hyperthyroidism, including PTPN11, PIK3CD, EGFR, HRAS, PIK3CA, AKT1, SRC, PIK3CB, and PIK3R1. They were mainly enriched in the biological processes of positive regulation of gene expression, positive regulation of MAPK cascade, signal transduction, protein phosphorylation, negative regulation of apoptotic process, positive regulation of protein kinase B signaling and positive regulation of MAP kinase activity; and molecular functions of identical protein binding, protein serine/threonine/tyrosine kinase activity, protein kinase activity, RNA polymerase II transcription factor activity, ligand-activated sequence-specific DNA binding and protein binding. A total of 185 signaling pathways enriched in the 214 common targets were associated with cell proliferation and angiogenesis. Conclusion: HYD exerts a pharmacological effect on hyperthyroidism via inhibiting pathological angiogenesis in the thyroid and rebalancing immunity. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that results in death within 2–5 years of diagnosis. Respiratory failure is the most common cause of death in ALS. Mutations in the transactive response DNA binding protein 43 (TDP-43) encoded by the TARDBP gene are associated with abnormal cellular aggregates in neurons of patients with both familial and sporadic ALS. The role of these abnormal aggregates on breathing is unclear. Since respiratory failure is a major cause of death in ALS, we sought to determine the role of TDP-43 mutations on the respiratory motor unit in the Prp-hTDP43A315T mouse model – a model that expresses human TDP-43 containing the A315T mutation. We assessed breathing using whole-body plethysmography, and investigated neuropathology in hypoglossal and phrenic respiratory motor units. Postmortem studies included quantification of hypoglossal and putative phrenic motor neurons, activated microglia and astrocytes in respiratory control centers, and assessment of hypoglossal and phrenic nerves of TDP43A315T mice. The male TDP43A315T mice display an early onset of rapid progression of disease, and premature death (less than 15 weeks) compared to control mice and compared to female TDP43A315T mice who die between 20 and 35 weeks of age. The TDP43A315T mice have progressive and profound breathing deficits at baseline and during a respiratory challenge. Histologically, hypoglossal and putative phrenic motor neurons of TDP43A315T mice are decreased and have increased microglial and astrocyte activation, indicating pronounced neurodegeneration and neuroinflammation. Further, there is axonopathy and demyelination in the hypoglossal and phrenic nerve of TDP43A315T mice. Thus, the TDP-43A315T mice have significant respiratory pathology and neuropathology, which makes them a useful translatable model for the study of novel therapies on breathing in ALS. [ABSTRACT FROM AUTHOR]

Memory effect refers to the phenomenon where past events influence a system's current and future states or behaviors. In biology, memory effects often arise from intra-or intermolecular interactions, leading to temporally correlated behaviors. Single-molecule studies have shown that enzymes and DNA-binding proteins can exhibit time-correlated behaviors of their activity. While memory effects are well documented and studied in vitro, no such examples exist in cells to our knowledge. Combining single-molecule tracking (SMT) and single-cell protein quantitation, we find in living Escherichia coli cells distinct temporal correlations in the binding/unbinding events on DNA by MerR-and Fur-family metalloregulators, manifesting as memory effects with timescales of ~1 s. These memory effects persist irrespective of the type of the metalloregulators or their metallation states. Moreover, these temporal correlations of metalloregulator-DNA interactions are associated with spatial confinements of the metalloregulators near their DNA binding sites, suggesting microdomains of ~100 nm in size that possibly result from the spatial organizations of the bacterial chromosome without the involvement of membranes. These microdomains likely facilitate repeated binding events, enhancing regulator-DNA contact frequency and potentially gene regulation efficiency. These findings provide unique insights into the spatiotemporal dynamics of protein-DNA interactions in bacterial cells, introducing the concept of microdomains as a crucial player in memory effect-driven gene regulation. [ABSTRACT FROM AUTHOR]

DNA methylation plays a critical role in gene regulation by modulating the DNA binding of transcription factors (TFs). This study integrates TFs' ChIP-seq profiles with WGBS profiles to investigate how DNA methylation affects protein interactions. Statistical methods and a 5-letter DNA motif calling model have been developed to characterize DNA sequences bound by proteins, while considering the effects of DNA modifications. By employing these methods, 79 significant universal "stripe" TFs and cofactors (USFs), 2360 co-binding protein pairs, and distinct protein modules associated with various DNA methylation states have been identified. The USFs hint a regulatory hierarchy within these protein interactions. Proteins preferentially bind to non-CpG sites in methylated regions, indicating binding affinity is not solely CpG-dependent. Proteins involved in methylation-specific USFs and cobinding pairs play essential roles in promoting and sustaining DNA methylation through interacting with DNMTs or inhibiting TET binding. These findings underscore the interplay between protein binding and methylation, offering insights into epigenetic regulation in cellular biology. Investigation on the effect of DNA methylation on the cooperation of transcription factors. [ABSTRACT FROM AUTHOR]

