The emerging data indicates that non-coding RNAs (ncRNAs) epresent more than the “junk sequences” of the genome. Both miRNAs and long non-coding RNAs (lncRNAs) are involved in fundamental biological processes, and their deregulation may lead to oncogenesis and other diseases. As an important RNA-binding protein (RBP), heterogeneous nuclear ribonucleoprotein K (hnRNPK) is known to regulate gene expression through the RNA-binding domain involved in various pathways, such as transcription, splicing, and translation. HnRNPK is a highly conserved gene that is abundantly expressed in mammalian cells. The interaction of hnRNPK and ncRNAs defines the novel way through which ncRNAs affect the expression of protein-coding genes and form autoregulatory feedback loops. This review summarizes the interactions of hnRNPK and ncRNAs in regulating gene expression at transcriptional and post-transcriptional levels or by changing the genomic structure, highlighting their involvement in carcinogenesis, glucose metabolism, stem cell differentiation, virus infection and other cellular functions. Drawing connections between such discoveries might provide novel targets to control the biological outputs of cells in response to different stimuli. In the human genome, less than 2% of the sequence has proved to be protein-coding genes—the sequence mainly comprises noncoding segments which were once considered “junk sequences” . RecentlAs one group of trans-acting factors, RNA-binding proteins (RBPs) are typically thought of as proteins that bind RNA through one or multiple RNA-binding domains (RBDs), such as the RNA recognition motif (RRM), the hnRNPK homology domain (KH) or the DEAD box helicase domain, and have been accepted as key regulators in transcriptional and post-transcriptional events . RBPs wic-Src, c-Myc, p21, eIF4E, r15-LOX, UCP2 that encode proteins involved in cell proliferation, apoptosis, and differentiation [Heterogeneous nuclear ribonucleoproteins (hnRNPs) are a large family of RBPs that are crucial for multiple aspects of RNA metabolism, while hnRNPK is the most extensively studied member of the hnRNP family involved in both physiological and pathological processes, such as spermatogenesis, nervous system and ovary development, erythroid differentiation, organogenesis, and carcinogenesis ,17,18. Hntiation . RecentlAs mentioned above, lncRNAs are the biggest class of ncRNAs. They are greater than 200 nt in length and are emerging as the key regulators of many important biological processes impacting differentiation, development, and disease in every branch of life ,22,23. TLncRNA-p21, ~3.0 kb in length, has been identified as located in and transcribed from the cell-cycle regulator gene p21 locus; it is a novel regulator of cell proliferation, apoptosis and DNA damage response, and plays a key role in development and diseases [LncRNA-p21 regulates gene expression by associating with the RNA-binding protein hnRNPK. Two groups have confirmed that the p53-inducible lncRNA-p21 bound to hnRNPK to mediate gene repression in response to DNA damage [lncRNA-p21 deletion and indicated that lncRNA-p21 deficiency could reduce p21 expression and promoted the proliferation of mouse embryonic fibroblasts (MEFs) [lncRNA-p21 functions in concert with hnRNPK to promote p53-mediated expression of neighboring gene, p21, predominantly in cis, and they support a model in which lncRNA-p21 and hnRNPK act as transcriptional coactivators for local p53-mediated transcription. However, Huarte et al. did not detect any diminution in the expression of p21 upon lncRNA-p21 knockdown in MEFs using siRNA. They suggested that lncRNA-p21 is a downstream effector of p53-dependent transcriptional response and is shown to mediate its function in trans by associating with hnRNPK; this interaction is needed for the proper localization of hnRNPK at loci of repressed genes and the regulation of p53-mediated apoptosis. A potential explanation for the discrepancy between the two studies is the different approaches used to knockdown lincRNA-p21. There could be a more complicated function mechanism for hnRNPK–lncRNA-p21 interactions in apoptosis and DNA damage response, and additional experiments are needed to clarify this issue in the future.diseases ,51. LncRA damage ,26,27. Hs (MEFs) . They deLincRNA-p21 also plays a role in regulating somatic reprogramming through interacting with hnRNPK [lincRNA-p21 blocks reprogramming by forming a complex with hnRNPK and either H3K9 methyltransferase SETDB1 or DNA methyltransferase DNMT1. In combination, the two factors form repressive complexes containing either SETDB1 or DNMT1, preserving the high levels of H3K9me3 or CpG methylation at the pluripotency gene promoters. Nanog and Lin28a are two important pluripotency regulators. LincRNA-p21 may block somatic cell reprogramming by CpG methylation at the promoters of a subset of genes  and H3K9me3 maintenance at the promoters of another set of pluripotency genes , which are both repressed by hnRNPK-mediated transcriptional regulation. Moreover, Lin28a and Nanog are significantly upregulated by hnRNPK or lincRNA-p21 knockdown. Therefore, lincRNA-p21 exerts its role by reprogramming mainly through hnRNPK, which may constitute the essential functional unit in somatic cell reprogramming regulatory networks.h hnRNPK . MechaniTUNA), shows remarkable sequence conservation in vertebrates; it is required for the maintenance of pluripotency and is specifically expressed in the central nervous system (CNS) of zebrafish, mice, and humans. Indeed, TUNA expression was increased when mESCs differentiated toward the neural lineages, and TUNA depletion inhibited the neural differentiation of ESCs. TUNA formed an RNA–multiprotein complex with hnRNPK and PTBP1 that was specifically enriched at the promoters of Sox2, Nanog and Fgf4, and activated these pluripotency genes directly [TUNA depletion. Thus, TUNA represents a lncRNA that is important for at least two cell states  and probably operates through multiple molecular mechanisms. In addition, Lncenc1, a highly abundant long noncoding RNA in nESC (naive embryonic stem cells), also interacts with hnRNPK and PTBP1, which regulate the transcription of glycolytic genes, thereby maintaining the self-renewal of nESCs [Lncenc1, the expression of glycolysis-associated genes is significantly reduced and the glycolytic activity is substantially impaired, as indicated by more than 50% reduction in the levels of glucose consumption, lactate production, and the extracellular acidification rate. The functions of Lncenc1-mediated hnRNPK and PTBP1 recruitment in linking energy metabolism and the naive state of ESCs may enhance the understanding of the molecular basis underlying naive pluripotency. These findings suggest that hnRNPK–lncRNAs interactions may be key regulators of complexity in the networks controlling stem cell biology and disease pathophysiology. LncRNAs are emerging as important players in many biological processes including embryonic development, stem cell self-renewal, and differentiation . Recentldirectly . Importaof nESCs . Upon thpancEts-1 (Ets-1 promoter-associated noncoding RNA) as a novel 1395 nt lncRNA associated with the poor outcome of NB through the mining of a public microarray dataset [PancEts-1 is up-regulated in NB tissues and cell lines and promotes the growth, invasion, and metastasis of NB cells in vitro and in vivo. In addition, PancEts-1 bound to the KH2 domain of hnRNPK to facilitate its physical interaction with β-catenin, while hnRNPK stabilized the β-catenin by inhibiting proteasome-mediated degradation, before increasing the nuclear translocation and activity of β-catenin, resulting in transcriptional alteration of the target genes associated with NB progression. The knockdown of hnRNPK abolished changes in the biological features of NB cells induced by pancEts-1, which indicates that the pancEts-1/hnRNPK/β-catenin axis may be of potential value as a novel therapeutic target for NB.Neuroblastoma (NB) is a malignant tumor arising from primitive neural crests, and has a poor prognosis. There is a need to explore novel therapies . Recent  dataset . PancEtsCASC11), located in the chromosome 8q24 gene desert, ~2.1 kb upstream of c-Myc, is overexpressed in colorectal cancer (CRC) tissues [CASC11 enhances cell growth and metastasis by interacting with hnRNPK, which can promote the nuclear accumulation of β-catenin and activate the Wnt/β-catenin pathway to accelerate growth and metastasis in CRC cells. In addition, c-Myc, one of Wnt’s target genes, can directly bind to the promoter regions of CASC11 and increase promoter histone acetylation to enhance CASC11 expression. Therefore, CASC11 is able to regulate metastatic potential in vitro and in vivo via the above pathway, and the interaction between hnRNPK and CASC11 plays a pivotal role in the function of CASC11 in CRC pathogenesis as an activity regulator of Wnt/β-catenin signaling. In addition, another lncRNA, named lncRNA 91H, which is located at the position of the H19/IGF2 locus, was also observed with remarkably elevated levels in the serum of CRC patients and cancer lines [lncRNA 91H in CRC cells, and its expression level was closely related to lncRNA 91H expression. Thus, lncRNA 91H enhancing CRC metastasis by modifying hnRNPK expression might be an early plasma-based biomarker for CRC recurrence or metastasis. In summary, both CASC11 and lncRNA 91H participate in the development and progression of CRC by associating with hnRNPK, while they may exert their actions in CRC pathogenesis through distinctly different signaling pathways. These findings might provide a foundation for the development of an early diagnostic and prognostic biomarker for CRC and the determination of innovative therapeutic strategies.Wnt/β-catenin signaling is known to regulate a broad range of cellular processes through regulating the ability of the multifunctional β-catenin protein, and its activation was involved in various human cancers . lncRNA  tissues . Mechanier lines . FurtherLncRNA-277, also known as EWSAT1 (Ewing sarcoma associated transcript 1), is induced by EWS-FLI1 in bone marrow-derived mesenchymal progenitor cells that were isolated from pediatric patients [EWSAT1 expression facilitates the development of Ewing sarcoma via the repression of target genes, revealing that it plays an important role in the pathogenesis of Ewing sarcoma. Using protein arrays and RNA immunoprecipitation (RIP) assays, the authors found that hnRNPK can interplay with EWSAT1 specifically. Interestingly, there is a marked overlap in hnRNPK-repressed genes and those repressed by EWSAT1, suggesting that hnRNPK and EWSAT1 act together to repress the expression of a subset of target genes in the context of Ewing sarcoma. Thus, EWSAT1 may function, in part, by regulating the interaction of hnRNPK with