Genome-scale exon perturbation screens uncover exons critical for cell fitness.

CRISPR-Cas technology has transformed functional genomics, yet understanding of how individual exons differentially shape cellular phenotypes remains limited. Here, we optimized and conducted massively parallel exon deletion and splice-site mutation screens in human cell lines to identify exons that regulate cellular fitness. Fitness-promoting exons are prevalent in essential and highly expressed genes and commonly overlap with protein domains and interaction interfaces. Conversely, fitness-suppressing exons are enriched in nonessential genes, exhibiting lower inclusion levels, and overlap with intrinsically disordered regions and disease-associated mutations. In-depth mechanistic investigation of the screen-hit TAF5 alternative exon-8 revealed that its inclusion is required for assembly of the TFIID general transcription initiation complex, thereby regulating global gene expression output. Collectively, our orthogonal exon perturbation screens established a comprehensive repository of phenotypically important exons and uncovered regulatory mechanisms governing cellular fitness and gene expression.

Molecular insight into interactions between the Taf14, Yng1 and Sas3 subunits of the NuA3 complex.

The NuA3 complex is a major regulator of gene transcription and the cell cycle in yeast. Five core subunits are required for complex assembly and function, but it remains unclear how these subunits interact to form the complex. Here, we report that the Taf14 subunit of the NuA3 complex binds to two other subunits of the complex, Yng1 and Sas3, and describe the molecular mechanism by which the extra-terminal domain of Taf14 recognizes the conserved motif present in Yng1 and Sas3. Structural, biochemical, and mutational analyses show that two motifs are sandwiched between the two extra-terminal domains of Taf14. The head-to-toe dimeric complex enhances the DNA binding activity of Taf14, and the formation of the hetero-dimer involving the motifs of Yng1 and Sas3 is driven by sequence complementarity. In vivo assays in yeast demonstrate that the interactions of Taf14 with both Sas3 and Yng1 are required for proper function of the NuA3 complex in gene transcription and DNA repair. Our findings suggest a potential basis for the assembly of three core subunits of the NuA3 complex, Taf14, Yng1 and Sas3.

SETD7 Promotes Cell Proliferation and Migration via Methylation-mediated TAF7 in Clear Cell Renal Cell Carcinoma.

SET domain containing 7(SETD7), a member of histone methyltransferases, is abnormally expressed in multiple tumor types. However, the biological function and underlying molecular mechanism of SETD7 in clear cell renal cell carcinoma (ccRCC) remain unclear. Here, we explored the biological effects of SETD7-TAF7-CCNA2 axis on proliferation and metastasis in ccRCC. We identified both SETD7 and TAF7 were up-regulated and significantly promoted the proliferation and migration of ccRCC cells. Concurrently, there was a significant positive correlation between the expression of SETD7 and TAF7, and the two were colocalized in the nucleus. Mechanistically, SETD7 methylates TAF7 at K5 and K300 sites, resulting in the deubiquitination and stabilization of TAF7. Furthermore, re-expression of TAF7 could partially restore SETD7 knockdown inhibited ccRCC cells proliferation and migration. In addition, TAF7 transcriptionally activated to drive the expression of cyclin A2 (CCNA2). And more importantly, the methylation of TAF7 at K5 and K300 sites exhibited higher transcriptional activity of CCNA2, which promotes formation and progression of ccRCC. Our findings reveal a unique mechanism that SETD7 mediated TAF7 methylation in regulating transcriptional activation of CCNA2 in ccRCC progression and provide a basis for developing effective therapeutic strategies by targeting members of SETD7-TAF7-CCNA2 axis.

Taf1 knockout is lethal in embryonic male mice and heterozygous females show weight and movement disorders.