Simple Summary: DNA-dependent protein kinase (DNA-PK) is a key player in repairing DNA damage. Ku proteins detect DNA damage and activate DNA-PK. Blocking DNA-PK can make cancer treatments such as radiation more effective. We have developed Ku–DNA binding inhibitors (Ku-DBis) that stop DNA-PK activation, making cancer cells more sensitive to radiation. We recently discovered new Ku-DBis that enter cells better than predecessor compounds, allowing cellular and in vivo analyses. Some non-small cell lung cancer (NSCLC) cells, especially those lacking ATM, were very sensitive to these inhibitors and radiation. However, cells lacking BRCA1 were resistant, which reduced the effectiveness of radiation. In animal studies of NSCLC, Ku-DBi treatment inhibited DNA-PK and enhanced a radiation-dependent decrease in tumor cell proliferation. This is the first time a Ku-targeted inhibitor has been shown to work in living organisms, suggesting their potential application in cancer treatment. Background: DNA-dependent protein kinase (DNA-PK) is a validated cancer therapeutic target involved in DNA damage response (DDR) and non-homologous end-joining (NHEJ) repair of DNA double-strand breaks (DSBs). Ku serves as a sensor of DSBs by binding to DNA ends and activating DNA-PK. Inhibition of DNA-PK is a common strategy to block DSB repair and improve efficacy of ionizing radiation (IR) therapy and radiomimetic drug therapies. We have previously developed Ku–DNA binding inhibitors (Ku-DBis) that block in vitro and cellular NHEJ activity, abrogate DNA-PK autophosphorylation, and potentiate cellular sensitivity to IR. Results and Conclusions: Here we report the discovery of oxindole Ku-DBis with improved cellular uptake and retained potent Ku-inhibitory activity. Variable monotherapy activity was observed in a panel of non-small cell lung cancer (NSCLC) cell lines, with ATM-null cells being the most sensitive and showing synergy with IR. BRCA1-deficient cells were resistant to single-agent treatment and antagonistic when combined with DSB-generating therapies. In vivo studies in an NSCLC xenograft model demonstrated that the Ku-DBi treatment blocked IR-dependent DNA-PKcs autophosphorylation, modulated DDR, and reduced tumor cell proliferation. This represents the first in vivo demonstration of a Ku-targeted DNA-binding inhibitor impacting IR response and highlights the potential therapeutic utility of Ku-DBis for cancer treatment. [ABSTRACT FROM AUTHOR]

Simple Summary: Epithelial-to-mesenchymal transition (EMT) plays a critical role in cancer progression, contributing to the invasive and migratory abilities of tumor cells. In this study, we show that FBXO11 promotes the degradation of ZEB1, a key EMT regulator, via ubiquitination. Loss of FBXO11 increases ZEB1 levels, enhancing the invasiveness of lung cancer cells, while its overexpression reduces ZEB1 and suppresses invasion. Importantly, higher FBXO11 expression is associated with better prognosis in non-small cell lung cancer (NSCLC), highlighting its potential role as a therapeutic target for controlling EMT and cancer metastasis. Epithelial-to-mesenchymal transition (EMT) affects the invasion and migration of cancer cells. Here, we show that FBXO11 recognizes and promotes ubiquitin-mediated degradation of ZEB1. There is a strong association between FBXO11 and ZEB1 in non-small cell lung cancer (NSLC) in a clinical database. FBXO11 interacts with ZEB1, a core inducer of EMT. FBXO11 leads to increased ubiquitination and proteasomal degradation of ZEB1. Depletion of endogenous FBXO11 causes ZEB1 protein accumulation and EMT in A549 and H1299 cells, while overexpression of FBXO11 reduces ZEB1 protein abundance and cellular invasiveness. Importantly, the depletion of ZEB1 suppresses the increased migration and invasion of A549 and H1299 cells promoted by the depletion of FBXO11. The same results are shown in xenograft tumors. High FBXO11 expression is associated with a favorable prognosis in NSLC. Collectively, our study demonstrates that FBXO11 modulates EMT by mediating the stability of ZEB1 in lung cancer cells. [ABSTRACT FROM AUTHOR]