target genes, although EWSAT1 probably represses many genes through interactions with other yet to be identified proteins.RNA-binding protein EWS-FLI1 (EWS and transcription factor FLI1) can both activate and repress genes that are critical for cell transformation . LncRNA-patients . The exaSLINKY , is validated in an ethnically distinct dataset of ccRCC samples and provides prognostic information independent of the tumor stage and grade [SLINKY appears to be a novel lncRNA that is upregulated across several malignancies and that drives aggressiveness in ccRCCs by regulating cancer cell proliferation. Knockdown SLINKY in ccRCC cell lines reduces cell proliferation, causes cell-cycle arrest, and alters gene expression programs related to cell growth and survival. Interestingly, SLINKY enhances cancer cell proliferation—most likely through its interaction with hnRNPK. Furthermore, hnRNPK deficiency reproduces the effects of SLINKY knockdown on cell proliferation and altered gene expression (both the upregulation and downregulation), so the possible recruitment of hnRNPK to gene loci might serve to either activate or repress the genes. Taken together, SLINKY appears to be a novel lncRNA that is upregulated across several malignancies and that drives aggressiveness in ccRCCs by regulating cancer cell proliferation through the recruitment of hnRNPK.Clear cell renal cell carcinomas (ccRCCs) are the most common histologic subtype with a broad range of clinical behavior, and prognostic biomarkers are needed to stratify patients for appropriate management . Using vnd grade . SLINKY MYCLo-2, which represses p21 transcription by interacting with hnRNPK, and plays a crucial role in cancer transformation and tumorigenesis [MYCLo-2 function decreases colon inhibitory cancer cell transformation and tumorigenesis, indicating that MYCLo-2 is a new target of cancer and c-Myc-associated pathogenesis.C-Myc, as a transcription factor, not only regulates the transcription of many protein-coding genes but also noncoding RNAs such as miRNAs and lncRNAs, and the downstream genes are involved in various cellular processes including cell cycle, differentiation, cell growth, metabolism, cell adhesion, chromosome instability, and cell transformation . C-Myc-rigenesis . Loss oflncRNA-OG (osteogenesis-associated lncRNA) significantly promotes BM–MSC osteogenesis in vitro [LncRNA-OG is mainly located in the nucleus of BM–MSCs and is upregulated during BM–MSC osteogenic differentiation, which regulates the activation of the BMP (bone morphogenetic protein) signaling pathway via interactions with hnRNPK. Surprisingly, hnRNPK also positively regulates lncRNA-OG transcriptional activity by promoting H3K27 acetylation of the lncRNA-OG promoter. Thus, this study revealed a novel lncRNA with a positive function on BM–MSC osteogenic differentiation and provides a new insight into the relationship between hnRNPK and lncRNA.Bone formation is a complex process consisting of intramembranous ossification and endochondral ossification; bone marrow-derived mesenchymal stem cells (BM–MSCs) are the main source of osteoblasts in vivo . Recentlin vitro . LncRNA-SOX2, as a master regulator that maintains stemness in embryonic stem cells as well as the self-renewal of CSCs in several malignancies, also plays a critical role in drug resistance to anticarcinogen  in several cancers [lncRNA–LBCS is significantly downregulated in bladder cancer stem cells (BCSCs) and cancer tissue, and suppresses self-renewal and chemoresistance of BCSCs in vitro and in vivo [lncRNA-LBCS directly binds to the hnRNPK–EZH2 complex and guides it to the SOX2 promoter and induces H3K27me3 to inhibit SOX2 expression, contributing to the attenuation of bladder cancer initiation and chemoresistance. Therefore, the lncRNA–LBCS/hnRNPK–EZH2/SOX2 regulatory axis acts as a tumor suppressor in bladder tumorigenesis and progression, and could be considered as a therapeutic target for clinical intervention in chemoresistant bladder cancer. Cancer stem cells (CSCs) are responsible for the initiation, propagation, metastasis, drug resistance and relapse of cancers . SOX2, a cancers ,61. Rece in vivo . InteresSCAT7, termed S-phase cancer-associated transcripts 7, is one of the differentially expressed S-phase-enriched lncRNAs and is elevated in multiple cancers. Mechanistic investigations on SCAT7 in multiple cancer types using chromatin oligo-affinity precipitation (ChOP) and RNA immunoprecipitation (RIP) assays reveal that it interacts with the hnRNPK/YBX1 complex. Furthermore, the recruitment of the SCAT7/hnRNPK/YBX1 RNP complex at the promoter regions of FGFR2 and FGFR3 promotes transcriptional activation of FGF/FGFR and its downstream PI3K/AKT and MAPK pathways, leading to sustained cell proliferation and tumor development [SCAT7/hnRNPK/YBX1 RNP complex plays a crucial role in carcinogenesis, and SCAT7 may be used as a prognostic marker in risk assessments as well as potential therapeutic regimens in the treatment of cancer.The signaling pathways controlling accurate DNA replication during the S-phase are critical for normal cell proliferation, since replication errors could result in abnormal cell proliferation . To undeelopment . Taken tMYU, named c-Myc upregulated lncRNA, is generated from the opposite strand of the VPS9D1 gene and is mainly localized in the cytoplasm [MYU, as a direct target gene of the Wnt/c-Myc pathway, is upregulated in most colon cancers and required for the tumorigenicity of colon cancer. The overexpression of MYU in colon cancer cells significantly promotes its proliferation and tumorigenesis characteristics. Conversely, MYU knockout reduces the cellular CDK6 (cyclin-dependent kinase 6), mRNA and protein levels. Further investigation of lncRNA-mediated CDK6 regulation uncovered the significant role of hnRNPK and miR-16 in fine-tuning CDK6 mRNA stability and translation. In human colorectal cancer cells, MYU interacts with hnRNPK to stabilize CDK6 expression and thereby promotes the G1-S transition of the cell cycle. Mechanistically, MYU, hnRNPK, and CDK6 mRNA form a complex in which hnRNPK prevents the binding of miR-16 to CDK6 mRNA 3′UTR, whereas the binding of miR-16 to CDK6 3′UTR results in decay of its mRNA in noncancerous cells [MYU renders specificity in regulating the stability of CDK6 mRNA by determining its binding with hnRNPK or miR-16. In addition, one group has identified and characterized a novel lncRNA linc00460, which is a cytoplasmic transcript with upregulated expression in non-small cell lung cancer and associated with a poor prognosis for NSCLC  patients, implying that linc00460 is important for lung cancer development [Linc00460 can also physically and specifically interact with hnRNPK. In lung cancer, elevated linc00460 binds with and translocates hnRNPK to the cytoplasm to participate in special mRNA stability and translation regulation, and also plays a key role in cell migration and invasion. However, the detailed molecular mechanism needs further investigation. Furthermore, Shin et al. reported that a lncRNA named PTOV1-AS1 (prostate tumor overexpressed 1 antisense transcript 1), which directly interacts with hnRNPK and is directly regulated by hnRNPK, regulates the proto-oncogene heme oxygenase 1 (HMOX1) [PTOV1-AS1 and HMOX1 mRNA have the same miRNA response element (MRE) of miR-1270-5p. Therefore, hnRNPK-mediated PTOV1-AS1 regulation acts as a decoy for miR-1207-5p to modulate HMOX1 expression, which plays a critical role in the proliferation and metastasis of various cancers. LncRNAs control gene expression at not only the transcriptional but also the post-transcriptional level. In this section, we will discuss a few lncRNAs that regulate mRNA stability and translation. The aberrant expression of Wnt/β-catenin signaling enhances c-Myc and leads to tumorigenesis, especially in colon cancer. ytoplasm . MYU, asus cells . In thiselopment . Linc004 (HMOX1) . Both PTMY, linc00460 and PTOV1-AS1, which promote the translation of target mRNAs, treRNA   reduces the level of E-cadherin protein (EMT) without affecting the levels of its mRNA [TreRNA is increased in metastatic breast cancer and controls the metastatic potential by regulating a subset of EMT markers. Nevertheless, there is no sequence homology between treRNA and the 3′UTR of E-cadherin mRNA. Mechanically, treRNA interacts with hnRNPK and four other RNA-binding proteins  promoting the formation of a treRNA-associated protein (treRNP) complex, which interacts, directly or indirectly, with the 3′UTR of E-cadherin mRNA and reduces the translation efficiency. Altogether, these findings emphasize the role of lncRNAs acting as molecular anchors that fine-tune cell signaling by recruiting hnRNPK, resulting in tumorigenesis by the repression or activation of specific genes.Unlike lncRNA its mRNA . TreRNA GFP mRNA in MCF7 cells. Meanwhile, upon the siRNA-mediated knockdown of hnRNPK in MCF7 and HeLa cells, subcellular localization of lncRNAs was disturbed; specifically, SIRLOIN-containing RNAs showed decreased nuclear localization, which suggests that hnRNPK binding drives nuclear enrichment mediated by SIRLOIN elements [LncRNAs are more commonly localized in the nuclear fraction, whereas mRNAs are usually exported to and enriched in the cytoplasm, although several reports have described that a small number of mRNAs still exist in the nucleus ,64. Howeelements . OverallXist) gene, as a 17 kb lncRNA, was first identified as a candidate for the master switch locus required in cis for the process of X chromosome inactivation, which can organize a repressive chromosome compartment as a physical scaffold [Xist-mediated silencing [Xist [Xist-mediated silencing in ESCs by assaying the silencing of genes located in cis to an inducible Xist transgene [Xist from the inactive X-chromosome, suggesting that hnRNPK is required for Xist RNA localization on the inactive X-chromosome. For further confirmation that hnRNPK interacts directly with Xist RNA, Cirillo et al. define a strong peak precisely corresponding to the cytidine-rich B-repeat of Xist RNA using hnRNPK iCLIP  analysis [Xist-mediated chromatin modifications [Xist/hnRNPK/PRC1 pathway for recruitment to the inactive X [Xist RNA-lacking B-repeat is sufficient for Xist-dependent Polycomb recruitment. Given the potential importance of the interaction between lncRNA Xist and hnRNPK in guiding chromatin modification, this mechanism provides a model for further studies on lncRNA function both in development and in disease.LncRNAs also play a central role in regulating chromatin structure and nuclear architecture ,66. The scaffold ,68,69. Hilencing . Severalng [Xist ,45,46,47ications . MoreoveNuclear Enriched Abundant Transcript 1 (NEAT1), a lncRNA with five known splice variants, is an essential structural constituent of paraspeckle nuclear bodies [NEAT1_1 and 23.7-kb NEAT1_2 are the two most abundant isoforms of NEAT1 that are produced by alternative 3′-end processing. The short-isoform NEAT1_1 is canonically polyadenylated, whereas the long-isoform NEAT1_2 is stabilized by a triple helix structure processed by RNase P cleavage at its 3′ end. Interestingly, the production of the essential NEAT1_2 isoform is regulated by hnRNPK, which can bind to the short pyrimidine stretch located between the canonical polyadenylation signal for NEAT1_1 and the upstream CPSF6/NUDT21 (CFIm) complex binding cluster and interferes with NEAT1_1 3′-end processing, resulting in the preferential synthesis of NEAT1_2 [NEAT1_1, leading to the preferential accumulation of NEAT1_2 and initiated paraspeckle construction. Furthermore, hnRNPK is also one of the essential paraspeckle proteins through binding NEAT1_2 [NEAT1 3′-end processing is required for NEAT1_2 accumulation and the next paraspeckle formation. Further studies designed in order to understand the NEAT1–hnRNPK interaction and its dynamics could aid in understanding of the molecular mechanism underlying the dynamic nature of paraspeckles under normal or disease conditions.Paraspeckles are unique lncRNA-directed nuclear substructures that are usually detected in cultured cell lines as a variable number of foci found in close proximity to the nuclear speckles, which play an important role in the regulation of gene expression through various mechanisms including mRNA retaining, mRNA breakage, A-to-I editing and protein seizing ,71,72. Pr bodies . 