The TATA box-binding protein-associated factor 1 (TAF1) is a ubiquitously expressed protein and the largest subunit of the basal transcription factor TFIID, which plays a key role in initiation of RNA polymerase II-dependent transcription. TAF1 missense variants in human males cause X-linked intellectual disability, a neurodevelopmental disorder, and TAF1 is dysregulated in X-linked dystonia-parkinsonism, a neurodegenerative disorder. However, this field has lacked a genetic mouse model of TAF1 disease to explore its mechanism in mammals and treatments. Here, we generated and validated a conditional cre-lox allele and the first ubiquitous Taf1 knockout mouse. We discovered that Taf1 deletion in male mice was embryonically lethal, which may explain why no null variants have been identified in humans. In the brains of Taf1 heterozygous female mice, no differences were found in gross structure, overall expression and protein localisation, suggesting extreme skewed X inactivation towards the non-mutant chromosome. Nevertheless, these female mice exhibited a significant increase in weight, weight with age, and reduced movement, suggesting that a small subset of neurons was negatively impacted by Taf1 loss. Finally, this new mouse model may be a future platform for the development of TAF1 disease therapeutics.

circZNF532 promotes endothelial-to-mesenchymal transition in diabetic retinopathy by recruiting TAF15 to stabilize PIK3CD.

Endothelial-to-mesenchymal transition (EndMT) is a pivotal event in diabetic retinopathy (DR). This study explored the role of circRNA zinc finger protein 532 (circZNF532) in regulating EndMT in DR progression. Human retinal microvascular endothelial cells (HRMECs) were exposed to high glucose (HG) to induce the DR cell model. Actinomycin D-treated HRMECs were used to confirm the mRNA stability of phosphoinositide-3 kinase catalytic subunit δ (PIK3CD). The interaction between TATA-box-binding protein-associated factor 15 (TAF15) and circZNF532/PIK3CD was subsequently analyzed using RNA immunoprecipitation (RIP), RNA pull-down. It was found that HG treatment accelerated EndMT process, facilitated cell migration and angiogenesis, and enhanced PIK3CD and p-AKT levels in HRMECs, whereas si-circZNF532 transfection neutralized these effects. Further data showed that circZNF532 recruited TAF15 to stabilize PIK3CD, thus elevating PIK3CD expression. Following rescue experiments suggested that PIK3CD overexpression partially negated the inhibitory effect of circZNF532 silencing on EndMT, migration, and angiogenesis of HG-treated HRMECs. In conclusion, our results suggest that circZNF532 recruits TAF15 to stabilize PIK3CD, thereby facilitating EndMT in DR.

[Clinical features and genetic analysis of 17 Chinese pedigrees affected with X-linked intellectual disability].

OBJECTIVE: To analyze the clinical features and genetic etiology of 17 Chinese pedigrees affected with X-linked intellectual disability (XLID). METHODS: Seventeen pedigrees affected with unexplained intellectual disability which had presented at Henan Provincial People's Hospital from May 2021 to May 2023 were selected as the study subjects. Clinical data of the probands and their pedigree members were collected. Trio-whole exome sequencing (Trio-WES), Sanger sequencing and X chromosome inactivation (XCI) analysis were carried out. Pathogenicity of candidate variants was predicted based on the guidelines from the American College of Medical Genetics and Genomics and co-segregation analysis. RESULTS: The 17 probands, including 9 males and 8 females with an age ranging from 0.6 to 8 years old, had all shown mental retardation and developmental delay. Fourteen variants were detected by genetic testing, which included 4 pathogenic variants (MECP2: c.502C>T, MECP2: c.916C>T/c.806delG, IQSEC2: c.1417G>T), 4 likely pathogenic variants (MECP2: c.1157_1197del/c.925C>T, KDM5C: c.2128A>T, SLC6A8: c.1631C>T) and 6 variants of uncertain significance (KLHL15: c.26G>C, PAK3: c.970A>G/c.1520G>A, GRIA3: c.2153C>G, TAF1: c.2233T>G, HUWE1: c.10301T>A). The PAK3: c.970A>G, GRIA3: c.2153C>G and TAF1: c.2233T>G variants were considered as the genetic etiology for pedigrees 12, 14 and 15 by co-segregation analysis, respectively. The proband of pedigree 13 was found to have non-random XCI (81:19). Therefore, the PAK3: c.1520G>A variant may underlie its pathogenesis. CONCLUSION: Trio-WES has attained genetic diagnosis for the 17 XLID pedigrees. Sanger sequencing and XCI assay can provide auxiliary tests for the diagnosis of XLID.

TAF1 is needed for the proliferation and maturation of thyroid follicle cells via Notch signaling.