DNA methylation is an important epigenetic mark that modulates gene expression through the inhibition of transcriptional proteins binding to DNA. As in many other omics experiments, the issue of missing values is an important one, and appropriate imputation techniques are important in avoiding an unnecessary sample size reduction as well as to optimally leverage the information collected. We consider the case where relatively few samples are processed via an expensive high-density whole genome bisulfite sequencing (WGBS) strategy and a larger number of samples is processed using more affordable low-density, array-based technologies. In such cases, one can impute the low-coverage (array-based) methylation data using the high-density information provided by the WGBS samples. In this paper, we propose an efficient Linear Model of Coregionalisation with informative Covariates (LMCC) to predict missing values based on observed values and covariates. Our model assumes that at each site, the methylation vector of all samples is linked to the set of fixed factors (covariates) and a set of latent factors. Furthermore, we exploit the functional nature of the data and the spatial correlation across sites by assuming some Gaussian processes on the fixed and latent coefficient vectors, respectively. Our simulations show that the use of covariates can significantly improve the accuracy of imputed values, especially in cases where missing data contain some relevant information about the explanatory variable. We also showed that our proposed model is particularly efficient when the number of columns is much greater than the number of rows—which is usually the case in methylation data analysis. Finally, we apply and compare our proposed method with alternative approaches on two real methylation datasets, showing how covariates such as cell type, tissue type or age can enhance the accuracy of imputed values. [ABSTRACT FROM AUTHOR]

Cohesins promote proper chromosome segregation, gene transcription, genomic architecture, DNA condensation, and DNA damage repair. Mutations in either cohesin subunits or regulatory genes can give rise to severe developmental abnormalities (such as Robert Syndrome and Cornelia de Lange Syndrome) and also are highly correlated with cancer. Despite this, little is known about cohesin regulation. Eco1 (ESCO2/EFO2 in humans) and Rad61 (WAPL in humans) represent two such regulators but perform opposing roles. Eco1 acetylation of cohesin during S phase, for instance, stabilizes cohesin-DNA binding to promote sister chromatid cohesion. On the other hand, Rad61 promotes the dissociation of cohesin from DNA. While Eco1 is essential, ECO1 and RAD61 co-deletion results in yeast cell viability, but only within a limited temperature range. Here, we report that eco1 rad61 cell lethality is due to reduced levels of the cohesin subunit Mcd1. Results from a suppressor screen further reveals that FDO1 deletion rescues the temperature-sensitive (ts) growth defects exhibited by eco1 rad61 double mutant cells by increasing Mcd1 levels. Regulation of MCD1 expression, however, appears more complex. Elevated expression of MBP1 , which encodes a subunit of the MBF transcription complex, also rescues eco1 rad61 cell growth defects. Elevated expression of SWI6 , however, which encodes the Mbp1 -binding partner of MBF, exacerbates eco1 rad61 cell growth and also abrogates the Mpb1-dependent rescue. Finally, we identify two additional transcription factors, Fkh1 and Fkh2 , that impact MCD1 expression. In combination, these findings provide new insights into the nuanced and multi-faceted transcriptional pathways that impact MCD1 expression. [ABSTRACT FROM AUTHOR]

Intraflagellar transport (IFT) proteins have been reported to regulate cell growth and differentiation as the essential functional component of primary cilia. The effects of IFT80 on early bone healing of extraction sockets have not been well studied. To investigate whether deletion of Ift80 in alveolar bone‐derived mesenchymal stem cells (aBMSCs) affected socket bone healing, we generated a mouse model of specific knockout of Ift80 in Prx1 mesenchymal lineage cells (Prx1Cre;IFT80f/f). Our results demonstrated that deletion of IFT80 in Prx1 lineage cells decreased the trabecular bone volume, ALP‐positive osteoblastic activity, TRAP‐positive osteoclastic activity, and OSX‐/COL I‐/OCN‐positive areas in tooth extraction sockets of Prx1Cre; IFT80f/f mice compared with IFT80f/f littermates. Furthermore, aBMSCs from Prx1Cre; IFT80f/f mice showed significantly decreased osteogenic markers and downregulated migration and proliferation capacity. Importantly, the overexpression of TAZ recovered significantly the expressions of osteogenic markers and migration capacity of aBMSCs. Lastly, the local administration of lentivirus for TAZ enhanced the expression of RUNX2 and OSX and promoted early bone healing of extraction sockets from Prx1Cre; IFT80f/f mice. Thus, IFT80 promotes osteogenesis and early bone healing of tooth sockets through the activation of TAZ/RUNX2 pathway. [ABSTRACT FROM AUTHOR]