3.7-kb  NEAT1_2 ,49. Mech NEAT1_2 . Thus, tC/EBPα, which is a master regulator of myeloid cell differentiation and survival [C/EBPα myeloid differentiation [MiRNAs are a class of endogenous, noncoding single-stranded ~22 nt RNA, which are evolutionarily conserved and contribute to the control of cellular processes such as proliferation, differentiation, growth, death, inflammation, and development ,8,13. Basurvival ,79. Intentiation . In addintiation ,81. Fan ntiation . Thus, iNcRNAs, such as lncRNAs and miRNAs, are involved in cellular homeostasis affecting cell proliferation, cell growth, differentiation and apoptosis, while their disruption leads to malignant transformation of the cells. HnRNPK is a highly conserved gene that is abundantly expressed in mammalian cells. The interaction of hnRNPK and ncRNAs defines the novel way through which ncRNAs affect chromatin structure, the expression of protein-coding genes, and modulate pathways contributing to development, differentiation and oncogenesis. Both lncRNAs and miRNAs physically interact with hnRNPK and this leads to the regulation of RNA stability or translation. In addition, lncRNAs and miRNAs are able to cross-talk with hnRNPK to change the expression of their target genes competitively or cooperatively. Furthermore, various cancer-associated hnRNPK–lncRNAs axes function to regulate the cancer-associated phenotype through modulating target genes. In this review, we summarized the role of ncRNAs and hnRNPK interactions in regulating gene expression and cellular functions and emphasized gene regulation via the interaction of hnRNPK with ncRNAs, such as lncRNAs and miRNAs, at transcriptional and post-transcriptional levels. The mechanisms of action can mainly be categorized into the following groups: (1) lncRNAs recruit hnRNPK and turn on or off gene transcription by binding to gene promoters; (2) lncRNAs and hnRNPK interaction promotes lncRNAs nuclear enrichment; (3) lncRNAs, hnRNPK and mRNA form a complex that controls mRNA stability or translation; (4) hnRNPK acts as an endogenous decoy for miRNAs, affecting their distribution on their target genes; (5) lncRNAs regulate genome organization by interacting with hnRNPK, indirectly affecting gene expression; and (6) hnRNPK regulate the alternative splicing of lncRNAs .Despite the progress in the ncRNAs field in recent years, research into the hnRNPK–ncRNAs interaction’s involvement in biological processes and human diseases is yet to be elucidated. To better understand the hnRNPK–ncRNAs regulatory network, there are several areas that warrant further investigation. Firstly, there is an urgent need for the systematical identification of hnRNPK-interplayed ncRNAs including lncRNAs, miRNAs and emerging circRNAs. Current research is still scattered, and a more systematic screen may be able to provide a more comprehensive picture of hnRNPK–ncRNAs interaction. To detect this possibility, a global search by RIP-seq with the hnRNPK antibody under various conditions, including physiological or pathogenetic conditions, would be one good approach. Secondly, the regulation of the hnRNPK–ncRNA interaction should be further investigated. The location and magnitude of the interaction for a specific ncRNA and hnRNPK must be associated to regulate the change of target gene expression and cellular function. For instance, the mechanisms that regulate the nucleo-cytoplasmic shuttling of hnRNPK and ncRNA complexes are important to investigate. Thirdly, Statello et al. recently discovered interplay between RNA-binding proteins and RNAs by packaging into exosomes, which could be important for the transport of RNAs into exosomes and the maintenance of RNAs inside exosomes . These s


Increased lower body fat is associated with reduced cardiometabolic risk. The molecular basis for depot-specific differences in gluteofemoral (GF) compared with abdominal (A) subcutaneous adipocyte function is poorly understood. In the current report, we used a combination of Assay for Transposase-Accessible Chromatin followed by sequencing (ATAC-seq), RNA-seq, and chromatin immunoprecipitation (ChIP)-qPCR analyses that provide evidence that depot-specific gene expression patterns are associated with differential epigenetic chromatin signatures.-shaped women were used to perform transcriptome analysis by RNA-seq and assess accessible chromatin regions by ATAC-seq. We measured mRNA expression and performed ChIP-qPCR experiments for histone modifications of active (H3K4me3) and repressed chromatin (H3K27me3) regions respectively on the promoter regions of differentially expressed genes.Preadipocytes cultured from A and GF adipose tissue obtained from premenopausal appleRNA-seq experiments revealed an A-fat and GF-fat selective gene expression signature, with 126 genes upregulated in abdominal preadipocytes and 90 genes upregulated in GF cells. ATAC-seq identified almost 10-times more A-specific chromatin-accessible regions. Using a combined analysis of ATAC-seq and global gene expression data, we identified 74 of the 126 abdominal-specific genes (59%) with A-specific accessible chromatin sites within 200 kb of the transcription start site (TSS), including HOXA3, HOXA5, IL8, IL1b, and IL6. Interestingly, only 14 of the 90 GF-specific genes (15%) had GF-specific accessible chromatin sites within 200 kb of the corresponding TSS, including HOXC13 and HOTAIR, whereas 25 of them (28%) had abdominal-specific accessible chromatin sites. ChIP-qPCR experiments confirmed that the active H3K4me3 chromatin mark was significantly enriched at the promoter regions of HOXA5 and HOXA3 genes in abdominal preadipocytes, while H3K27me3 was less abundant relative to chromatin from GF. This is consistent with their A-fat specific gene expression pattern. Conversely, analysis of the promoter regions of the GF specific HOTAIR and HOXC13 genes exhibited high H3K4me3 and low H3K27me3 levels in GF chromatin compared to A chromatin.Global transcriptome and open chromatin analyses of depot-specific preadipocytes identified their gene expression signature and differential open chromatin profile. Interestingly, A-fat-specific open chromatin regions can be observed in the proximity of GF-fat genes, but not vice versa.NCT01745471. Registered 5 December 2012.Clinicaltrials.gov, The online version of this article (10.1186/s13148-018-0582-0) contains supplementary material, which is available to authorized users. As body weight increases, the distribution of body fat becomes an important factor that determines metabolic health . Excess Multiple studies, including our own work, reported that the homeobox (HOX) family and HOX domain-encoding genes (SHOX2 and IRBX2) are differentially expressed between the two major subcutaneous tissue depots, abdominal and GF –17. InteCurrent knowledge is limited concerning the chromatin status of adipose tissue from different anatomical locations. To date, only one study investigated the DNA methylome of the human A- and GF-fat by using DNA methylation array in a heterogenous group of subjects . Chromat2 were recruited in Orlando using advertisements approved by an institutional review board. Subjects were excluded if they reported a history of chronic disease , recent weight loss or gain (> 3 kg over the past 8 weeks), had abnormal blood or urine values, or use of oral contraceptives or hormone replacement therapy. Adipose tissue biopsies were collected with a three-hole 2.5-mm liposuction cannula from the mid-abdomen approximately 5–8 cm lateral to the umbilicus. Gluteofemoral adipose tissue was collected 10–20 cm below the greater trochanter on the most lateral side of the upper thigh. Samples were cleaned at the bedside; a fraction of it was snap frozen in liquid nitrogen, and the rest was immediately used for preadipocyte isolation.Six healthy premenopausal, weight-stable females aged 20–40 years with a body mass index (BMI)  between 27 and 40 kg/mThe characteristics of the study group are presented in Table Stromal vascular fractions were isolated from scWAT abdominal and GF depot, plated, and grown as previously described  with theRNA was isolated using RNeasy Mini Kit (Qiagen) from abdominal and GF preadipocytes. Approximately 2 μg was used for library preparation with TruSeq RNA Sample Preparation Kit (Illumina). Following purification, RNA was fragmented with divalent cations at 85 °C, and then cDNA was generated by random primers and SuperScript II enzyme (Life Technologies). Second-strand synthesis was performed followed by end repair, single “A” base addition, and ligation of barcode indexed adaptors to the DNA fragments. Adapter-specific PCRs were performed to generate sequencing libraries. Libraries were size selected with E-Gel EX 2% agarose gels (Life Technologies) and purified by QIAquick Gel Extraction Kit (Qiagen). The result is libraries with inserts ranging in size from 120 to 210 bp with a median size of 155 bp. Libraries were sequenced on HiSeq 2500 instrument.ATAC-seq was carried out as described earlier . After tTotal RNA from adipose tissue and preadipocytes were extracted using the lipid RNeasy kit and the RNeasy mini kit (Qiagen), respectively. Gene expression was assessed by real-time PCR using a ViiA7 sequence detection system (Life Technologies) and Taqman technology suitable for