Thyroid dysgenesis (TD) is the common pathogenic mechanism of congenital hypothyroidism (CH). In addition, known pathogenic genes are limited to those that are directly involved in thyroid development. To identify additional candidate pathogenetic genes, we performed forward genetic screening for TD in zebrafish, followed by positional cloning. The candidate gene was confirmed in vitro using the Nthy-ori 3.1 cell line and in vivo using a zebrafish model organism. We obtained a novel zebrafish line with thyroid dysgenesis and identified the candidate pathogenetic mutation TATA-box binding protein associated Factor 1 (taf1) by positional cloning. Further molecular studies revealed that taf1 was needed for the proliferation of thyroid follicular cells by binding to the NOTCH1 promoter region. Knockdown of TAF1 impaired the proliferation and maturation of thyroid cells, thereby leading to thyroid dysplasia. This study showed that TAF1 promoted Notch signaling and that this association played a pivotal role in thyroid development.NEW & NOTEWORTHY In our study, we obtained a novel zebrafish line with thyroid dysgenesis (TD) and identified the candidate pathogenetic mutation TATA-box binding protein associated Factor 1 (taf1). Further researches revealed that taf1 was required for thyroid follicular cells by binding to the NOTCH1 promoter region. Our findings revealed a novel role of TAF1 in thyroid morphogenesis.

Therapeutic targeting of BET bromodomain and other epigenetic acetylrecognition domain-containing factors.

Development of cancer therapies targeting chromatin modifiers and transcriptional regulatory factors is rapidly expanding to include new targets and novel targeting strategies. At the same time, basic molecular research continues to refine our understanding of the epigenetic mechanisms regulating transcription, gene expression, and oncogenesis. This mini-review focuses on cancer therapies targeting the chromatin-associated factors that recognize histone lysine acetylation. Recently reported safety and efficacy are discussed for inhibitors targeting the bromodomains of bromodomain and extraterminal domain (BET) family proteins. In light of recent results indicating that the transcriptional regulator BRD4-PTEFb can function independently of BRD4's bromodomains, the clinical trial performance of these BET inhibitors is placed in a broader context of existing and potential strategies for targeting BRD4-PTEFb. Recently developed therapies targeting bromodomain-containing factors within the SWI/SNF (BAF) family of chromatin remodeling complexes are discussed, as is the potential for targeting the bromodomain-containing transcription factor TAF1 and the YEATS acetylrecognition domain-containing factor GAS41. Recent findings regarding the selectivity and combinatorial specificity of acetylrecognition are highlighted. In conclusion, the potential for further development is discussed with a focus on proximity-based therapies targeting this class of epigenetic factors.

TATA-binding associated factors have distinct roles during early mammalian development.

Early embryonic development is a finely orchestrated process that requires precise regulation of gene expression coordinated with morphogenetic events. TATA-box binding protein-associated factors (TAFs), integral components of transcription initiation coactivators like TFIID and SAGA, play a crucial role in this intricate process. Here we show that disruptions in TAF5, TAF12 and TAF13 individually lead to embryonic lethality in the mouse, resulting in overlapping yet distinct phenotypes. Taf5 and Taf12 mutant embryos exhibited a failure to implant post-blastocyst formation, and Taf5 mutants have aberrant lineage specification within the inner cell mass. In contrast, Taf13 mutant embryos successfully implant and form egg-cylinder stages but fail to initiate gastrulation. Strikingly, we observed a depletion of pluripotency factors in TAF13-deficient embryos, including OCT4, NANOG and SOX2, highlighting an indispensable role of TAF13 in maintaining pluripotency. Transcriptomic analysis revealed distinct gene targets affected by the loss of TAF5, TAF12 and TAF13. Thus, we propose that TAF5, TAF12 and TAF13 convey locus specificity to the TFIID complex throughout the mouse genome.

Phosphorylation of the compartmentalized PKA substrate TAF15 regulates RNA-protein interactions.