Abnormal cytoplasmic localization and accumulation of pathological transactive response DNA binding protein of 43 kDa (TDP-43) underlies several devastating diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP). A key element is the correlation between disease progression and spatio-temporal propagation of TDP-43-mediated pathology in the central nervous system. Several lines of evidence support the concept of templated aggregation and cell to cell spreading of pathological TDP-43. To further investigate this mechanism in vivo, we explored the efficacy of capturing and masking the seeding-competent region of extracellular TDP-43 species. For this, we generated a novel monoclonal antibody (mAb), ACI-6677, that targets the pathogenic protease-resistant amyloid core of TDP-43. ACI-6677 has a picomolar binding affinity for TDP-43 and is capable of binding to all C-terminal TDP-43 fragments. In vitro, ACI-6677 inhibited TDP-43 aggregation and boosted removal of pathological TDP-43 aggregates by phagocytosis. When injecting FTLD-TDP brain extracts unilaterally in the CamKIIa-hTDP-43NLSm mouse model, ACI-6677 significantly limited the induction of phosphorylated TDP-43 (pTDP-43) inclusions. Strikingly, on the contralateral side, the mAb significantly prevented pTDP-43 inclusion appearance exemplifying blocking of the spreading process. Taken together, these data demonstrate for the first time that an immunotherapy targeting the protease-resistant amyloid core of TDP-43 has the potential to restrict spreading, substantially slowing or stopping progression of disease. [ABSTRACT FROM AUTHOR]

Objectives To investigate the potential role of shelterin dysfunction in naïve CD4+ T cells in the pathogenesis of Behçet's disease (BD). Methods Naïve CD4+ T cells were isolated from 40 BD patients and 40 sex- and age-matched healthy controls (HC). Senescent profiles, shelterin subunits expression, telomere length, telomerase activity and critical DNA damage response (DDR) were evaluated. Telomere repeat factor-2 (TRF2) silencing was conducted for further validation. Results Compared with HC, BD patients had significantly decreased naïve CD4+ T cells, increased cell apoptosis, senescence, and productions of TNF-α and IFN-γ upon activation. Notably, BD naïve CD4+ T cells had shortened telomere, impaired telomerase activity, and expressed lower levels of shelterin subunits TRF2, TRF1- and TRF2-Interacting Nuclear Protein 2 (TIN2) and Repressor/Activator Protein 1 (RAP1). Furthermore, BD naïve CD4+ T cells exhibited significantly increased DDR, evidenced by elevated phosphorylated ataxia telangiectasia (AT) mutated (pATM), phosphorylated p53 (pp53) and p21. Finally, TRF2 silencing markedly upregulated DDR, apoptosis and proinflammatory cytokines production in HC naïve CD4+ T cells. Conclusion Our study demonstrated that TRF2 deficiency in BD naïve CD4+ T cells promoted cell apoptosis and senescence, leading to proinflammatory cytokines overproduction. Therefore, restoring TRF2 might be a promising therapeutic strategy for BD. [ABSTRACT FROM AUTHOR]

Nucleotide excision repair (NER) is a major DNA repair system and hereditary defects in this system cause critical genetic diseases (e.g. xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy). Various proteins are involved in the eukaryotic NER system and undergo several post-translational modifications. Damaged DNA-binding protein 2 (DDB2) is a DNA damage recognition factor in the NER pathway. We previously demonstrated that DDB2 was SUMOylated in response to UV irradiation; however, its physiological roles remain unclear. We herein analysed several mutants and showed that the N-terminal tail of DDB2 was the target for SUMOylation; however, this region did not contain a consensus SUMOylation sequence. We found a SUMO-interacting motif (SIM) in the N-terminal tail that facilitated SUMOylation. The ubiquitination of a SUMOylation-deficient DDB2 SIM mutant was decreased, and its retention of chromatin was prolonged. The SIM mutant showed impaired NER, possibly due to a decline in the timely handover of the lesion site to XP complementation group C. These results suggest that the SUMOylation of DDB2 facilitates NER through enhancements in ubiquitination. [ABSTRACT FROM AUTHOR]