relative gene expression quantification using the following parameters: 1 cycle of 95 °C for 10 min, followed by 40 cycles at 95 °C for 10 s and 60 °C for 1 min. All samples were normalized to PPIA showing low variability between A and GF samples  were performed and analyzed as described . BrieflyChIP-PCR primers Table  were desp ≤ 0.05 in both subjects were used to further analyses  by using TopHat v2.0.7). The fragments per kilobase of transcript per million mapped reads (FPKM) values were calculated by Cufflinks (v2.0.2) with default parameters . Differe.0.7. ThemakeTagDirectory program of the Hypergeometric Optimization of Motif EnRichment (HOMER) package (v4.2) [Raw sequence reads were aligned to the hg19 reference genome assembly (GRCh37) by using the Burrows-Wheeler Alignment (BWA) tool (v07.10) , and thee (v4.2) .makeUCSCfile.pl program of HOMER. A consensus set was generated from those peaks which could be predicted from at least two samples, and “summit coverage” values (on the middle 50-bp bins) were calculated based on this set by annotatePeaks.pl (HOMER) with -hist 50 and -ghist parameters. Each value within the coverage files was divided with the median value of “summit coverage.” Upon this normalization, bedgraph files were converted to tiled data formats (.tdf) by using the toTDF command of the IGVtools program (v2.3.9) [annotatePeaks.pl. Histograms, created also with annotatePeaks.pl, show the average tag densities of the peaks relative to the transcription start site (TSS) ± 250 bp in 1-bp resolution. Overlapping and depot- or subject-specific peaks were determined using the intersectBed program of BedTools (v2.23.0) [Coverage (bedgraph) files were created by the (v2.3.9) . Median v2.23.0) .P ≤ 0.005 threshold were used for visualization and further analyses. For volcano plots, significance and log fold change values were calculated by DiffBind. ATAC-seq peaks within ± 100 kb to the TSS of depot-specific genes but not farther than the neighboring gene (BedTools) were used for further DiffBind analyses.Correlation heat maps of ATAC-seq peak densities were created by differential binding analysis (DBA) using the DiffBind package (v1.2.4) in R. Regions that were predicted from at least two samples (minOverlap parameter was set to 2) and were found differently opened between the depots according to a findMotifsGenome.pl (HOMER). It was performed on the ± 100 bp flanking regions of the peak summits or in the ± 250 bp regions of genes TSSs. The search lengths of the motifs were 8, 10, 12, and 14 bp. p values were calculated by comparing the enrichments within the target regions and those of a random set of regions (background) generated by HOMER.Motif enrichment analysis was carried out by Volcano plots, box plots, and histograms were created with GraphPad Prism 6. Coverage files were visualized by Integrative Genomics Viewer (IGV).t tests were used. ChIP-PCR data are presented as a percent of 5% input. All statistical analyses on clinical data were performed in SAS (V11.2.1).To compare A-fat vs. GF-fat gene expression and histone marks in obese subjects, individual data are presented and paired 2, waist to hip ratio = 0.93 ± 0.02). In order to gain a genome-wide view of accessible chromatin regions from the isolated preadipocytes, we utilized Assay for Transposase-Accessible Chromatin followed by sequencing (ATAC-seq). We sequenced two technical replicates from each individual’s A- and GF-derived preadipocytes yielding four samples from each depot. We identified 112,362 and 104,957 peaks in A and GF samples respectively, 91,022 peaks being common to both depots (81% of the A peaks and 87% of the GF peaks). Differential binding analysis of the ATAC-seq data and hierarchical clustering revealed that A and GF samples are clustered together as expected based on their distinct anatomical origin , using abdominal (A) and gluteofemoral (GF) depot-derived preadipocytes isolated from two obese apple-shaped women  as shown in Fig. After we determined the accessible chromatin regions of depot-specific preadipocytes, we investigated the associated gene expression profiles. In the first subject, 2117 genes were differentially expressed between abdominal and GF preadipocytes. The difference was much smaller for the second subject with only 536 differentially expressed genes  of the differentially expressed genes Fig. a. NormalTo provide a much more detailed view on chromatin openness on the abdominal- and GF-specific gene promoters, we calculated the ratio of the RPKM values of the ATAC-seq data from the two depots on each promoter for genes that showed depot-specific expression. This analysis allowed us to define more accessible promoter regions using a 1.5-fold change cutoff. Interestingly, more than 39% of abdominal- and close to 65% of GF-specific genes promoters did not follow the expression of the corresponding gene with regard to their chromatin openness  Fig. c. Motif n = 6 obese, apple-shaped women, including the two subjects previously studied), and carried out promoter-based ChIP-qPCR for a select group of histone modifications known to be associated with active (H3K4me3) or repressed (H3K27me3) chromatin. We also performed RT-qPCR on the whole tissue and on the preadipocytes to measure the mRNA levels of the depot-specific genes. ChIP-qPCR experiments confirmed that H3K4me3 was significantly enriched at the promoter regions of HOXA5 and HOXA3 genes in abdominal preadipocytes, while H3K27me3 was less abundant  Figs. a, isolat A substantial number of studies have focused on identifying extrinsic regulators of preadipocytes and adipocytes, whereas comparatively very little is known about the chromatin structure differences that might control their identity and/or functions. A recent study showed the importance of chromatin modifications in the maintenance of the preadipocyte state by keeping the levels of the adipogenic master regulatory genes (PPARγ and C/EBPα) low. Similarly, high expression levels of these transcription factors upon adipocyte differentiation were also dependent on the presence of certain chromatin modifications , suggestUsing RNA-seq, we identified 126 genes upregulated in abdominal cells and 90 genes upregulated in GF cells. In accordance with our previous studies , 22, theThe strong association observed between open chromatin and active gene expression in A-fat supports the role of differential chromatin signatures in the transcriptional regulation of abdominal-specific genes. Inversely, GF-specific genes displayed a loss in GF chromatin accessibility within 200 kb of the TSS, reflecting a more complex situation in lower body fat depots. Surprisingly, we observed abdominal-specific ATAC-seq signals around the TSS of 25 GF-specific genes, which may suggest the presence of active transcriptional repressors at these open chromatin regions. Transcription factor motif analysis confirmed the presence of the DNA-binding motif for the transcriptional repressors, NF1, and/or ZNF263 at these abdominal-dominant open chromatin regions. ATAC-seq regions identified in abdominal depot near the TSS of GF-specific genes were also enriched for H3K27me3, which is the marker of repressed chromatin territories (data not shown). Altogether, these observations suggest the presence of abdominal-specific repressive mechanisms that affect the chromatin landscape of GF-specific gene loci and gene expression signature, but further functional studies are required to provide evidence about the existence of such phenomenon.As described in our previous study , we obseSeveral studies revealed adipose tissue depot-specific expression of multiple HOX genes  and theiRegarding the limitations of our study, our genome-wide analysis was performed only on two apple-shaped women. Future studies must involve more subjects not only for validation purposes but also to extend our analysis and provide a more comprehensive view on the potential epigenomic programming of depot-specific adipocytes, especially on women presenting with a gynoid fat pattern. A previous analysis of adipogenesis regulator genes performed in human mesenchymal stem cells revealed dynamic changes in histone marks during the differentiation . HoweverIn conclusion, our analysis revealed that the open chromatin landscapes for abdominal vs. GF preadipocytes exhibit significant differences, suggesting depot-specific mechanisms that shape the cellular epigenome, according to their anatomical origin. In addition, the different open chromatin profile positively associates with a depot-specific gene expression signature, supporting a role for chromatin status in the regulation of the depot-selective patterns of scWAT gene expression. Finally, our gene-specific analyses show that our findings are reproducible with available technologies and highlight a strong positive correlation between chromatin openness and the presence of active and repressive chromatin marks in preadipocytes.Additional file 1:Additional figure with data on PPIA gene expression. Representation of the individual PPIA gene Ct value obtained by RT-qPCR in cells (white squares) and tissue (black squares). A = abdominal depot, G = GF depot. (PPTX 51 kb)Additional file 2:Table S1. Differentially expressed genes between abdominal and GF depot in subject 1, subject 2, and in both. Table S2. List of open chromatin regions in the proximity of the depot-specific genes. (ZIP 231 kb)Additional tables with data on RNA-seq and ATAC-seq list of genes. Additional file 3:Figure S1. Differential binding analysis identifies depot-specific chromatin accessibility in preadipocytes. a) Correlation plot of accessible chromatin regions defined by ATAC-seq from different subjects (Sub1 and Sub2), fat depots , and technical replicates (rep#1 and rep#2). b) Identification of differentially open chromatin regions from GF and abdominal preadipocytes using differential binding analysis (DiffBind). Heat map representation of the abdominal- (n = 7160) and the GF-specific (n = 780) open chromatin regions. Sub = subject, Rep = technical replicate. The pairwise correlation scores were used for hierarchical clustering . Figure S2. Depot-specific open chromatin regions of preadipocytes. IGV genome browser view of depot-specific ATAC-seq signals from abdominal (red) and GF (green) preadipocytes. Abdominal- (left) and GF-specific open chromatin regions are shown. Results are shown from two subjects and from 2-2 technical replicates. Chromosomal locations are indicated at the bottom for each genomic loci. Figure S3. Motif analysis on the 40 abdominal-specific open chromatin regions annotated to GF specific genes. Enriched motif matrices are presented along with the p values, the percentages of each motif found in the target (Target%) and background (Bg%) genomic regions. (PPTX 146 kb)Additional figures with data on ATAC-seq.


