Acute lung injury (ALI) is a major cause of death in bacterial sepsis due to endothelial inflammation and endothelial permeability defects. Mitochondrial dysfunction is recognized as a key mediator in the pathogenesis of sepsis-induced ALI. Sirtuin 3 (SIRT3) is a histone protein deacetylase involved in preservation of mitochondrial function, which has been demonstrated in our previous study. Here, we investigated the effects of SIRT3 deficiency on impaired mitophagy to promote lung endothelial cells (ECs) pyroptosis during sepsis-induced ALI. We found that 3-TYP aggravated sepsis-induced ALI with increased lung ECs pyroptosis and enhanced NLRP3 activation. Mitochondrial reactive oxygen species (mtROS) and extracellular mitochondrial DNA (mtDNA) released from damaged mitochondria could be exacerbated in SIRT3 deficiency, which further elicit NLRP3 inflammasome activation in lung ECs during sepsis-induced ALI. Furthermore, Knockdown of SIRT3 contributed to impaired mitophagy via downregulating Parkin, which resulted in mitochondrial dysfunction. Moreover, pharmacological inhibition NLRP3 or restoration of SIRT3 attenuates sepsis-induced ALI and sepsis severity in vivo. Taken together, our results demonstrated SIRT3 deficiency facilitated mtROS production and cytosolic release of mtDNA by impaired Parkin-dependent mitophagy, promoting to lung ECs pyroptosis through the NLRP3 inflammasome activation, which providing potential therapeutic targets for sepsis-induced ALI.

Myotonic dystrophy type 1 (DM1) is a multisystemic disorder caused by a CTG triplet repeat expansion within the 3' untranslated region of the gene. Expression of the expanded allele generates RNA containing long tracts of CUG repeats (CUGexp RNA) that form hairpin structures and accumulate in nuclear RNA foci; however, the factors that control expression and the formation of CUGexp RNA foci remain largely unknown. We performed an unbiased small molecule screen in an immortalized human DM1 skeletal muscle myoblast cell line and identified HSP90 as a modifier of endogenous RNA foci. Small molecule inhibition of HSP90 leads to enhancement of RNA foci and upregulation of mRNA levels. Knockdown and overexpression of HSP90 in undifferentiated DM1 myoblasts validated the impact of HSP90 with upregulation and downregulation of mRNA, respectively. Furthermore, we identified p-STAT3 as a downstream mediator of HSP90 impacting levels of mRNA and RNA foci. Interestingly, differentiated cells exhibited an opposite effect of HSP90 inhibition displaying downregulation of mRNA through a mechanism independent of p-STAT3 involvement. This study has revealed a novel mediator for mRNA and foci regulation in DM1 cells with the potential to identify targets for future therapeutic intervention.

Myogenesis is a highly orchestrated process whereby muscle precursor cells, myoblasts, develop into muscle fibers to form skeletal muscle during embryogenesis and regenerate adult muscle. Here, we studied the RNA-binding protein FUS (fused in sarcoma), which has been implicated in muscular and neuromuscular pathologies but is poorly characterized in myogenesis. Given that FUS levels declined in human and mouse models of skeletal myogenesis, and that silencing FUS enhanced myogenesis, we hypothesized that FUS might be a repressor of myogenic differentiation. Interestingly, overexpression of FUS delayed myogenesis, accompanied by slower production of muscle differentiation markers. To identify the mechanisms through which FUS inhibits myogenesis, we uncovered RNA targets of FUS by ribonucleoprotein immunoprecipitation (RIP) followed by RNA-sequencing (RNA-seq) analysis. Stringent selection of the bound transcripts uncovered mRNA, encoding troponin T1 (TNNT1), as a major effector of FUS influence on myogenesis. We found that in myoblasts, FUS retained mRNA in the nucleus, preventing TNNT1 expression; however, reduction of FUS during myogenesis or by silencing FUS released mRNA for export to the cytoplasm, enabling TNNT1 translation and promoting myogenesis. We propose that FUS inhibits myogenesis by suppressing TNNT1 expression through a mechanism of nuclear mRNA retention.