The plant AT protein and zinc-binding protein (PLATZ) genes, a novel cluster of plant-specific zinc-finger-dependent DNA-binding proteins, play a crucial role in regulating stress response and plant development. However, there has been little study focus on the role of the cucumber PLATZ family in assimilating loading in leaves. (1) In this study, a total of 12 PLATZ genes were identified from the cucumber genome. The cucumber PLATZ genes were clustered into five groups, and unevenly distributed on five chromosomes. A single pair of cucumber PLATZ genes underwent segmental duplication. (2) The results of genome-wide expression analysis suggested that the cucumber PLATZ genes were widely expressed in a wide range of cucumber tissues, with three PLATZ (PLATZ2, PLATZ6, and PLATZ12) genes exhibiting high expression in the vascular tissues of cucumber leaves. PLATZ2, PLATZ6, and PLATZ12 proteins were primarily located in cytomembrane and nucleus. (3) In VIGS-PLATZ6 plants, the expression of Galactinol synthase 1 (GolS1) and STACHYOSE SYNTHASE (STS), two genes involved in the synthesis of raffinose family oligosaccharides (RFOs) were observed to be decreased in cucumber leaves. In conclusion, the comprehensive analysis of the cucumber PLATZ family and the preliminary functional verification of PLATZ6 lay the foundation for the molecular and physiological functions of cucumber PLATZ genes. [ABSTRACT FROM AUTHOR]

Endocervical adenocarcinoma (ECA) is reported increasingly often in young women, and this aggressive disease lacks effective methods of targeted therapy. Since mismatch repair deficiency (dMMR) is an important biomarker for predicting response to immune checkpoint inhibitors, it is important to investigate the clinicopathological features and immune microenvironment of dMMR ECAs. We assessed 617 ECAs from representative tissue microarray sections, gathered clinicopathologic information, reviewed histological characteristics, and performed immunohistochemical staining for MMR, programmed cell death 1 (PD-L1), and other immune markers. Of 617 ECA samples, 20 (3.2%) cases had dMMR. Among them, loss of MMR-related proteins expression was observed in 17/562 (3.0%) human papilloma virus-associated (HPVA) adenocarcinoma and 3/55 (5.5%) non-HPV-associated (NHPVA) adenocarcinoma. In NHPVA cohort, dMMR status was observed in 3 (3/14, 15.0%) patients with clear cells. dMMR ECAs had a higher tendency to have a family history of cancer, larger tumor size, p16 negative, HPV E6/E7 mRNA in situ hybridization (HPV E6/E7 RNAscope) negative, and lower ki-67 index. Among the morphological variables evaluated, poor differentiation, necrosis, stromal tumor-infiltrating lymphocytes, peritumoral lymphocytes, and lymphoid follicles were easily recognized in the dMMR ECAs. In addition, dMMR ECAs had higher CD3+, CD8+, CD38+, CD68+ and PD-1+ immune cells. A relatively high prevalence of PD-L1 expression was observed in dMMR ECAs. dMMR ECAs were significantly more likely to present with a tumor-infiltrating lymphocytes -high/PD-L1-positive status. In conclusion, dMMR ECAs have some specific morphological features and a critical impact on the immune microenvironment, which may provide insights into improving responses to immunotherapy-included comprehensive treatment for ECAs in the future. [ABSTRACT FROM AUTHOR]

Introduction: The successful outcome of a pregnancy depends on the proper functioning uterine epithelium. DNA damage binding protein 1 and cullin 4-associated factor 2 (DCAF2), a conserved substrate receptor for the cullin 4-RING E3 ubiquitin ligase (CRL4) complex, is essential for maintaining genome stability by facilitating ubiquitin-mediated degradation of substrates. Methods: To better understand the physiological role of DCAF2 in female reproduction, we conducted a study using mice with conditional knockout (cKO) of DCAF2 in the uterus using the progesterone receptor Cre (PgrCre/+) mouse model. Results: Our results showed the cKO mice were completely infertile, despite having ovarian function. The cKO mice exhibited severely thin uteri, demonstrating notable defects in both the uterine epithelium and a lack of glands. In addition, there were impaired proliferation and differentiation of epithelial cells in the cKO mice, ultimately resulting in failed implantation. Moreover, through deciphering the uterine transcriptome of cKO mice, we revealed crucial differentially expressed genes associated with steroid signaling. Further experiments have demonstrated cKO mice exhibit elevated uterine PGR signaling and reduced estrogen receptor signaling, although the levels of progesterone and estrogen remained unaltered. These alterations may contribute to defects in epithelium. Discussion: Overall, our findings highlight a previously unrecognized but indispensable role for DCAF2 in the development of uterine luminal and glandular epithelium by orchestrating PGR and estrogen receptor responses. Its deficiency in the uterus leads to mouse infertility. [ABSTRACT FROM AUTHOR]