The expansion of long non-coding RNAs (lncRNAs) in organismal genomes has been associated with the emergence of sophisticated regulatory networks that may have contributed to more complex neuronal processes, such as higher-order cognition. In line with the important roles of lncRNAs in the normal functioning of the human brain, dysregulation of lncRNA expression has been implicated in aging and age-related neurodegenerative disorders. In this paper, we discuss the function and expression of known neuronal-associated lncRNAs, their impact on epigenetic changes, the contribution of transposable elements to lncRNA expression, and the implication of lncRNAs in maintaining the 3D nuclear architecture in neurons. Moreover, we discuss how the complex molecular processes that are orchestrated by lncRNAs in the aged brain may contribute to neuronal pathogenesis by promoting protein aggregation and neurodegeneration. Finally, this review explores the possibility that age-related disturbances of lncRNA expression change the genomic and epigenetic regulatory landscape of neurons, which may affect neuronal processes such as neurogenesis and synaptic plasticity. Aging of the human brain often leads to cognitive decline , reducedAmong non-coding transcripts, long non-coding RNAs (lncRNAs) have recently emerged as key regulators of the molecular processes that underlie age-associated phenotypes ,8. lncRNThis review proposes ways by which lncRNAs may contribute to neural aging and how their functions can be altered across the human lifespan. We discuss that antisense (AS) lncRNAs can regulate pathological protein aggregation and that subnuclear compartment specific (SCS) lncRNAs can regulate neuronal splicing, transcription, and sponging of ion channels in aging. Other pre- and post-transcriptional regulatory roles performed by lncRNAs are also discussed in the context of cognition, neurogenesis, and neurodegeneration in aging, including the possible influence of lncRNAs on the maintenance of the 3D nuclear architecture.Neurogenesis is the process by which new functional neurons are generated from neural stem cells (NSCs) throughout life. In the mammalian adult brain, NSCs persist in the subgranular zone of the dentate gyrus of the hippocampus and in the subventricular zone (SVZ) of the lateral ventricles ,17. The MALAT1; BCYRN1; MIAT; SOX2-OT, TUG1, and RMST) [Dlx1as [Six3os [Pnky [Although lncRNAs have been found to play crucial roles in the developing mammalian brain, little is known about their function in post-natal and adult neurogenesis. It is conceivable that the same lncRNA pool that regulates NSC behavior in the embryonic stage is equally important in adults, since neuronal development pathways are highly conserved among embryonic, early post-natal, and adult neurogenesis . A studynd RMST)  were alsnd RMST) . Dlx1as  [Dlx1as , Six3os  [Six3os  and Pnkyos [Pnky  are amonos [Pnky ,27.Taken together, studies on the developing and adult mammalian brain suggest that the relative abundance of individual lncRNAs in the total NSC pool may determine the course of neurogenesis. lncRNAs are key modulators of NSC maintenance, lineage commitment/differentiation, and telomere maintenance . TherefoSix3os in the SVZs of adult mice NSCs resulted in a two-fold decrease in Tuj1-positive cells  and increased GFAP-positive cells (activated NSC marker) [Dlx1as knockdown in adult murine SVZ of NSCs caused a three-fold decrease in Tuj1-positive neuroblasts, an increase of nearly 60% in GFAP-positive cells, and a decrease in the expression of Dlx1 and Dlx2, which are two transcription factors that play major roles in neuronal development [Six3os and Dlx1as are important for the amplification of intermediate progenitors and neuroblast generation. In contrast, Pnky knockdown in post-natal NSCs potentiates neuronal lineage commitment and expands the transit-amplifying cell population, increasing neuron production by several-fold [Pnky is important for NSC maintenance.In adult neurogenesis, activated NSCs give rise to transit-amplifying cells, which in turn generate neuroblasts . lncRNAs marker) . Moreoveelopment . These rral-fold . TherefoDLX1AS expression in the SVZ is not altered during aging [MALAT1, GOMAFU, NEAT1, and TUG1 in the human SVZ significantly increases with age [It is likely that subtle alterations in the levels of these lncRNAs have dramatic effects on destabilizing the dynamic balance that is established between NSC proliferation, intermediate amplification, and differentiation into neuroblasts, potentially leading to impaired neurogenesis in aging. Importantly, ng aging . Howeverwith age . This mawith age  and senewith age .Six3os lncRNA resulted in three-fold fewer cells expressing the oligodendrocyte marker OLIG2 [(lnc-OPC) depletion resulted in a significant decrease in the expression of oligodendrocyte precursor cell (OPC) markers  and O4+ (oligodendrocyte surface marker), demonstrating that lnc-OPC plays a role in oligodendrogenesis [In addition to the neuronal cell lineage, NSCs also generate glial lineages, which give rise to astrocytes, oligodendrocytes, and ependymal cells . Many lner OLIG2 , suggestogenesis .RMST blocks neuronal differentiation and is required for the binding of SOX2 to promoter regions of neurogenic transcription factors [lncRNAs may also direct a neurogenic fate in NSCs. For example, knockdown of the lncRNA  factors . Interes factors . The autTelomeres are repetitive DNA elements that cap the ends of chromosomes and protect their integrity. Throughout life, telomeres of somatic cells shorten at every round of DNA replication until the progressive and cumulative loss of telomere sequences ultimately triggers cellular senescence . For thiTERC (telomerase RNA component) forms a ribonucleoprotein complex with TERT, acting as a scaffold that brings the protein subunits of telomerase together, but also serves as a template for the synthesis of new telomeric repeats [TERC are essential for telomere maintenance and elongation, as shown by their respective knockout mice models—which display short telomeres, instability, and premature aging [TERC is the limiting factor for telomerase activity as TERT heterozygote mice show no defects in telomere elongation, while TERC homozygotes do [TERC is not only crucial for telomerase activity, but also modulates it to promote and maintain telomere length. Interestingly, Klapper et al., 2001, observed that the temporal pattern of telomerase activity does not reflect the observed decrease in TERT transcript levels throughout pre- and post-natal neuronal development in mice [TERC regulates neurogenesis, with high levels of TERT and TERC being responsible for NSC proliferation, low levels of TERC and high levels of TERT inducing differentiation, and low levels of both lncRNAs resulting in cell death. While the relative abundance of TERC and TERT in adult and aged NSCs remains unknown, this theory raises the question of whether a dysfunctional balance between TERC and TERT may trigger processes observed in aged NSCs, such as telomere de-protection (ultimately leading to senescence) or compromised cellular viability (leading to apoptosis).lncRNAs play key roles in telomere dynamics in stem cells. The lncRNA  repeats . Both TEre aging ,42. Impo in mice . MoreoveTERRAs (telomeric repeat containing RNAs) are also key regulators of telomere dynamics. TERRA molecules are transcribed from the subtelomeric region of chromosomes and can be actively displaced to chromosome ends, the nucleosome [TERRA transcripts can base-pair with complementary DNA, forming RNA:DNA hybrid structures that regulate telomere length [TERRA transcripts remains unclear. On one hand, in vitro experiments using TERRA-mimicking oligonucleotides suggest that TERRAs inhibit telomerase activity by directly binding to both TERT and TERC [TERRAs transcription is induced at short telomeres and form TERRA-telomerase RNA clusters in the early S phase. These are later recruited to short telomeres from which the TERRAs originated, triggering telomere elongation [TERRAs levels decline at telomeres [TERRA molecules throughout the cell cycle is a crucial factor that sustains and regulates telomere length [TERRAs are also proposed to regulate telomere length via heterochromatin formation at chromosome ends [TERRA subtelomeric region knockouts in three human cell lines  are often lethal and lead to a dramatic loss of telomere sequences and a massive induction of the DNA damage response [Additionally, lncRNAs named cleosome ,45, or tcleosome . At chroe length ,48. In cand TERC . On the elomeres . Therefoe length ,51. Besiome ends , cappingome ends ,54,55, aome ends . MoreoveTERRAs are involved in multiple functions that mediate genomic instability, cell survival, and cellular senescence. As these functions are extensively affected through life, a role for TERRAs in aging can be reasonably inferred  have constantly elevated levels of TERRA transcripts [TERRA overexpression is found in proliferating progenitor cells in the developing mouse brain that exhibits TERRA foci [TERRA expression is coupled with cellular proliferation in progenitor cells. Therefore, TERRA expression in the presence of reduced telomerase activity—which is typical of aging—could lead to NSC cellular arrest and eventual senescence. The state of NSCs in aging is likely dependent on the interplay that is established between TERC, TERRA, and TERT. A shift in the abundance or activity of one of these molecules, both overall and in a cell-cycle-stage specific manner, could be implicated in the progression of aging.Taken together, these studies suggest that d e.g., ,57). Int. IntTERRd , a gene involved in learning and long-term memory with no homologue in mammals, is performed by an AS lncRNA with an expression that is reduced in aging [SRN-AS within a neuron becomes asymmetric in aging. Hence, we hypothesize that a decreased generation of AS lncRNAs that are capable of dynamic gene regulation or the aberrant spatial distribution of sense/antisense transcripts in an aged neuron, might underlie local protein level defects and, consequently, synaptic plasticity deficiency.While some groundbreaking studies showed that antisense regulation of gene expression in synapses plays important roles in neuronal plasticity, its impact in aging remains unknown. Interestingly, in in aging . This stlncRNAs also mediate the splicing of pre-mRNAs with retained introns. It has become increasingly clear that a dynamic regulation of splicing in the nervous system is critical for neuronal development and for the establishment and maintenance of neuronal networks . A recenMALAT1 is specifically enriched in nuclear speckles, where it is proposed to act as a splicing factor sponge [MALAT1 was shown to modulate the recruitment of SR family pre-mRNA-splicing factors to an active transcription site of a reporter gene locus [MALAT1 in neuroblastoma cells affected not only the expression of genes involved in nuclear processes, but also that of genes in synapse function and dendrite development [MALAT1 knockdown in cultured hippocampal neurons significantly decreased the synaptic density, whereas its overexpression resulted in increased synaptic density [MALAT1, in a similar way to the RNA-binding protein, SLM2, modulates the expression and splicing of genes involved in synapse function and maintenance. While this mechanism is not fully understood, further insights from studies of the lncRNA GOMAFU help to draw a possible working model.As an example of lncRNAs located in subnuclear compartments, the lncRNA r sponge . Accordine locus . Surpriselopment . In line density . Hence, MALAT1, GOMAFU is retained in the nucleus and is localized in another specific nuclear