Spatiotemporal-controlled second messengers alter molecular interactions of central signaling nodes for ensuring physiological signal transmission. One prototypical second messenger molecule which modulates kinase signal transmission is the cyclic-adenosine monophosphate (cAMP). The main proteinogenic cellular effectors of cAMP are compartmentalized protein kinase A (PKA) complexes. Their cell-type specific compositions precisely coordinate substrate phosphorylation and proper signal propagation which is indispensable for numerous cell-type specific functions. Here we present evidence that TAF15, which is implicated in the etiology of amyotrophic lateral sclerosis, represents a novel nuclear PKA substrate. In cross-linking and immunoprecipitation experiments (iCLIP) we showed that TAF15 phosphorylation alters the binding to target transcripts related to mRNA maturation, splicing and protein-binding related functions. TAF15 appears to be one of multiple PKA substrates that undergo RNA-binding dynamics upon phosphorylation. We observed that the activation of the cAMP-PKA signaling axis caused a change in the composition of a collection of RNA species that interact with TAF15. This observation appears to be a broader principle in the regulation of molecular interactions, as we identified a significant enrichment of RNA-binding proteins within endogenous PKA complexes. We assume that phosphorylation of RNA-binding domains adds another layer of regulation to binary protein-RNAs interactions with consequences to RNA features including binding specificities, localization, abundance and composition.

Insights into Molecular Diversity within the FUS/EWS/TAF15 Protein Family: Unraveling Phase Separation of the N-Terminal Low-Complexity Domain from RNA-Binding Protein EWS.

The FET protein family, comprising FUS, EWS, and TAF15, plays crucial roles in mRNA maturation, transcriptional regulation, and DNA damage response. Clinically, they are linked to Ewing family tumors and neurodegenerative diseases such as amyotrophic lateral sclerosis. The fusion protein EWS::FLI1, the causative mutation of Ewing sarcoma, arises from a genomic translocation that fuses a portion of the low-complexity domain (LCD) of EWS (EWSLCD) with the DNA binding domain of the ETS transcription factor FLI1. This fusion protein modifies transcriptional programs and disrupts native EWS functions, such as splicing. The exact role of the intrinsically disordered EWSLCD remains a topic of active investigation, but its ability to phase separate and form biomolecular condensates is believed to be central to EWS::FLI1's oncogenic properties. Here, we used paramagnetic relaxation enhancement NMR, microscopy, and all-atom molecular dynamics (MD) simulations to better understand the self-association and phase separation tendencies of the EWSLCD. Our NMR data and mutational analysis suggest that a higher density and proximity of tyrosine residues amplify the likelihood of condensate formation. MD simulations revealed that the tyrosine-rich termini exhibit compact conformations with unique contact networks and provided critical input on the relationship between contacts formed within a single molecule (intramolecular) and inside the condensed phase (intermolecular). These findings enhance our understanding of FET proteins' condensate-forming capabilities and underline differences between EWS, FUS, and TAF15.

Unveiling hormone-stimulated gene mechanisms in prostate cancer: A prognostic model, immune infiltration analysis, and drug sensitivity study.

Hormones promote the progression of prostate cancer (PRCA) through the activation of a complex regulatory network. Inhibition of hormones or modulation of specific network nodes alone is insufficient to suppress the entire oncogenic network. Therefore, it is imperative to elucidate the mechanisms underlying the occurrence and development of PRCA in order to identify reliable diagnostic markers and therapeutic targets. To this end, we used publicly available data to analyze the potential mechanisms of hormone-stimulated genes in PRCA, construct a prognostic model, and assess immune infiltration and drug sensitivity. The single-cell RNA-sequencing data of PRCA were subjected to dimensionality reduction clustering and annotation, and the cells were categorized into two groups based on hormone stimulus-related scores. The differentially expressed genes between the two groups were screened and incorporated into the least absolute shrinkage and selection operator machine learning algorithm, and a prognostic model comprising six genes (ZNF862, YIF1A, USP22, TAF7, SRSF3, and SPARC) was constructed. The robustness of the model was validation through multiple methods. Immune infiltration scores in the two risk groups were calculated using three different algorithms. In addition, the relationship between the model genes and immune cell infiltration, and that between risk score and immune cell infiltration were analyzed. Drug sensitivity analysis was performed for the model genes and risk score using public databases to identify potential candidate drugs. Our findings provide novel insights into the mechanisms of hormone-stimulated genes in PRCA progression, prognosis, and drug screening.

TAF15 amyloid filaments in frontotemporal lobar degeneration.