Context: The MEIS proteins, originally described as the "helper groups" of HOX proteins, are now becoming recognized as significant and incredibly diverse controllers of cellular behavior. The MEIS agents can incorporate a variety of epigenetic modulators into chromatin to aid in embryonic growth or tissue homeostasis by adult stem cells. The MEIS gene's expression, binding, stability, post-translational protein modification, and controlled intracellular availability are all fully regulated, and they all react to external stimuli. Objectives: We explored the diverse roles of MEIS proteins in cellular behavior and epigenetic regulation and investigated the potential of MEIS proteins as diagnostic and therapeutic biomarkers in cancers. Results: The MEIS proteins can be diagnostic and therapeutic biomarkers for cancer. The MEIS-PBX heterotrimeric is involved in neurogenesis processes, and the formation of the MEIS2 heterodimer with PAX6 and DLX2 can proliferate periplomerular dopaminergic neurons in the olfactory bulb. The estrogen receptor in breast cancer upregulates MEIS1 and FOXP3. The estrogen receptor pathway's cancer-related genes can express themselves more strongly when MEIS1-FOXP3 interacts with a positive feedback mechanism. Because MEIS1 expression is decreased in clear renal cell carcinoma (ccRCC) cell lines, MEIS1 may function as a tumor suppressor in the development of ccRCC. Conclusions: MEIS1 is recognized as one of the main contributors to the development of leukemias. The MEIS proteins influence the c-MYC signaling pathway, cell proliferation, and invasion of PC by contributing to its development. [ABSTRACT FROM AUTHOR]

Aldehyde exposure has been shown to lead to the formation of DNA damage comprising of DNA-protein crosslinks (DPCs), base adducts and interstrand or intrastrand crosslinks. DPCs have recently drawn more attention because of recent advances in detection and quantification of these adducts. DPCs are highly deleterious to genome stability and have been shown to block replication forks, leading to wide-spread mutagenesis. Cellular mechanisms to prevent DPC-induced damage include excision repair pathways, homologous recombination, and specialized proteases involved in cleaving the covalently bound proteins from DNA. These pathways were first discovered in formaldehyde-treated cells, however, since then, various other aldehydes have been shown to induce formation of DPCs in cells. Defects in DPC repair or aldehyde clearance mechanisms lead to various diseases including Ruijs-Aalfs syndrome and AMeD syndrome in humans. Here, we discuss recent developments in understanding how aldehydes form DPCs, how they are repaired, and the consequences of defects in these repair pathways. [ABSTRACT FROM AUTHOR]

Esophageal squamous cell carcinoma (ESCC) is one of the most prevalent malignancies with a 5‐year survival rate of only 15% in patients with advanced diseases. Tumor protein 63 (TP63), a master transcription factor (TF) in ESCC, cooperates with other TFs to regulate enhancers and/or promoters of target oncogenes, which in turn promotes tumorigenesis. TAR‐DNA‐binding protein‐43 (TDP‐43) is an RNA/DNA binding protein with elevated expression in several neoplasms. However, it remains unclear how TDP‐43 contributes to ESCC progression. In this study, TDP‐43 is identified as a novel oncogene with markedly upregulated expression in ESCC tissues through profiling expression levels of one hundred and fifty canonical RNA binding protein (RBP) genes in multiple ESCC patient cohorts. Importantly, TDP‐43 boosted TP63 expression via post‐transcriptionally stabilizing TP63 mRNAs as a RBP and promoting TP63 transcription as a TF binding to the TP63 promoter in ESCC cells. In contrast, the master TF TP63 also bound to the TDP‐43 promoter, accelerated TDP‐43 transcription, and caused a noticeable increase in TDP‐43 expression in ESCC cells. The findings highlight TDP‐43 as a viable therapeutic target for ESCC and uncover a hitherto unrecognized TDP‐43/TP63 circuit in cancer. [ABSTRACT FROM AUTHOR]

We analyzed genomic data from the prostate cancer of African- and European American men to identify differences contributing to racial disparity of outcome. We also performed FISH-based studies of Chromodomain helicase DNA-binding protein 1 (CHD1) loss on prostate cancer tissue microarrays. We created CHD1-deficient prostate cancer cell lines for genomic, drug sensitivity and functional homologous recombination (HR) activity analysis. Subclonal deletion of CHD1 was nearly three times as frequent in prostate tumors of African American than in European American men and it associates with rapid disease progression. CHD1 deletion was not associated with HR deficiency associated mutational signatures or HR deficiency as detected by RAD51 foci formation. This was consistent with the moderate increase of olaparib and talazoparib sensitivity with several CHD1 deficient cell lines showing talazoparib sensitivity in the clinically relevant concentration range. CHD1 loss may contribute to worse disease outcome in African American men. [ABSTRACT FROM AUTHOR]