compartment [GOMAFU in human iPSC-derived neurons lead to alternative splicing patterns [DISC1 [ERBB4 [DRD2 [GOMAFU transcript levels are downregulated while MALAT1 levels remain unchanged [GOMAFU regulates plasticity-related, activity-dependent alternative splicing. Thus, the authors suggest a model in which GOMAFU acts as a splicing factor scaffold in the nuclear compartments of an inactivated neuron. Upon neuronal activation, GOMAFU expression is downregulated, allowing for the release of splicing factors into the nucleoplasm, where they can modulate the splicing of transcripts involved in synaptic and dendritic growth, morphology, and function [MALAT1 works in a similar way, but that its expression is regulated by stimuli other than the KCl-induced depolarization of neurons.Like partment , where ipartment ,94. Losspatterns  in the ss [DISC1 ,96,97,981 [ERBB4 ,100, andB4 [DRD2 ,102. Intnchanged . This sufunction . AlthougTaken together, these studies demonstrate that lncRNAs can act as RNA-binding proteins in specific subnuclear compartments and are dynamically regulated in response to neuronal activity, allowing for the splicing of genes involved in neuronal plasticity. Even though this mechanism remains poorly characterized, it would be very interesting to test these ideas in aging models in vivo.NEAT1 is retained in the nucleus, where it aggregates into paraspeckles structures [NEAT1 expression level is dynamically regulated by neuronal activity and binds potassium channel-interacting proteins, including KCNAB2 and KCNIP [NEAT1 appears to be particularly important in this process as its transient downregulation in response to neuronal activity induces the release of potassium channel proteins, such as KCNAB2, from the nucleus into the cytosol [NEAT1 transcript induces a neuronal hyper-potentiation phenotype in iPSC-derived human cortical neurons [NEAT1 is also involved in the transcriptional regulation of ion channel components as its knockdown in activated neurons drives a significant increase in ion channel gene expression [NEAT1 activity may be involved in this phenotype.The lncRNA ructures . NEAT1 end KCNIP . The modnd KCNIP  and othend KCNIP  regulate cytosol . Once in neurons . NEAT1 ipression . Interespression . AdditioMEG3, MEG8, MEG9, and RTL1-AS—in primary cortical neurons following glycine stimulation occurred in an N-Methyl-d-aspartate receptor (NMDAR)-dependent manner [MEG3 knockdown blocked the glycine-induced increase of the GluA1 subunit of AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors at the plasma membrane by regulating its trafficking, which shows that MEG3 may have a role in the regulation of LTP [A recent study reported that the expression of a lncRNA cluster—consisting of t manner . MEG3 knn of LTP .Bc1 and BC200 may leak into the cytoplasm, counteracting their neuronal activity-dependent regulation. The loss of cell nucleocytoplasmic compartmentation also poses a threat for nuclear lncRNAs. Firstly, cytoplasmic proteins and RNAs that leak into the nucleoplasm can compete with endogenous factors for binding sites in scaffolding lncRNAs, interfering with the specificity of their regulation. Secondly, the activity-dependent binding of ion channel-interacting proteins in the nucleus may also be compromised by their leakage into the cytoplasm. This would most likely give rise to hyperexcitability phenotypes, which have been found in aged CA3 pyramidal neurons [While we have focused on alterations in the expression of lncRNAs that directly modulate the synaptic response, factors intrinsic to the cellular organization of an aged neuron may also modify lncRNA-associated synaptic activity. Nuclear and cytoplasmic  cellular neurons . AdditioThe transcriptomes of mammalian brains show widespread changes during aging . In addiWe suggest that changes in the expression levels of lncRNAs, caused by age-dependent epigenetic alterations, may impair or attempt to compensate for processes of adult neurogenesis, cognition, and neurodegeneration. The insights from age-related neurodegenerative disorders  imply thMore than half of mammalian coding genes have complementary non-coding AS transcription . AS lncRBACE1-AS hybridizes to BACE1 mRNA [BACE1-AS is upregulated in the brains of AD patients, consequently leading to the overexpression of BACE1 and to an increase in Aβ generation [SORL1-AS, which is also upregulated in the brains of AD patients, induces the synthesis of pathogenic splicing isoforms of SORL1, which are associated with increased Aβ levels in cultured human neuronal cells [UCHL1-AS targets UCHL1 mRNA to heavy polysomes for translation, resulting in increased UCHL1 protein levels [UCHL1 is a Parkinson’s disease (PD) and AD risk gene that is believed to prevent pathological protein aggregation by promoting its (or its precursors) ubiquitination [UCHL1-AS is downregulated in PD [LRP1-AS directly binds to HMGB2, preventing it from enhancing LRP1 transcription, thereby reducing its expression [LRP1-AS is upregulated in AD [In age-related neurodegenerative disorders, the dysregulation of AS lncRNAs plays crucial roles in pathological protein aggregation. In Alzheimer’s disease (AD), AS lncRNAs contribute to amyloid-beta (Aβ) aggregation by modulating the expression and/or splicing of proteins involved in the generation and trafficking of Aβ. For example, the lncRNA CE1 mRNA , a proteneration   or cause their decay . In the nucleus, besides sequestering chromatin regulatory molecules, AS expression may also recruit transcriptional repressors to chromatin-modifying complexes that are close to gene promoters, thus silencing gene expression. A recent study showed that 20% of lncRNAs (out of a cohort of 3300) often associate with the polycomb repressive complex 2 (PRC2), a histone methyltransferase that catalyzes repressive H3K27 methylation [BDNF-AS recruits PRC2 to the BDNF promoter, resulting in transcriptional repression [HOTAIR) binds and targets PRC2 in trans to the HOXD cluster [HOTAIR can also interact with multiple regulatory complexes simultaneously. HOTAIR binds both PRC2 and the LSD1/coREST/REST complex—which catalyzes H3K4 demethylation—into a single ribonucleic complex [HOTAIR can specify the pattern of histone modifications on target genes  are included in the ~2000 genes that are differentially expressed across aging in all human tissues [HOTAIR is overexpressed in a mouse model of PD [CDKN2B–CDKN2A locus have been associated with AD pathology [ANRIL regulates the expression of CDKN2B that accumulates in neurofibrillary tangles and amyloid plaques in the brains of AD patients [Besides regulating protein aggregation, cytoplasmic AS lncRNAs can also regulate the stability of transcripts from genes linked to neurodegenerative disorders and cognition . The bihylation . For exapression , which p cluster  and to m cluster . HOTAIR  complex . By funcet genes E. AS lnche locus ,137. Int tissues . Importael of PD  and singathology . Furtherpatients .Alu TEs during adult human stem cell aging promotes senescence, whereas knockdown of Alu lncRNAs reverses senescence [Transposonable elements (TEs) are repetitive DNA elements that account for nearly 50% of the human genome . TEs arenescence .(1)INK4 and HOX loci coordinate the expression of genes involved in cell cycle regulation and developmental patterning, respectively. These loci also contain the lncRNAs ANRIL and HOTAIR, respectively, which have altered expression in human tissues during aging [ANRIL and HOTAIR may function in cis for loci regulation, and are likely to play roles in post-mitotic neuronal processes, their dysregulation may also trigger the aberrant re-activation of cell cycle and developmental transcriptional programs—changes that are typically found in neuronal aging [As previously mentioned, many lncRNAs bind to chromatin-modifying proteins and recruit their catalytic activity in cis or trans to specific gene loci, thereby modulating chromatin states and impacting gene expression . Modulatng aging . While Aal aging ,158.(2)MALAT1 causes differential expression of several genes that localize away from the MALAT1 locus [MALAT1-associated epigenetic-regulation of genes was found to happen exclusively in cis [NEAT1 impairs paraspeckles’ assembly [FIRRE, show that it forms punctate compartments in the nucleus that include not only its own locus, but also specific loci from several other chromosomes [lncRNAs influence the nuclear architecture directly by organizing the dynamic assembly and disassembly of subnuclear compartments in the periphery of active chromatin regions , and by T1 locus , and becy in cis , it is tassembly  and alsoassembly . In factomosomes . This fiomosomes . Another(3)An emerging view is that the act of lncRNA transcription possibly defines or affects the nuclear architecture . This moHigher level chromatin organization within the nuclear space is termed nuclear architecture . OrganizIt follows that long-term maintenance of the nuclear architecture by lncRNAs is vital for neuronal functioning, and that lncRNA-associated age-related disturbances have a broad impact on 3D nuclear architecture and may lead to neuronal dysfunction.In summary, we have discussed multiple examples whereby lncRNA activity is involved in aspects of neuronal function , such as


Drosophila larvae and human cells reported here show that PWP1 associates with the chromatin at the 5S rDNA loci and is needed for nutrient-induced expression of 5S rRNA. Similar to the Pol I target rDNAs, PWP1 epigenetically maintains 5S rDNA in a transcription competent state. Thus, as a common regulator of Pol I and Pol III, PWP1 might contribute to coordinated control of ribosomal gene expression in response to nutrition.PWP1 is a chromatin binding protein with an important role in animal growth control downstream of mTOR-mediated nutrient sensing. PWP1 has been shown to control tissue growth by promoting the transcription of 5.8S, 18S and 28S ribosomal RNAs (rRNAs) by RNA polymerase I (Pol I). Concomitantly with Pol I, RNA Polymerase III (Pol III) contributes to ribosome biogenesis by transcribing 5S rRNA in the nucleoplasm. Pol III activity is also closely controlled by nutrient-dependent signaling, however, how the activities of Pol I and Pol III are coordinated in response to nutrient-derived signals remains insufficiently understood. Experiments in This article has an associated First Person interview with the first author of the paper. Summary: We report that a chromatin-binding protein PWP1 binds to 5S ribosomal DNA, regulates its epigenetic status and controls nutrient-dependent RNA polymerase III-mediated transcription of 5S ribosomal RNA. Animal growth is dynamically regulated in response to environmental factors, including nutrients. Ribosome biogenesis serves as a limiting factor for growth capacity and it is tightly controlled by nutrient-responsive signaling, including the mTOR (mechanistic target of rapamycin) pathway . The ribDrosophila mutant larvae lacking PWP1 show a prominent growth retardation. In addition to the role in Pol I regulation, we observed earlier that pwp1 mutant Drosophila larvae display reduced expression of RNA polymerase III targets, including 5S rRNA  controls the expression of Pol III-dependent 5S rRNA in this setting. This was indeed the case, as control larvae displayed strongly elevated 5S rRNA expression upon refeeding on protein-rich yeast food following protein starvation, but this effect was blunted in pwp1nclb2 null mutant larvae . As previously shown . We focused our attention on the division 56E, which contains Pol III targets, including the cluster of 5S rDNA as well as several tRNA-encoding loci  in combination with ChIP to analyze Histone 4 lysine 12 (H4K12) acetylation, which