Cellular senescence is a complex biological process that leads to irreversible cell-cycle arrest. Various extrinsic and intrinsic insults are associated with the onset of cellular senescence and frequently accompany genomic or epigenomic alterations. Cellular senescence is believed to contribute to tumor suppression, immune response, and tissue repair as well as aging and age-related diseases. Long noncoding RNAs (lncRNAs) are >200 nucleotides long, poorly conserved, and transcribed in a manner similar to that of mRNAs. They are tightly regulated during various cellular and physiological processes. Although many lncRNAs and their functional roles are still undescribed, the importance of lncRNAs in a variety of biological processes is widely recognized. RNA-binding proteins (RBPs) have a pivotal role in posttranscriptional regulation as well as in mRNA transport, storage, turnover, and translation. RBPs interact with mRNAs, other RBPs, and noncoding RNAs (ncRNAs) including lncRNAs, and they are involved in the regulation of a broad spectrum of cellular processes. Like other cell fate regulators, lncRNAs and RBPs, separately or cooperatively, are implicated in initiation and maintenance of cellular senescence, aging, and age-related diseases. Here, we review the current understanding of both lncRNAs and RBPs and their association with oxidative stress, senescence, and age-related diseases. Hayflick and Moorehead firstly described replicative senescence (RS), which is an exhaustion of replicative potential in human diploid fibroblasts after continuous cultivation , 2. Moreocessing . In the ocessing . Currentocessing . Althougocessing . SASP caocessing –10. Aginocessing –14. Acti disease . Schemat disease –18.The lncRNAs are transcripts more than 200 nucleotide long that have no protein-coding potential. Moreover, they are poorly conserved, transcribed from the intergenic and intronic regions of genome primarily by polymerase II, 5′ methyl-capped, and polyadenylated in manner similar to that of mRNAs . The lncLincRNA-p21 is 3.1 kb long and is transcribed from the opposite strand to p21 (CDKN1A) in a p53-dependent manner [LincRNA-p21, which is also induced by hypoxia and/or hypoxia inducible factor-1α (HIF-1α), is able to bind HIF-1α and VHL, and it disrupts the VHL–HIF-1α interaction. This disassociation attenuates VHL-mediated HIF-1α ubiquitination and causes HIF-1α accumulation. These results indicate a positive feedback loop between HIF-1α and lincRNA-p21 under hypoxia [LincRNA-p21 is highly inducible by UVB through a p53-dependent pathway and plays key role in the UVB-induced apoptotic pathway [t manner . LincRNA pathway .LincRNA-RoR, a 2.6 kb long transcript, was first described as having potentially important functions in embryonic stem cells and induced pluripotent stem cells (iPSCs) [LincRNA-RoR regulates genes involved in the p53 response, such as responses to oxidative stress and DNA damage [lincRNA-RoR. LincRNA-RoR dramatically represses DNA damage-induced p53 compared to that in unstressed cells. Depletion of lincRNA-RoR did not regulate p53 mRNA levels, suggesting posttranscriptional regulation of p53. Mechanistically, lincRNA-RoR has a 28-base heterogeneous nuclear ribonucleoprotein I- (hnRNP I-) binding motif and directly interacts with phosphorylated hnRNP I in the cytoplasm. The interaction between lincRNA-RoR and phosphorylated hnRNP I directly represses p53 translation and results in the modulation of cell-cycle progression and apoptosis. Thus, lincRNA-RoR and p53 act within an autoregulatory feedback loop in response to cellular stress [lincRNA-RoR can epigenetically regulate the expression of TESC by recruiting G9A methyltransferase in the TESC promoter [ (iPSCs) . LincRNAA damage . Depletir stress . A recenpromoter .Pint (p53-induced noncoding transcript), previously named lincRNA-Mkln1, has highly conserved canonical p53-binding motifs in the promoter and is a transcriptional target of p53 [Pint is a nuclear lincRNA and is transcribed from an intergenic region on mouse