A significant number of the genetic alterations observed in cancer patients lie within nonprotein-coding segments of the genome, including regions coding for long noncoding RNAs (lncRNAs). LncRNAs display aberrant expression in breast cancer (BrCa), but the functional implications of this altered expression remain to be elucidated. By performing transcriptome screen in a triple negative BrCa (TNBC) isogenic 2D and 3D spheroid model, we observed aberrant expression of >1000 lncRNAs during BrCa progression. The chromatin-associated lncRNA MANCR shows elevated expression in metastatic TNBC. MANCR is upregulated in response to cellular stress and modulates DNA repair and cell proliferation. MANCR promotes metastasis as MANCR-depleted cells show reduced cell migration, invasion, and wound healing in vitro, and reduced metastatic lung colonization in xenograft experiments in vivo. Transcriptome analyses reveal that MANCR modulates expression and pre-mRNA splicing of genes, controlling DNA repair and checkpoint response. MANCR promotes the transcription of NET1, a Rho-GEF that regulates DNA damage checkpoint and metastatic processes in , by differential promoter usage. Experiments suggest that MANCR regulates the expression of cancer-associated genes by modulating the association of various transcription factors and RNA-binding proteins. Our results identified the metastasis-promoting activities of MANCR in TNBC by -regulation of gene expression.

LSG1 is a conserved GTPase involved in ribosome assembly. It is required for the eviction of the nuclear export adapter NMD3 from the pre-60S subunit in the cytoplasm. In human cells, LSG1 has also been shown to interact with vesicle-associated membrane protein-associated proteins (VAPs) that are found primarily on the endoplasmic reticulum. VAPs interact with a large host of proteins which contain FFAT motifs (two phenylalanines (FF) in an acidic tract) and are involved in many cellular functions including membrane traffic and regulation of lipid transport. Here, we show that human LSG1 binds to VAPs via a noncanonical FFAT-like motif. Deletion of this motif specifically disrupts the localization of LSG1 to the ER, without perturbing LSG1-dependent recycling of NMD3 or modulation of LSG1 GTPase activity .

The histone variant H2A.Z plays important functions in the regulation of gene expression. In mammals, it is encoded by two genes, giving rise to two highly related isoforms named H2A.Z.1 and H2A.Z.2, which can have similar or antagonistic functions depending on the promoter. Knowledge of the physiopathological consequences of such functions emerges, but how the balance between these isoforms regulates tissue homeostasis is not fully understood. Here, we investigated the relative role of H2A.Z isoforms in intestinal epithelial homeostasis. Through genome-wide analysis of H2A.Z genomic localization in differentiating Caco-2 cells, we uncovered an enrichment of H2A.Z isoforms on the bodies of genes which are induced during enterocyte differentiation, stressing the potential importance of H2A.Z isoforms dynamics in this process. Through a combination of in vitro and in vivo experiments, we further demonstrated the two isoforms cooperate for stem and progenitor cells proliferation, as well as for secretory lineage differentiation. However, we found that they antagonistically regulate enterocyte differentiation, with H2A.Z.1 preventing terminal differentiation and H2A.Z.2 favoring it. Altogether, these data indicate that H2A.Z isoforms are critical regulators of intestine homeostasis and may provide a paradigm of how the balance between two isoforms of the same chromatin structural protein can control physiopathological processes.