is associated with active transcription, as well as H3K9 dimethylation, a repression-associated modification. Similar to the earlier observations on the Pol I target loci  in human cell lines. A significant enrichment of PWP1 was observed in the 5S rDNA gene region . PWP1 waget loci , knockdoget loci A, while get loci B. To conget loci C.Fig. 3Drosophila fat body shows PWP1 localization in both nucleolus and nucleoplasm  and pwp1nclb2  were cultured at 37°C in Dulbecco's modified Eagle's medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Life Technologies), GlutaMax (Life Technologies), and penicillin/streptomycin (Life Technologies). siRNA used was SMART pool: siGENOME PWP1. The transfection of siRNA was performed using Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer's protocol.Drosophila larvae or U2OS cells using the Nucleospin RNA II kit (Macherey-Nagel), and cDNA was synthesized using the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) or the SensiFAST™ cDNA Synthesis Kit (Bioline) according to the manufacturer's protocol. qPCR was performed with the Light cycler480 Real-Time PCR System (Roche) using SensiFAST™ SYBR No-ROX kit (Bioline). The primers used are: human 5S rRNA , human GAPDH , human PWP1  Drosophila 5S rRNA , Drosophila CDK7 .Total RNA was extracted from Antibodies used for chromatin immunoprecipitation are anti-PWP1 , anti-Histone H3 , anti-Histone H4 (acetyl K12)  and anti-Histone H3 (di methyl K9) . The chromatin of HeLa and U2OS cells were collected as previous described , and theSalivary glands were dissected in PBS from wandering third instar larvae and fixed for 3 min in a mixture of 3.7% formaldehyde and 45% acetic acid. Fixed salivary glands were spread in a drop of fixative on a siliconized cover slip which was placed on top of a slide and the coverslip was pressed strongly against it. The slide was quickly submerged into liquid nitrogen, the coverslip was removed and the slide was stored in 67% glycerol in PBS. For immunostaining the preparations were washed in cold PBS 0.1% Tween, followed by 1 h blocking in 5% normal goat serum. The samples were then incubated overnight with the primary antibody anti-dPWP1 (1:500)  at 4°C a


Deciphering the molecular and cellular processes involved in foam cell formation is critical to understanding the pathogenesis of atherosclerosis. Interferon regulatory factor 1 (IRF1) was first identified as a transcriptional regulator of type-I interferons (IFNs) and IFN inducible genes. Our study aims to explore the role of IRF1 in atherosclerotic foam cell formation and understand the functional diversity of IRF1 in various cell types contributing to atherosclerosis.Methods: We induced experimental atherosclerosis in ApoE-/-IRF1-/- mice and evaluated the effect of IRF1 on disease progression and foam cell formation.Results: IRF1 expression was increased in human and mouse atherosclerotic lesions. IRF1 deficiency inhibited modified lipoprotein uptake and promoted cholesterol efflux, along with altered expression of genes implicated in lipid metabolism. Gene expression analysis identified scavenger receptor (SR)-AI as a regulated target of IRF1, and SR-AI silencing completely abrogated the increased uptake of modified lipoprotein induced by IRF1. Our data also explain a mechanism underlying endotoxemia-complicated atherogenesis as follows: two likely pro-inflammatory agents, oxidized low-density lipoprotein (ox-LDL) and bacterial lipopolysaccharide (LPS), exert cooperative effects on foam cell formation, which is partly attributable to a shift of IRF1-Ubc9 complex to IRF1- myeloid differentiation primary response protein 88 (Myd88) complex and subsequent IRF1 nuclear translocation. Additionally, it seems that improved function of vascular smooth muscle cells (VSMCs) also accounts for the diminished and more stable atherosclerotic plaques observed in ApoE-/-IRF1-/- mice.Conclusions: Our findings demonstrate an unanticipated role of IRF1 in the regulation of gene expression implicated in foam cell formation and identify IRF1 activation as a new risk factor in the development, progression and instability of atherosclerotic lesions. Written informed consent was obtained from all participants according to the declaration of Helsinki. The investigations were approved by the Ethical Committee of Huazhong University of Science and Technology.-/- on a C57BL/6 background) were kindly provided by Dr Hongliang Li  -/-IRF1-/- mice, we generally crossbred the IRF1-/- with ApoE-/- to get ApoE+/-IRF1+/- heterozygous mice. ApoE-/-IRF1-/- mice and the control ApoE-/- littermates were obtained by inbreeding between ApoE+/-IRF1+/- heterozygous mice. At the end of the study, Mice were euthanized via intraperitoneal injection of pentobarbital (150 mg/ kg). All the procedures involving mouse experiments were approved by the Ethics Committee of Union Hospital, Huazhong University of Science and Technology, China, and were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals.IRF1 global knockout mice , for collagen content with Masson's trichrome staining (Masson), for elastic fibers with elastica van Gieson staining (EVG). The relative content of macrophages and smooth muscle cells were detected by immunohistochemistry.Lipid accumulation of thoracoabdominal aorta was determined by Replication-defective recombinant adenovirus carrying the entire coding sequence of IRF1 (AdIRF1) or shRNA against IRF1 (AdshIRF1) were constructed with Adenovirus Expression Vector Kit . Amplification and purification of recombinant adenovirus was performed according to the manufacturer's instructions (Takara Bio).Quantitative polymerase chain reaction (PCR) amplification was performed according to the manufacturer's instructions. The real-time PCR primer sequences are shown in Supplementary Table Immunoprecipitation was performed to determine protein complex formation and coimmunoprecipitated proteins were separated with SDS-PAGE followed by mass spectrometry or Western blot.Luciferase reporter constructs  were co-transfected with an internal control plasmid pRL-TK  into HEK293T cells. The luciferase activity was determined with Dual Luciferase Reporter Assay Kit (Promega) according to the manufacturer's instruction.ChIP assay was performed according to the instructions  using monoclonal antibodies against IRF1. DNA samples recovered after immunoprecipitation were subjected to PCR.Macrophages were incubated with 50 μg/ml ox-LDL for different times, fixed with ethanol, and stained with Oil Red O as described Peritoneal macrophages were isolated from C57BL/6 mice P < 0.05 was considered statistically significant. Randomization and blinding strategy was used whenever possible. Animal cohort sizes were determined on the basis of similar previous studies.GraphPad Prism software  was used for statistical analyses. Band intensity in western blot images was quantified with Image J Software. Values are expressed as means ± SEM of at least three independent experiments. Student's t test was used to assess the statistical significance of the differences between two groups. Benjamini-Hochberg corrections for multiple variables were used in transcriptional studies. For multiple groups, significance was evaluated by one-way ANOVA with Bonferroni test (homogeneity of variance) or Tamhanes's T2 test (heterogeneity of variance). -/- mice and normal C57BL/6 mice. As expected, IRF1 protein level in aorta of ApoE-/- mice was significantly increased after 8 weeks of western diet feeding compared to that on standard chow diet or C57BL/6 mice . As shown in Figure -/- and ApoE-/-IRF1-/- mice  content, smooth muscle cell (α-SMC- positive) and collagen (Masson's Trichrome stain) content, fibrous cap area and necrotic croe area, and the destruction of the elastic laminae (Verhoeff's Van Gieson stain), was significantly altered by IRF1 deficiency Figure E. Compare Figure F-K. Thes-/-IRF1-/- mice compared with ApoE-/- mice  was also observed in ApoE-/-IRF1-/- mice  or silence (AdshIRF1 and control AdshRNA) the expression of IRF1. Equal number of cells were seeded into 12-well plates and cultured for 72 hours in medium containing 10% fetal bovine serum (FBS). As expected, knockdown of IRF1 significantly increased the cell number at 48 and 72 hours, whereas overexpression of IRF1 had the opposite effects , which was relatively conserved across vertebrate species, and found an IRF1 binding site, also known as IFN- stimulated response element (ISRE). Mutation of this site completely abrogated the effect of IRF1 on SR-AI promoter activation  and Poly (I:C), which is a synthetic double-stranded RNA (TLR3 agonist), also exerted a similar effect on macrophage lipid accumulation  e Figure B and C, n Figure D. Furthes Figure E and imms Figure F. This it Figure G. Moreovt Figure A. In cont Figure E and G. Although the association between Myd88 and IRF1 clearly led to IRF1 translocation from cytoplasm to nucleus, Myd88 itself remained in cytoplasm and had not been recruited to SR-AI promoter in response to LPS stimulation , also led to IRF1 translocation and lipid accumulation in macrophages incubated with ox-LDL. This is consistent with the fact that all TLRs, except for TLR3, require MyD88 for their downstream signaling Metabolic diseases, such as atherosclerosis, nonalcoholic fatty liver (NAFL) and type 2 diabetes, were more likely to be complicated with subclinical endotoxemia -/-IRF1-/- mice, which correlated with increased growth and inhibited apoptosis of SMCs. Studies in primary VSMCs in vitro confirmed these results and indicated that IRF1 is a negative regulator of VSMC proliferation and migration, and increases its susceptibility to apoptosis. The anti-proliferative and pro-apoptotic mechanisms initiated by IRF1 have been partially characterized previously in different cell systems. Consistent with our current study, Wessely et al. identified IRF1 as a potential inhibitor of the proliferation and migration of the coronary artery smooth muscle cells (CASMC), as well as in neointima formation in vivo in a murine model of neointimal hyperplasia  et al. proved that IRF1 has bi-directional effects on high glucose-induced proliferation of VSMCs through different regulation of cell cycle related proteins Vulnerable plaques contain fewer SMCs and less collagen in fibrous caps than stable plaques, and increased apoptotic SMCs are viewed as a hallmark of plaque instability Compromised endothelial function is a hallmark of early atherosclerosis It is noteworthy that the protective role of IRF1 deficiency in atherosclerosis is multifactorial, and cell type-selective genetic manipulation should be taken into account in our future studies to elaborate on cell specific effects of IRF1 in pro-atherosclerotic mice. Besides, only male mice were used in the present study because of its high success rate and good repeatability of modeling. However, the cardiovascular characteristics, such as blood pressure, serum lipid profile, endothelial function, and abundance of thrombotic plaques of female sex are different from male sex In summary, our study identifies a prominent role of IRF1 in the development, progression and instability of atherosclerotic lesions, and provides a mechanism explaining the link between subclinical endotoxemia and atherosclerosis, which is partly attributable to the acceleration of foam cell formation induced by nuclear translocation of IRF1 and activation of genes implicated in cholesterol metabolism. These findings provide an impetus for further exploration of the roles of IRF1 in other metabolic or inflammatory diseases, and to consider IRF1 as a target for therapeutic intervention in cardiovascular disease.Supplementary methods and figures.Click here for additional data file.