Background: The medial temporal lobe (MTL) is hypothesized to be relatively spared in early-onset Alzheimer's disease (EOAD). Yet, detailed examination of MTL subfields and drivers of atrophy in amnestic EOAD is lacking. Methods: BioFINDER-2 participants with memory impairment, abnormal amyloid-β and tau-PET were included. Forty-one amnestic EOAD individuals ≤65 years and, as comparison, late-onset AD (aLOAD, ≥70 years, n = 154) and amyloid-β-negative cognitively unimpaired controls were included. MTL subregions and biomarkers of (co-)pathologies were measured. Results: AD groups showed smaller MTL subregions compared to controls. Atrophy patterns were similar across AD groups: aLOAD showed thinner entorhinal cortices than aEOAD; aEOAD showed thinner parietal regions than aLOAD. aEOAD showed lower white matter hyperintensities than aLOAD. No differences in MTL tau-PET or transactive response DNA binding protein 43-proxy positivity were found. Conclusions: We found evidence for MTL atrophy in amnestic EOAD and overall similar levels to aLOAD of MTL tau pathology and co-pathologies. [ABSTRACT FROM AUTHOR]

The progressive degeneration of motor neurons in amyotrophic lateral sclerosis (ALS) is accompanied by the formation of a broad array of cytoplasmic and nuclear neuronal inclusions (protein aggregates) largely containing RNA-binding proteins such as TAR DNA-binding protein 43 (TDP-43) or fused in sarcoma/translocated in liposarcoma (FUS/TLS). This process is driven by a liquid-to-solid phase separation generally from proteins in membrane-less organelles giving rise to pathological biomolecular condensates. The formation of these protein aggregates suggests a fundamental alteration in the mRNA expression or the levels of the proteins involved. Considering the role of the epigenome in gene expression, alterations in DNA methylation, histone modifications, chromatin remodeling, non-coding RNAs, and RNA modifications become highly relevant to understanding how this pathological process takes effect. In this review, we explore the evidence that links epigenetic mechanisms with the formation of protein aggregates in ALS. We propose that a greater understanding of the role of the epigenome and how this inter-relates with the formation of pathological LLPS in ALS will provide an attractive therapeutic target. [ABSTRACT FROM AUTHOR]

Two new bis-Schiff base metal(II) complexes viz., bis{N′-[(6-methoxynaphthalen-2-yl)methylidene]-N-methylcarbamohydrazonothioato}-copper(II) complex [Cu(L)2](1) and bis{N′-[(6-methoxynaphthalen-2-yl)methylidene]-N-methylcarbamohydrazonothioato}-nickel(II) complex [Ni(L)2](2), were synthesized and designed from a bidentate-N,S donor Schiff base ligand (HL), and characterized via a single crystal X-ray diffraction (SC-XRD) study, FT-IR, UV-visible and fluorescence spectroscopy, thermal analysis and DFT calculations. Single crystal X-ray diffraction analysis established the perfect square-planar geometry around the metal(II) ions obtained by the coordination of the two N and two S atoms from the two deprotonated HL ligands. Non-covalent intermolecular interactions, which are the main features of the supramolecular assembly, were investigated via Hirshfeld surface (HSs) analysis and 2D-fingerprint plots (FPs) for both the complexes. The supramolecular architecture within the crystal packing is stabilized by C–H⋯S, C–H⋯O and N–H⋯O type hydrogen bonds for the copper(II) complex [Cu(L)2](1), while C–H⋯S, C–H⋯N and N–H⋯O connecting units stabilize the supramolecular assembly for the nickel(II) complex [Ni(L)2](2). The thermal properties of the synthesized complexes offer valuable details on the stability of the complexes. The intermolecular interaction energies have been estimated and their topologies were explored using energy framework analysis. Their interactions with calf-thymus DNA (CT-DNA) and bovine serum albumin (BSA) proteins were also evaluated using UV-visible and fluorescence spectroscopic methods. Absorption titration studies revealed that they interacted strongly with CT-DNA. To gain insight into the potential antiviral applications, we examined the molecular docking of the copper(II) complex [Cu(L)2](1) and nickel(II) complex [Ni(L)2](2) with SARS-CoV-2 RNA binding protein (PDB ID: 6WKP), the Omicron RBD protein (PDB ID: 7WRL) and HIV-1 protease (PDB ID: 8CI7) viruses. They bind to the active positions of viral proteins and show good inhibition performance with target viral proteins. A stronger binding was predicted between the complexes and the SARS-CoV-2 RNA, Omicron and HIV-1 viruses through the negative values of binding affinity. The binding free energies computed using a molecular docking approach were analyzed in detail and the importance of specific interactions was outlined. Furthermore, molecular docking calculations of the complexes against DNA binding protein (PDB ID: 7UV6) were also evaluated. The calculated docking results suggested that they could be adopted as potential inhibitors for the development of new anti-SARS-CoV-2 Omicron and anti-HIV drugs. [ABSTRACT FROM AUTHOR]