Restricting the localization of evolutionarily conserved histone H3 variant CENP-A to the centromere is essential to prevent chromosomal instability (CIN), an important hallmark of cancers. Overexpressed CENP-A mislocalizes to non-centromeric regions and contributes to CIN in yeast, flies, and human cells. Centromeric localization of CENP-A is facilitated by the interaction of Mis18β with CENP-A specific chaperone HJURP. Cellular levels of Mis18β are regulated by β-transducin repeat containing protein (β-TrCP), an F-box protein of SCF (Skp1, Cullin, F-box) E3-ubiquitin ligase complex. Here, we show that defects in β-TrCP-mediated proteolysis of Mis18β contributes to the mislocalization of endogenous CENP-A and CIN in a triple-negative breast cancer (TNBC) cell line, MDA-MB-231. CENP-A mislocalization in β-TrCP depleted cells is dependent on high levels of Mis18β as depletion of Mis18β suppresses mislocalization of CENP-A in these cells. Consistent with these results, endogenous CENP-A is mislocalized in cells overexpressing Mis18β alone. In summary, our results show that β-TrCP-mediated degradation of Mis18β prevents mislocalization of CENP-A and CIN. We propose that deregulated expression of Mis18β may be one of the key mechanisms that contributes to chromosome segregation defects in cancers.

One of the primary reasons behind the pathogenesis of T cell acute lymphoblastic leukemia (T-ALL) is the deregulation of the transcription factor . The exon 4 of harbors several driver mutations, which abolishes its DNA-binding ability. The high frequency of C > T or G > A conversion in close vicinity of AID (Activation-induced cytidine deaminase)-hotspot motifs in the deregulated gene prompted us to investigate the role of AID in mutagenesis. Our results reveal that AID is expressed in T-ALL patient-derived cells, binds to fragile region (FR) in exon 4 of T cells in vivo, and generates a signature mutation pattern in this region. The mutation frequency in could be modulated upon overexpression of the AID gene in the knockout background, further suggesting the involvement of AID in mutagenesis. Importantly, various lines of experimentation reveal that could fold into parallel G-quadruplex, triplex, and hairpin structures, which could act as a replication/transcription block, causing mutagenesis. Thus, our results suggest that AID binds to exon 4 due to non-B DNA formation, causing U:G mismatches or replication blocks, which, when repaired erroneously, generates deleterious mutations, resulting in loss of functionality of , and thus becomes the cause of T-ALL.

Histone 3 lysine 4 methylation (H3K4me) is a highly evolutionary conserved chromatin modification associated with active transcription, and its three methylation states-mono, di, and trimethylation-mark distinct regulatory elements. However, whether H3K4me plays functional roles in transcriptional regulation or is merely a by-product of histone methyltransferases recruited to actively transcribed loci is still under debate. Here, we outline the studies that have addressed this question in yeast, , and mammalian systems. We review evidence from histone residue mutation, histone modifier manipulation, and epigenetic editing, focusing on the relative roles of H3K4me1 and H3K4me3. We conclude that H3K4me1 and H3K4me3 may have convergent functions in establishing open chromatin and promoting transcriptional activation during cell differentiation.

Mutations in the tumor suppressor gene are the most abundant genetic occurrences in cancer. Some of these mutations lead to loss of function of p53 protein, some are gain of function, and some variants are hypomorphic (partially functional). Currently, there is no clinical distinction between different p53 mutations and cancer therapy or prognosis. Mutations in the oligomerization domain of p53 appear to be quite distinct in function, compared to mutations in the DNA binding domain. Here we show that, like other p53 oligomerization domain mutants, the Ashkenazi-specific G334R mutant accumulates to very high levels in cells and is significantly impaired for the transactivation of canonical p53 target genes. Surprisingly, we find that this mutant retains the ability to bind to consensus p53 target sites. A mouse model reveals that mice containing the G334R variant show increased predisposition to cancer, but only a fraction of these mice develop late-onset cancer. We show that the G334R variant retains the ability to interact with the SP1 transcription factor and contributes to the transactivation of joint SP1-p53 target genes. The combined evidence indicates that G334R is a unique oligomerization domain mutant that retains some tumor suppressor function.