IRF1 (Interferon Regulatory Factor-1) is the prototype of the IRF family of DNA binding transcription factors. IRF1 protein expression is regulated by transient up-regulation in response to external stimuli followed by rapid degradation via the ubiquitin-proteasome system. Here we report that DNA bound IRF1 turnover is promoted by GSK3β (Glycogen Synthase Kinase 3β) via phosphorylation of the T181 residue which generates a phosphodegron for the SCF (Skp-Cul-Fbox) ubiquitin E3-ligase receptor protein Fbxw7α (F-box/WD40 7). This regulated turnover is essential for IRF1 activity, as mutation of T181 results in an improperly stabilized protein that accumulates at target promoters but fails to induce RNA-Pol-II elongation and subsequent transcription of target genes. Consequently, the anti-proliferative activity of IRF1 is lost in cell lines expressing T181A mutant. Further, cell lines with dysfunctional Fbxw7 are less sensitive to IRF1 overexpression, suggesting an important co-activator function for this ligase complex. As T181 phosphorylation requires both DNA binding and RNA-Pol-II elongation, we propose that this event acts to clear ‘spent’ molecules of IRF1 from transcriptionally engaged target promoters. IRF1 is a transcription factor essential for regulating a number of cellular responses including, immunity, apoptosis and DNA repair . IRF1 isIRF1 is a highly unstable protein with a half-life of around 30 minutes  that canCrosstalk between phosphorylation and the Ub machinery is important for regulating protein quantity, activity and interactions ,20. In sWhile IRF1 is known to be extensively modified, relatively little is known about how IRF1 activity is modulated at the posttranslational level. In this study we focused on a pair of previously uncharacterized phosphorylation sites and uncovered a novel mechanism by which cells mark IRF1 as ‘spent’ at the end of the transcriptional cycle.l-glutamine (d-ribofuranosylbenzimidazole), Dox and CHX (Cycloheximide) were from Sigma Aldrich, GSK3 inhibitors BIO (6-bromoindirubin-3′ oxime) and methyl-BIO were from Merck. Details of antibodies used can be found in Cells were maintained in the recommended growth media supplemented with 10% FBS, 50 U/ml Penicillin-Streptomycin and 2 mM lutamine . H3396 dReporter assays; cells were seeded (30 000/well) for 24 h in 24 well plates followed by transfection with reporter construct, IRF1 and internal control CMV-βGAL. Lysis was carried out 48 h post-transfection essentially according to manufacturer's instructions (Applied Biosystems). Luminescence was detected on a Berthold Orion micro-plate luminometer. For analysis of protein degradation, CHX chase assays were performed as follows; cells were seeded on six-well plates for 24 h, transfected with 2.5 μg/well of IRF1 and 24 h later cells treated with 25 μg/ml CHX for the indicated times followed by lysis and immunoblot against IRF1 and β-actin loading control.N-ethylmaleimide (NEM). After pre-clearing for 1 h with Protein G beads, the lysates were incubated overnight at 4°C with the appropriate antibody, washed three times with RIPA buffer and eluted in loading buffer with boiling. Co-immunoprecipitations (1 mg lysate) between FLAG-IRF1 and GSK3β-HA were carried out as above but with washes in TNE buffer (10 mM Tris (pH 7.4), 1 mM EDTA and 200 mM NaCl). For GST-F-box co-immunoprecipitations extracts were made in NP40 lysis buffer . 0.5 mg of lysate was incubated at 4°C with glutathione–sepharose beads  for 3 h, followed by 3× washes in NP40 buffer and elution in loading buffer.For immunoprecipitations, cell lysates (0.5 mg) were diluted in RIPA buffer  supplemented with protease and phosphatase inhibitor cocktail (SIGMA), 1 mM DTT and 100 mM in vitro pulldown assays, GST or GST-IRF1 (1 μg) conjugated GSH beads were diluted in NETN buffer . In vitro translated 35S-labelled proteins (TnT Promega) were added to GST beads and incubated overnight at 4°C followed by 3× washes with NETN. Boiled eluates were separated by SDS-PAGE and the gel was fixed in fixing solution (10% acetic acid 10% methanol) for 30 min before being treated with amplifier solution (Amersham) for 30 min with gentle rocking. Gels were dried and exposed to film at −80°C.For For ubiquitination assays, 60% confluent 10 cm dishes of HEK293 were transfected with 6× His-myc-Ub (2.5 μg) or HA-Ub and FLAG IRF1 (2.5 μg). Forty hours later, MG132 (10 μM) was added for 5 hr. Duplicate transfected plates were treated with 0.01% DMSO as vehicle control. 6xHis nickel pulldowns were carried out essentially as described .Recombinant GSK3β (New England Biolabs) was diluted to 20 ng/μl in kinase buffer and incubated with 4 μg of GST-IRF1 or GST. The final concentration of ATP was 250 μM, in 25 μl. The reaction was carried out at 37°C for 1 h, and terminated by the addition of 6 μl of 5× Laemmli buffer (with 100 mM DTT). Samples were separated by SDS-PAGE and immunoblotted with indicated antibodies.All RNA extractions, reverse transcription, real-time PCR reactions and chromatin immunoprecipitations were performed essentially as in (2).Proliferation assays were performed in H3396 stable cell lines expressing murine IRF1 in pBabeSIN puro under the control of a tetracycline inducible promoter. These vectors were used to transduce H3396 together with a retrovirus encoding the Tet transactivator in a Tet-on configuration selectable with hygromycin and a Tet-repressor, which consists of a Tet DNA binding domain and a KRAB domain selectable with neomycin in order to minimize the background expression. Proliferation assays were carried out by plating the cells at 300 cells/well on a 96-well plate and then treating with Dox at 3 μg/ml for the indicated times. Cells were then trypsinized, re-suspended in PBS/trypan blue and counted by light microscope using the trypan blue exclusion method. All cell counts were done in quadruplicate. For proliferation of other lines, cells were transduced with pBabeSIN puro, IRF1 WT or IRF1 T181A for 48 hr prior to selection with 1 μg/ml puromycin to remove non-transduced cells. To assess clonal growth, cells were counted and plated at low dilution (100–500 cells) on 48-well plates and left to grow for 10 days prior to staining with crystal violet (0.05% in 50% methanol). For short-term growth assays, cells were plated in 24-well plates following puromycin selection and allowed to grow for 4 days. Cells were counted in triplicate after trypan blue staining.P value <0.05 as detected by Student's t-test **P < 0.01, ***P < 0.005.Unless stated otherwise, all quantitative or semi-quantitative assays  were performed in three independent experiments with three technical repeats. *denotes statistical significance with a in vitro kinase assays with recombinant GSK3β and murine GST-IRF1. The reaction mix was subjected to western blotting using an antibody raised against phospho-threonine-proline dipeptide. As murine IRF1 contains only a single threonine-proline dipeptide sequence, we could therefore use this antibody to assess IRF1 T181 phosphorylation. In addition to a migration shift in the GST-IRF1 protein, we detected phosphorylation of IRF1 after exposure to GSK3β indicating that GSK3β could directly modify IRF1  that shares similarity to a subset of consensus GSK3 target sites. This sequence is conserved in mammalian species and comprises a threonine residue (T181) with an upstream ‘priming’ residue (S185) Figure . To dete1 Figure . To supp1 Figure .We noted that the sequence surrounding T181 (i.e. 181-TPALSP-186) is conserved in a subset of phosphoproteins  includin180/181/Ser184/185 phosphorylation by GSK3β.Taken together, these data provide evidence that both exogenous murine and endogenous human IRF1 proteins are subjected to dual Thrin vitro was confirmed by GST-IRF1 pulldown of 35 fall into this category . FuIRF1 has previously been shown to be poly-ubiquitinated in cells  which pr181/Ser185 in both basal and GSK3β overexpression conditions . HEK293 lysates expressing GST-Fbxw7 isoforms were subjected to GST pulldowns and demonstrated that IRF1 preferentially interacts with the nuclear form; Fbxw7α . The intWT and UbK48O (K48 only) pulldowns of IRF1 with GSK3β or Fbxw7α overexpression and detected an increase in IRF1 ubiquitination when either of these proteins were overexpressed  . Conversd Figure . This sud Figure . Next weα Figure . These mresidues . The Fbxresidues , F and lresidues . CollectThe anti-proliferative action of IRF1 is well studied and contributes to tumour suppressor activity . To inveWe measured proliferation rates in vector, WT and T181A expressing H3396 stable cell lines. Induction of WT IRF1 resulted in a marked decrease in growth of H3396. In contrast, the T181A mutant showed no inhibition of growth similar to empty vector control Figure . We nextHere we report a novel mechanism for IRF1 transcriptional control centring on phosphorylation-dependent degradation. This tightly controlled clearance is important for IRF1 dependent RNA-Pol-II elongation, mRNA generation and anti-proliferative activity.Several reports have identified Ub E3 ligases that target IRF1. The CHIP E3 ligase was shown to ubiquitinate IRF1 using both K48 and K63 linkages . SubsequDe novo IRF1 protein is induced following stimulation with agents such as IFNγ. The steady state levels of non-DNA bound IRF1 are maintained by E3 ligases (such as CHIP), which target this pool for degradation. IFNγ signalling induces a widespread recruitment of IRF1 to target genes. DNA-bound IRF1 is then protected from degradation as lysines in the DBD are shielded from Ub E3 ligases. The difference in half-life of nucleoplasmic versus DNA engaged IRF1 most likely reflects the ‘time’ required for other signalling/remodelling/recruiting events to occur prior to successful transcriptional elongation. We propose that following firing of RNA polymerase II into the elongation phase, GSK3β dependent phosphorylation marks IRF1 as ‘spent’, which then targets SCFFbxw7 to ubiquitinate C-terminal lysine residues and promote proteasome dependent degradation. Essentially, this represents a transcriptional time clock . SUMOylation of C-terminal lysines has been shown to stabilize IRF1 by competing for ubiquitination. Hyper-SUMOylation of IRF1 has been detected in ovarian cancers, and is known to disrupt IRF1 transcriptional activity .Loss of function in the GSK3β-Fbxw7α axis occurs in several cancer types . This isSupplementary DataClick here for additional data file.