chromosome 6. Pint has three p53 response elements and is directly regulated by p53 upon DNA damage [Pint significantly decreases cell proliferation, and overexpression of Pint conversely increases cell growth. Pint directly interacts with Polycomb repressive complex 2 (PRC2) and represses expression of PRC2 targeting genes via H3K27 trimethylation. PINT, the pint human ortholog, is also regulated by p53. However, overexpressed PINT diminishes tumor cell proliferation, indicating both analogy and difference between murine Pint and human ortholog PINT [t of p53 . Pint isA damage . Depletilog PINT .PANDA (P21-associated ncRNA DNA damage-activated) was identified at the CDKN1A locus [PANDA is specifically induced by DNA damage in a p53-dependent manner. PANDA is a 5′-capped, polyadenylated lncRNA located approximately 4.5 kb upstream of the CDKN1A transcriptional start site. In human fibroblasts treated with doxorubicin, PANDA prevents NF-YA activation, through its association with NF-YA, finally suppressing transcription of proapoptotic genes. Thus, PANDA induced by DNA damage impedes apoptosis through recruitment of NF-YA [PANDA can stabilize p53 proteins in response to DNA damage [PANDA causes G1 arrest via an increase in the mRNA level of cyclin-dependent kinase inhibitor p18 [To detect functional noncoding RNAs (ncRNAs) in the regulatory region of human cell-cycle genes, ultrahigh-density array fabrication was performed and the lncRNA 1A locus . PANDA iof NF-YA . AnotherA damage . In addiitor p18 .LncRNA-JADE is induced by ATM-NF-кB signaling and is mainly localized in the nucleus in the DNA damage response (DDR) [lncRNA-JADE interacts with breast cancer type 1 susceptibility protein (Brca1) and induces expression of Jade1, a major component of human acetylase binding to ORC 1 (HBO1) histone acetylation complex. Consequently, depletion of lncRNA-JADE renders sensitivity to DNA damaging drugs through the functional link between DDR and histone H4 acetylation in the DDR. [se (DDR) . In respthe DDR. .H19 was first described as an imprinted ncRNA transcript at the Igf2 locus. A number of studies have reported that H19 is upregulated in both primary and metastatic tumors and is closely involved in migration, angiogenesis, and inflammatory diseases [α and p53 are involved in the upregulation of H19 in hypoxic cancer cells [H19 expression is elevated under hypoxic conditions in mesenchymal stem cells [H19 in diabetic rats can attenuate oxidative stress, inflammation, and apoptosis [diseases . HIF-1α er cells . Recentlem cells . In addipoptosis .ANRIL (antisense noncoding RNA in the INK locus) is transcribed in the antisense direction to the INK4B-ARF-INK4A locus and is transcriptionally upregulated by the transcription factor E2F1 in an ATM-dependent manner after DNA damage. Such elevated levels of ANRIL suppress the expression of INK4A-ARF-INK4B in the late-DDR stage, allowing negative feedback to the DDR. Thus, ANRIL helps the cell to return to a normal status at the completion of DNA repair [A repair .LncRNA-LET (lncRNA low expression in tumor) transcripts are underexpressed in tumor tissues compared to their expression in paired nontumor tissues [LncRNA-LET is downregulated by hypoxia-induced histone deacetylase 3 (HDAC3) under hypoxic conditions. LncRNA-LET is associated with degradation of nuclear factor of activated T cells 90 kDa (NF90) protein via the regulation of ubiquitin-proteasome pathway. Since NF90 stabilizes HIF-1α mRNA without altering HIF-1α transcriptional activity, lncRNA-LET finally decreases HIF-1α stability due to its association with NF90. These findings illustrate that lncRNA-LET could be a key regulator of hypoxia signaling [ tissues . Moreoveignaling .LINK-A (long intergenic noncoding RNA for kinase activation) is 1.5 kb long and mainly localized in the cytoplasm [LINK-A facilitates breast tumor kinase (BRK) activation through the recruitment of BRK to the EGFR : GPNMB heterodimeric complex upon HB-EGF stimulation. Consequently, activated BRK induces phosphorylation