Frontotemporal lobar degeneration (FTLD) causes frontotemporal dementia (FTD), the most common form of dementia after Alzheimer's disease, and is often also associated with motor disorders1. The pathological hallmarks of FTLD are neuronal inclusions of specific, abnormally assembled proteins2. In the majority of cases the inclusions contain amyloid filament assemblies of TAR DNA-binding protein 43 (TDP-43) or tau, with distinct filament structures characterizing different FTLD subtypes3,4. The presence of amyloid filaments and their identities and structures in the remaining approximately 10% of FTLD cases are unknown but are widely believed to be composed of the protein fused in sarcoma (FUS, also known as translocated in liposarcoma). As such, these cases are commonly referred to as FTLD-FUS. Here we used cryogenic electron microscopy (cryo-EM) to determine the structures of amyloid filaments extracted from the prefrontal and temporal cortices of four individuals with FTLD-FUS. Surprisingly, we found abundant amyloid filaments of the FUS homologue TATA-binding protein-associated factor 15 (TAF15, also known as TATA-binding protein-associated factor 2N) rather than of FUS itself. The filament fold is formed from residues 7-99 in the low-complexity domain (LCD) of TAF15 and was identical between individuals. Furthermore, we found TAF15 filaments with the same fold in the motor cortex and brainstem of two of the individuals, both showing upper and lower motor neuron pathology. The formation of TAF15 amyloid filaments with a characteristic fold in FTLD establishes TAF15 proteinopathy in neurodegenerative disease. The structure of TAF15 amyloid filaments provides a basis for the development of model systems of neurodegenerative disease, as well as for the design of diagnostic and therapeutic tools targeting TAF15 proteinopathy.

TAF7L regulates early stages of male germ cell development in the rat.

Male germ cell development is dependent on the orchestrated regulation of gene networks. TATA-box binding protein associated factors (TAFs) facilitate interactions of TATA-binding protein with the TATA element, which is known to coordinate gene transcription during organogenesis. TAF7 like (Taf7l) is situated on the X chromosome and has been implicated in testis development. We examined the biology of TAF7L in testis development using the rat. Taf7l was prominently expressed in preleptotene to leptotene spermatocytes. To study the impact of TAF7L on the testis we generated a global loss-of-function rat model using CRISPR/Cas9 genome editing. Exon 3 of the Taf7l gene was targeted. A founder was generated possessing a 110 bp deletion within the Taf7l locus, which resulted in a frameshift and the premature appearance of a stop codon. The mutation was effectively transmitted through the germline. Deficits in TAF7L did not adversely affect pregnancy or postnatal survival. However, the Taf7l disruption resulted in male infertility due to compromised testis development and failed sperm production. Mutant germ cells suffer meiotic arrest at late zygotene/early pachynema stages, with defects in sex body formation. This testis phenotype was more pronounced than previously described for the subfertile Taf7l null mouse. We conclude that TAF7L is essential for male germ cell development in the rat.

Participation of the TBP-associated factors (TAFs) in cell differentiation.

The understanding of the mechanisms that regulate gene expression to establish differentiation programs and determine cell lineages, is one of the major challenges in Developmental Biology. Besides the participation of tissue-specific transcription factors and epigenetic processes, the role of general transcription factors has been ignored. Only in recent years, there have been scarce studies that address this issue. Here, we review the studies on the biological activity of some TATA-box binding protein (TBP)-associated factors (TAFs) during the proliferation of stem/progenitor cells and their involvement in cell differentiation. Particularly, the accumulated evidence suggests that TAF4, TAF4b, TAF7L, TAF8, TAF9, and TAF10, among others, participate in nervous system development, adipogenesis, myogenesis, and epidermal differentiation; while TAF1, TAF7, TAF15 may be involved in the regulation of stem cell proliferative abilities and cell cycle progression. On the other hand, evidence suggests that TBP variants such as TBPL1 and TBPL2 might be regulating some developmental processes such as germ cell maturation and differentiation, myogenesis, or ventral specification during development. Our analysis shows that it is necessary to study in greater depth the biological function of these factors and its participation in the assembly of specific transcription complexes that contribute to the differential gene expression that gives rise to the great diversity of cell types existing in an organism. The understanding of TAFs' regulation might lead to the development of new therapies for patients which suffer from mutations, alterations, and dysregulation of these essential elements of the transcriptional machinery.