P4-ATPases comprise a family of lipid flippases that translocate lipids from the exoplasmic (or luminal) to the cytoplasmic leaflet of biological membranes. Of the 14 known human P4-ATPases, ATP8B2 is a phosphatidylcholine flippase at the plasma membrane, but its physiological function is not well understood. Although ATP8B2 could interact with both CDC50A and CDC50B, it required only the CDC50A interaction for its exit from the endoplasmic reticulum and subsequent transport to the plasma membrane. Three de novo monoallelic missense variations of ATP8B2 were found in patients with intellectual disability. None of these variations affected the interaction of ATP8B2 with CDC50A or its localization to the plasma membrane. However, variations of either of two amino acid residues, which are conserved in all P4-ATPases, significantly reduced the phosphatidylcholine flippase activity of ATP8B2. Furthermore, mutations in the corresponding residues of ATP8B1 and ATP11C were found to decrease their flippase activities toward phosphatidylcholine and phosphatidylserine, respectively. These results indicate that the conserved amino acid residues are crucial for the enzymatic activities of the P4-ATPases.

Androgen receptor inhibitors are commonly used for prostate cancer treatment, but acquired resistance is a significant problem. Codeletion of RB and p53 is common in castration resistant prostate cancers, however they are difficult to target pharmacologically. To comprehensively identify gene loss events that contribute to enzalutamide response, we performed a genome-wide CRISPR knockout screen in LNCaP prostate cancer cells. This revealed novel genes implicated in resistance that are largely unstudied. Gene loss events that confer enzalutamide sensitivity are enriched for GSEA categories related to stem cell and epigenetic regulation. We investigated the myeloid lineage stem cell factor HOXA9 as a candidate gene whose loss promotes sensitivity to enzalutamide. Cancer genomic data reveals that HOXA9 overexpression correlates with poor prognosis and characteristics of advanced prostate cancer. In cell culture, HOXA9 depletion sensitizes cells to enzalutamide, whereas overexpression drives enzalutamide resistance. Combination of the HOXA9 inhibitor DB818 with enzalutamide demonstrates synergy. This demonstrates the utility of our CRISPR screen data in discovering new approaches for treating enzalutamide resistant prostate cancer.

Parkinson's disease (PD) is an age-related progressive neurodegenerative disease. Previously, we identified midnolin () as a genetic risk factor for PD. Although copy number loss increases the risk of PD, the molecular function of MIDN remains unclear. To investigate the role of MIDN in PD, we established monoclonal knockout (KO) PC12 cell models. KO inhibited neurite outgrowth and neurofilament light chain () gene expression. Although MIDN is mainly localized in the nucleus, it does not encode DNA-binding domains. We therefore hypothesized that MIDN might bind to certain transcription factors and regulate gene expression. Of the candidate transcription factors, we focused on early growth response 1 (EGR1) because it is required for neurite outgrowth and its target genes are downregulated by KO. An interaction between MIDN and EGR1 was confirmed by immunoprecipitation. Surprisingly, although EGR1 protein levels were significantly increased in KO cells, the binding of EGR1 to the promoter and resulting transcriptional activity were downregulated as measured by luciferase assay and chromatin immunoprecipitation quantitative real-time polymerase chain reaction. Overall, we identified the MIDN-dependent regulation of EGR1 function. This mechanism may be an underlying reason for the neurite outgrowth defects of KO PC12 cells.