of HIF-1α at Tyr565 and inhibits hydroxylation of HIF-1α, finally resulting in HIF-1α stabilization. In addition, LINK-A interacts with leucine-rich repeat kinase 2 (LRRK2) and enhances phosphorylation of HIF-1α at Ser797. Phosphorylation of Ser797 increases transcriptional activation of HIF-1α via HIF-1α–p300 interaction. These events illustrate the magnitude and diversity of cytoplasmic lncRNA LINK-A in signal transduction related to HIF-1α under normoxic conditions [ytoplasm . LINK-A nditions .7SL is a 300 bp long transcript and an RNA component of signal recognition proteins (SRP) [7SL is widely upregulated in cancer tissues and involved in cell proliferation. 7SL decreases p53 translation and accumulation by interacting with the 3′-untranslated region (3′-UTR) of TP53 mRNA, which encodes tumor suppressor p53. Depletion of 7SL increases the occupancy of HuR to TP53 mRNA and p53 production. 7SL-depleted cells undergo cellular senescence and autophagy, indicating that 7SL promotes cell growth via p53 suppression [ns (SRP) . 7SL is pression .HOTAIR (HOX antisense intergenic RNA) was first identified as HOX lncRNA located in the HOXC locus through transcriptomic analyses of HOX loci [HOTAIR enhances cancer progression and malignancy by leading to altered H3K27 methylation due to retargeting of PRC2 [HOTAIR induces cell-cycle arrest in various cancer types. In addition, HOTAIR can contribute to cellular senescence via a positive feedback loop cascade of an NF-кB–HOTAIR axis [HOX loci . This anHOX loci . HOTAIR  of PRC2 . DepletiAIR axis .UCA1 , an lncRNA with a length of 1.4 kb, was first identified in bladder cell carcinoma [UCA1, a direct target of coactivator of AP1 and estrogen receptor α (CAPERα)/T-box3 (TBX3) repression, sequesters hnRNP I, which suppresses transcription of CDKN2A and destabilizes CDKN2A mRNA [α/TBX3 corepressor and activates UCA1. CAPERα/TBX3 and UCA1 coordinately induce oncogene-induced senescence (OIS). In addition, UCA1 can bind with hnRNP I and competitively inhibit hnRNP I binding to p27 mRNA [p27 mRNA, and there is a negative correlation between p27 expression and UCA1 level.arcinoma . UCA1, aN2A mRNA . Oncogenp27 mRNA . hnRNP IlincRNA-p21 increases p21 expression at both mRNA and protein levels, and it impedes cell-cycle progression [LincRNA-p21 is necessary for the recruitment of hnRNP K to the p53 response element and for increasing the binding efficiency of p53 on the p21 promoter region. Moreover, lincRNA-p21 affects the G1/S checkpoint and p21 levels through deregulated expression and altered chromatin state of some Polycomb target genes. Thus, lincRNA-p21 is required for the positive regulation of p21 expression and finally is involved in cellular senescence [Overexpressed gression . LincRNAnescence .ANRIL is a 3.8 kb long lncRNA transcribed in an antisense orientation from the INK4B/ARF/INK4A gene cluster, and it overlaps with the promoter of p14/ARF and the two exons of p15/CDKN2B [ANRIL is required for the recruitment of chromobox (CBX7), a component of PRC1, to the INK4B/ARF/p16 gene locus. This complex exhibits high-affinity binding to methylated histone H3 at lysine 27 (H3K27me) and represses the transcription of INK4b/ARF/INK4a [ANRIL disrupts the binding of suppressor of zeste 12 protein homolog (Suz12), a component of PRC2, to INK4B locus, and increases the expression of p15 [ANRIL promotes silencing of KLF2 and P21 transcription via epigenetic silencing [ANRIL, which is associated with senescence, was reviewed by Aguilo et al. [5/CDKN2B . ANRIL iRF/INK4a . Moreoven of p15 , 50. Recilencing . Such epo et al. .ANRASSF1 (antisense intronic noncoding RASSF1) is an intronic lncRNA transcribed from the antisense to RAS-association domain family member 1A (RASSF1A) gene [RASSF1A, a tumor suppressor gene, is associated with cell-cycle arrest and senescence via p53-independent regulation of p21 [ANRASSF1 recruits PRC2 to the RASSF1A promoter and increases the H3K27me3 level, resulting in decreased RASSF1A expression. Therefore, ANRASSF1 mediates