Deletion of taf1 and taf5 in zebrafish capitulate cardiac and craniofacial abnormalities associated with TAFopathies through perturbations in metabolism.

Intellectual disability is a neurodevelopmental disorder that affects 2-3% of the general population. Syndromic forms of intellectual disability frequently have a genetic basis and are often accompanied by additional developmental anomalies. Pathogenic variants in components of TATA-binding protein associated factors (TAFs) have recently been identified in a subset of patients with intellectual disability, craniofacial hypoplasia, and congenital heart disease. This syndrome has been termed as a TAFopathy and includes mutations in TATA binding protein (TBP), TAF1, TAF2, and TAF6. The underlying mechanism by which TAFopathies give rise to neurodevelopmental, craniofacial, and cardiac abnormalities remains to be defined. Through a forward genetic screen in zebrafish, we have recovered a recessive mutant phenotype characterized by craniofacial hypoplasia, ventricular hypoplasia, heart failure at 96 h post-fertilization and lethality, and show it is caused by a nonsense mutation in taf5. CRISPR/CAS9 mediated gene editing revealed that these defects where phenocopied by mutations in taf1 and taf5. Mechanistically, taf5-/- zebrafish displayed misregulation in metabolic gene expression and metabolism as evidenced by RNA sequencing, respiration assays, and metabolite studies. Collectively, these findings suggest that the TAF complex may contribute to neurologic, craniofacial, and cardiac development through regulation of metabolism.

Hierarchical TAF1-dependent co-translational assembly of the basal transcription factor TFIID.

Large heteromeric multiprotein complexes play pivotal roles at every step of gene expression in eukaryotic cells. Among them, the 20-subunit basal transcription factor TFIID nucleates the RNA polymerase II preinitiation complex at gene promoters. Here, by combining systematic RNA-immunoprecipitation (RIP) experiments, single-molecule imaging, proteomics and structure-function analyses, we show that human TFIID biogenesis occurs co-translationally. We discovered that all protein heterodimerization steps happen during protein synthesis. We identify TAF1-the largest protein in the complex-as a critical factor for TFIID assembly. TAF1 acts as a flexible scaffold that drives the co-translational recruitment of TFIID submodules preassembled in the cytoplasm. Altogether, our data suggest a multistep hierarchical model for TFIID biogenesis that culminates with the co-translational assembly of the complex onto the nascent TAF1 polypeptide. We envision that this assembly strategy could be shared with other large heteromeric protein complexes.

lncRNA GMDS-AS1 restrains lung adenocarcinoma progression via recruiting TAF15 protein to stabilize SIRT1 mRNA.

Aim: To explore the roles of GMDS-AS1 in the epithelial-mesenchymal transition (EMT) of lung adenocarcinoma (LUAD). Materials & methods: Cell functions were detected by flow cytometry, cell counting kit-8, wound healing assays and transwell assays. RNA immunoprecipitation and pull-down assays were applied for determining the interaction among GMDA-AS1, TAF15 and SIRT1. A subcutaneous xenograft model was established. Results: GMDS-AS1 downregulation was associated with poor survival of LUAD patients. GMDS-AS1 repressed malignant phenotypes, tumor growth and EMT in vitro and in vivo. Mechanically, GMDS-AS1 recruited TAF15 protein to stabilize SIRT1 mRNA and thereby deacetylated p65 and reduced the recruitment of p65 to MMP-9 promoter, thus inhibiting MMP-9 expression. Conclusion: GMDS-AS1 represses EMT by recruiting TAF15 protein to stabilize SIRT1 mRNA and deacetylate p65, thus restraining LUAD progression.