Smyd1, a member of the Smyd lysine methyltransferase family, plays an important role in myofibrillogenesis of skeletal and cardiac muscles. Loss of Smyd1b (a Smyd1 ortholog) function in zebrafish results in embryonic death from heart malfunction. encodes two isoforms, Smyd1b_tv1 and Smyd1b_tv2, differing by 13 amino acids due to alternative splicing. While alternative splicing is evolutionarily conserved, the isoform-specific expression and function of Smyd1b_tv1 and Smyd1b_tv2 remained unknown. Here we analyzed their expression and function in skeletal and cardiac muscles. Our analysis revealed expression of predominately in cardiac and in skeletal muscles. Using zebrafish models expressing only one isoform, we demonstrated that Smyd1b_tv1 is essential for cardiomyocyte differentiation and fish viability, whereas Smyd1b_tv2 is dispensable for heart development and fish survival. Cellular and biochemical analyses revealed that Smyd1b_tv1 differs from Smyd1b_tv2 in protein localization and binding with myosin chaperones. While Smyd1b_tv2 diffused in the cytosol of muscle cells, Smyd1b_tv1 was localized to M-lines and essential for sarcomere organization in cardiomyocytes. Co-IP analysis revealed a stronger binding of Smyd1b_tv1 with chaperones and cochaperones compared with Smyd1b_tv2. Collectively, these findings highlight the nonequivalence of Smyd1b isoforms in cardiomyocyte differentiation, emphasizing the critical role of Smyd1b_tv1 in cardiac function.

Fibroblast growth factors (FGFs) are key orchestrators of development, tissue homeostasis and repair. FGF receptor (FGFR) deficiency in mouse keratinocytes causes an inflammatory skin phenotype with similarities to atopic dermatitis, but the human relevance is unclear. Therefore, we generated human keratinocytes with a CRISPR/Cas9-induced knockout of . Loss of this receptor promoted the expression of interferon-stimulated genes and pro-inflammatory cytokines under homeostatic conditions and in particular in response to different inflammatory mediators. Expression of FGFR2 itself was strongly downregulated in cultured human keratinocytes exposed to various pro-inflammatory stimuli. This is relevant , because bioinformatics analysis of bulk and single-cell RNA-seq data showed strongly reduced expression of in lesional skin of atopic dermatitis patients, which likely aggravates the inflammatory phenotype. These results reveal a key function of FGFR2 in human keratinocytes in the suppression of inflammation and suggest a role of FGFR2 downregulation in the pathogenesis of atopic dermatitis and possibly other inflammatory diseases.

The CK1 family are conserved serine/threonine kinases with numerous substrates and cellular functions. The fission yeast CK1 orthologues Hhp1 and Hhp2 were first characterized as regulators of DNA repair, but the mechanism(s) by which CK1 activity promotes DNA repair had not been investigated. Here, we found that deleting Hhp1 and Hhp2 or inhibiting CK1 catalytic activities in yeast or in human cells increased double-strand breaks (DSBs). The primary pathways to repair DSBs, homologous recombination and nonhomologous end joining, were both less efficient in cells lacking Hhp1 and Hhp2 activity. To understand how Hhp1 and Hhp2 promote DNA damage repair, we identified new substrates of these enzymes using quantitative phosphoproteomics. We confirmed that Arp8, a component of the INO80 chromatin remodeling complex, is a bona fide substrate of Hhp1 and Hhp2 important for DNA repair. Our data suggest that Hhp1 and Hhp2 facilitate DNA repair by phosphorylating multiple substrates, including Arp8.

N-terminal acetyltransferase B (NatB) is a major contributor to the N-terminal acetylome and is implicated in several key cellular processes including apoptosis and proteostasis. However, the molecular mechanisms linking NatB-mediated N-terminal acetylation to apoptosis and its relationship with protein homeostasis remain elusive. In this study, we generated mouse embryonic fibroblasts (MEFs) with an inactivated catalytic subunit of NatB () to investigate the impact of NatB deficiency on apoptosis regulation. Through quantitative N-terminomics, label-free quantification, and targeted proteomics, we demonstrated that NatB does not influence the proteostasis of all its substrates. Instead, our focus on putative NatB-dependent apoptotic factors revealed that NatB serves as a protective shield against UBR4 and UBR1 Arg/N-recognin-mediated degradation. Notably, MEFs exhibited reduced responsiveness to an extrinsic pro-apoptotic stimulus, a phenotype that was partially reversible upon UBR4 Arg/N-recognin silencing and consequent inhibition of procaspase-8 degradation. Collectively, our results shed light on how the interplay between NatB-mediated acetylation and the Arg/N-degron pathway appears to impact apoptosis regulation, providing new perspectives in the field including in therapeutic interventions.