DNA-protein interactions exert the fundamental structure of many pivotal biological processes, such as DNA replication, transcription, and gene regulation. However, accurate and efficient computational methods for identifying these interactions are still lacking. In this study, we propose a method ESM-DBP through refining the DNA-binding protein sequence repertory and domain-adaptive pretraining based the general protein language model. Our method considers the lacking exploration of general language model for DNA-binding protein domain-specific knowledge, so we screen out 170,264 DNA-binding protein sequences to construct the domain-adaptive language model. Experimental results on four downstream tasks show that ESM-DBP provides a better feature representation of DNA-binding protein compared to the original language model, resulting in improved prediction performance and outperforming the state-of-the-art methods. Moreover, ESM-DBP can still perform well even for those sequences with only a few homologous sequences. ChIP-seq on two predicted cases further support the validity of the proposed method. DNA-protein interactions form the fundamental structure of many pivotal biological processes. Here, authors propose ESM-DBP, which refines the DNA-binding protein sequence repertoire and domain-adaptive pretraining on general protein language models to predict DNA-binding protein-related tasks. [ABSTRACT FROM AUTHOR]

The majority of patients with Alzheimer's disease (AD) exhibit aggregates of Trans‐active response DNA binding protein 43 (TDP‐43) in their hippocampus, which is associated with a more aggressive disease progression. The TDP‐43 inclusions are commonly found in neurons, but also in astrocytes. The impact of the inclusions in astrocytes is less known. In the current study, we investigate the presence of phosphorylated TDP‐43 (pTDP‐43) inclusions in astrocytic endfeet and their potential association with blood–brain barrier (BBB) damage, glymphatic system dysfunction, and AD pathology. By staining postmortem hippocampal sections from AD patients and non‐demented controls against TDP‐43 and pTDP‐43 together with the astrocytic markers glial fibrillary acidic protein (GFAP), astrocytic endfeet marker Aquaporin‐4 (AQP4), and markers for BBB alterations (CD146) and leakiness (Immunoglobulin A), we demonstrate a close association between perivascular pTDP‐43 or TDP‐43 inclusions and GFAP or AQP4. These perivascular inclusions were more prominent in AD and correlated with the disease severity and loss of CD146 and AQP4. The findings indicate a relationship between pTDP‐43 accumulation in astrocytic endfeet and BBB and glymphatic system dysfunction, which may contribute to the downstream pathological events seen in AD patients and the aggressive disease progression. [ABSTRACT FROM AUTHOR]

Single-strand DNA-binding proteins SSB/RPA are ubiquitous and essential proteins that bind ssDNA in bacteria/eukaryotes and coordinate DNA metabolic processes such as replication, repair, and recombination. SSB protects ssDNA from degradation by nucleases, while also facilitating/regulating the activity of multiple partner proteins involved in DNA processes. Using Spi− assay, which detects aberrantly excised λ prophage from the E. coli chromosome as a measure of illegitimate recombination (IR) occurrence, we have shown that SSB inhibits IR in several DSB resection pathways. The conditional ssb-1 mutation produced a higher IR increase at the nonpermissive temperature than the recQ inactivation. A double ssb-1 recQ mutant had an even higher level of IR, while showing reduced homologous recombination (HR). Remarkably, the ssb gene overexpression complemented recQ deficiency in suppressing IR, indicating that the SSB function is epistatic to RecQ. Overproduced truncated SSBΔC8 protein, which binds to ssDNA, but does not interact with partner proteins, only partially complemented recQ and ssb-1 mutations, while causing an IR increase in otherwise wild-type bacteria, suggesting that ssDNA binding of SSB is required but not sufficient for effective IR inhibition, which rather entails interaction with RecQ and likely some other protein(s). Our results depict SSB as the main genome caretaker in E. coli, which facilitates HR while inhibiting IR. In enabling high-fidelity DSB repair under physiological conditions SSB is assisted by RecQ helicase, whose activity it controls. Conversely, an excess of SSB renders RecQ redundant for IR suppression. [ABSTRACT FROM AUTHOR]

The LysR-type transcriptional regulators (LTTRs) are DNA-binding proteins present in bacteria, archaea, and in algae. Knowledge about their distribution, abundance, evolution, structural organization, transcriptional regulation, fundamental roles in free life, pathogenesis, and bacteria–plant interaction has been generated. This review focuses on these aspects and provides a current picture of LTTR biology. [ABSTRACT FROM AUTHOR]