GMDS-AS1, a novel lncRNA, is involved in the progression of lung adenocarcinoma (LUAD), but the mechanism by which GMDS-AS1 regulates LUAD progression remains largely unknown. This study found that GMDS-AS1 was downregulated in LUAD patients, and its low expression was associated with advanced metastasis and poor survival. Overexpression of GMDS-AS1 significantly impaired tumor cell viability, proliferation, migration, invasion and epithelial­mesenchymal transition but enhanced apoptosis. In addition, GMDS-AS1 repressed tumor growth in mice. GMDS-AS1 functioned as a tumor suppressor in LUAD by recruiting TAF15 protein to stabilize SIRT1 mRNA and deacetylate p65, thus decreasing the recruitment of p65 to MMP-9 promoter. These findings have uncovered a novel mechanism underlying LUAD progression and suggested potential prognostic biomarkers and therapeutic targets.

m6A-enriched lncRNA LINC00839 promotes tumor progression by enhancing TAF15-mediated transcription of amine oxidase AOC1 in nasopharyngeal carcinoma.

Dysregulation of long noncoding RNAs (lncRNAs) contributes to tumorigenesis by modulating specific cancer-related pathways, but the roles of N6-methyladenosine (m6A)-enriched lncRNAs and underlying mechanisms remain elusive in nasopharyngeal carcinoma (NPC). Here, we reanalyzed the previous genome-wide analysis of lncRNA profiles in 18 pairs of NPC and normal tissues as well as in ten paired samples from NPC with or without post-treatment metastases. We discerned that an oncogenic m6A-enriched lncRNA, LINC00839, which was substantially upregulated in NPC and correlated with poor clinical prognosis, promoted NPC growth and metastasis both in vitro and in vivo. Mechanistically, by using RNA pull-down assay combined with mass spectrometry, we found that LINC00839 interacted directly with the transcription factor, TATA-box binding protein associated factor (TAF15). Besides, chromatin immunoprecipitation and dual-luciferase report assays demonstrated that LINC00839 coordinated the recruitment of TAF15 to the promoter region of amine oxidase copper-containing 1 (AOC1), which encodes a secreted glycoprotein playing vital roles in various cancers, thereby activating AOC1 transcription in trans. In this study, potential effects of AOC1 in NPC progression were first proposed. Moreover, ectopic expression of AOC1 partially rescued the inhibitory effect of downregulation of LINC00839 in NPC. Furthermore, we showed that silencing vir-like m6A methyltransferase-associated (VIRMA) and insulin-like growth factor 2 mRNA-binding proteins 1 (IGF2BP1) attenuated the expression level and RNA stability of LINC00839 in an m6A-dependent manner. Taken together, our study unveils a novel oncogenic VIRMA/IGF2BP1-LINC00839-TAF15-AOC1 axis and highlights the significance and prognostic value of LINC00839 expression in NPC carcinogenesis.

TAF15 exacerbates nonalcoholic steatohepatitis progression by regulating lipid metabolism and inflammation via FASN and p65 NF-κB.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) consists of a broad spectrum of conditions, and nonalcoholic steatohepatitis (NASH) is the advanced form of NAFLD. TAF15 is a DNA and RNA binding protein and is involved in crucial inflammatory signalling pathways. We aimed to investigate the role of TAF15 in the progression of NASH and the underlying molecular mechanism. METHODS: We generated mice with hepatocyte-specific knockdown and overexpression of TAF15 using a specific adeno-associated virus (AAV). NASH models were established by feeding mice high-fat and high-cholesterol diets and methionine- and choline-deficient diets. Cleavage under targets and tagmentation and dual-luciferase reporter assays were performed to investigate the effect of TAF15 on FASN transcription. Coimmunoprecipitation and immunofluorescence assays were conducted to explore the interaction of TAF15 and p65. In vitro coculture systems were established to study the interactions of hepatocytes, macrophages and HSCs. RESULTS: TAF15 was significantly increased in the livers of mouse NASH models and primary hepatocyte NASH model. Knockdown of TAF15 inhibited steatosis, inflammation and fibrosis, while overexpression of TAF15 promoted NASH phenotypes. Mechanistically, TAF15 bound directly to the promoter region of FASN to facilitate its expression, thereby promoting steatosis. Moreover, TAF15 interacted with p65 and activated the NF-κB signalling pathway, increasing the secretion of proinflammatory cytokines and triggering M1 macrophage polarization. Treatment with the FASN inhibitor orlistat partially reversed the phenotypes. CONCLUSIONS: These results suggested that TAF15 exacerbated NASH progression by regulating lipid metabolism and inflammation via transcriptional activation of FASN and interacting with p65 to activate the NF-κB signalling pathway.