Osteoarthritis (OA) is a chronic degenerative disease characterized by subchondral osteosclerosis, mainly due to osteoblast activity. This research investigates the function of Sik1, a member of the AMP-activated protein kinase family, in OA. Proteomic analysis was conducted on clinical samples from 30 OA patients, revealing a negative correlation between Sik1 expression and OA. In vitro experiments utilized BMSCs to examine the effect of Sik1 on osteogenic differentiation. BMSCs were cultured and induced toward osteogenesis with specific media. Sik1 overexpression was achieved through lentiviral transfection, followed by analysis of osteogenesis-associated proteins using Western blotting, RT-qPCR, and alkaline phosphate staining. In vivo experiments involved destabilizing the medial meniscus in mice to establish an OA model, assessing the therapeutic potential of Sik1. The CT scans and histological staining were used to analyze subchondral bone alterations and cartilage damage. The findings show that Sik1 downregulation correlates with advanced OA and heightened osteogenic differentiation in BMSCs. Sik1 overexpression inhibits osteogenesis-related markers in vitro and reduces cartilage damage and subchondral osteosclerosis in vivo. Mechanistically, Sik1 modulates osteogenesis and subchondral bone changes through Runx2 activity regulation. The research emphasizes Sik1 as a promising target for treating OA, suggesting its involvement in controlling bone formation and changes in the subchondral osteosclerosis.

Protein tyrosine phosphatases (PTPs) play central roles in the regulation of cell signaling, organismal development, cellular differentiation and proliferation, and cancer. In the immune system, PTPs regulate the activation, differentiation and effector function of lymphocytes and myeloid cells whilst single-nucleotide polymorphisms (SNPs) in PTP-encoding genes have been identified as risk factors for the development of autoimmunity. In this review we describe the roles for PTP nonreceptor type 22 (PTPN22) in the regulation of T lymphocyte signaling and activation in autoimmunity, infection and cancer. We summarize recent progress in our understanding of the regulation of PTPN22 activity, the impact of autoimmune disease-associated SNPs on T cell responses and describe approaches to harness PTPN22 as a target to improve T cell-based immunotherapies in cancer.

Rab11 family interacting protein 4 (Rab11-FIP4) regulates endocytic trafficking. A possible role for Rab11-FIP4 in the regulation of lysosomal function has been proposed, but its precise function in the regulation of cellular homeostasis is unknown. By mRNA array and protein analysis, we found that Rab11-FIP4 is downregulated in the lysosomal storage disease cystinosis, which is caused by genetic defects in the lysosomal cystine transporter, cystinosin. Rescue of Rab11-FIP4 expression in fibroblasts re-established normal autophagosome levels and decreased LC3B-II expression in cystinotic cells. Furthermore, Rab11-FIP4 reconstitution increased the localization of the chaperone-mediated autophagy receptor LAMP2A at the lysosomal membrane. Treatment with genistein, a phytoestrogen that upregulates macroautophagy, or the CMA activator QX77 (CA77) restored Rab11-FIP4 expression levels in cystinotic cells supporting a cross-regulation between two independent autophagic mechanisms, lysosomal function and Rab11-FIP4. Improved cellular homeostasis in cystinotic cells rescued by Rab11-FIP4 expression correlated with decreased endoplasmic reticulum stress, an effect that was potentiated by Rab11 and partially blocked by expression of a dominant negative Rab11. Restoring Rab11-FIP4 expression in cystinotic proximal tubule cells increased the localization of the endocytic receptor megalin at the plasma membrane, suggesting that Rab11-FIP4 reconstitution has the potential to improve cellular homeostasis and function in cystinosis.