cellular senescence through the epigenetic inactivation of the RASSF1A gene [1A) gene . RASSF1An of p21 . Highly PANDA (P21 associated ncRNA DNA damage-activated) is capable of interacting with scaffold-attachment-factor A (SAFA) [PANDA interaction recruits Polycomb repressive complex 1 (PRC1) and PRC2 complexes to senescence target genes including CDKN1A in order to silence their expression [PANDA depletion leads to senescence phenotypes by derepression of p21 due to disruption of SAFA-PANDA-PRC interactions. However, in senescent cells, PANDA sequesters transcription factor NF-YA and limits the expression of NF-YA-E2F-coregulated proliferation-promoting genes. Therefore, PANDA levels modulate cell fates to enter or exit from senescence [A (SAFA) . SAFA isA (SAFA) . In prolpression . Thus, Pnescence .FAL1  was identified from a genome-wide analysis of somatic copy number alterations [FAL1 interacts with epigenetic repressor BMI1 protein, a subcomponent of PRC1, and increases BMI1 stability. Thus, FAL1 can negatively regulate a large number of genes such as CDKN1A, FAS, and BTG2. In addition, FAL1 promotes tumor proliferation and represses senescence primarily by decreasing CDKN1A transcription [erations . FAL1 incription .MIR31HG lncRNA is upregulated during OIS, its depletion promotes p16-dependent senescence phenotypes [MIR31HG interacts with the INK4A and MIR31HG genomic loci and mediates repression of the INK4A locus with Polycomb group (PcG) proteins. MIR31HG plays a role during OIS as a transcriptional regulator of p16 via direct interaction with PcG proteins [Whereas enotypes . MIR31HGproteins .SALNR (senescence-associated long noncoding RNA) expression is downregulated in senescent human fibroblasts. SALNR interacts with NF90, a RNA-binding protein involved in microRNA (miRNA) biogenesis, and regulates its nuclear localization. SALNR and the NF90 complexes impede premature senescence through the regulation of senescence-associated miRNAs, specifically miR-181a and miR-22 [d miR-22 .VAD is a vlincRNA (very long intergenic ncRNA) that is differentially expressed in RAF-induced senescence and is localized in the chromatin. VAD is involved in the maintenance of senescence features. Mechanistically, VAD modulates chromatin structure in cis and increases the expression of INK4 genes in trans. VAD decreases the occupancy of the repressive histone variant H2A.Z at INK4 promoters during senescence induction [nduction .(1) Alzheimer's Disease. Alzheimer's disease (AD) is the most common neurodegenerative disease and accounts for the majority of dementia cases. Amyloid β (Aβ) plaques and neurofibrillary tangles are the two primary pathological hallmarks of AD. The amyloid cascade hypothesis suggests that deposition of Aβ might be cause of neuronal dysfunction and death of brain tissue in AD. Recently, the cleavage patterns of amyloid precursor protein (APP) to Aβ peptides (Aβ1–40 and Aβ1–42) by secretases, small oligomers of Aβ (2~12 peptides), Aβ concentration, and Aβ stability have been proposed as important factors in AD [rs in AD .BC200 is a 200 bp long RNA pol III-transcribed lncRNA that is predominantly expressed in the brain [BC200 is downregulated in normal aged brains, but BC200 is significantly upregulated in AD brains. Specifically, BC200 is highly expressed in AD-related regions  compared to its expression in nonrelated regions  [he brain . BC200 iarea 17) .BACE1-AS (BACE1-antisense transcript) is a 2 kb long transcript from the antisense strand of β-secretase-1 (BACE1) and is a crucial enzyme in AD pathology. BACE1-AS regulates BACE1 mRNA and protein expression in vitro and in vivo. In response to cell stress, elevated BACE1-AS increases BACE1 mRNA and protein levels due to RNA duplex formation, generating additional Aβ 1–42 peptides [BACE1 and the BACE1-AS transcript can participate in the alteration of oligomeric Aβ aggregation pattern and Aβ-related hippocampal neurogenesis [peptides . Moreoveogenesis .NDM29 (neuroblastoma differentiation marker 29) is a cytoplasmic lncRNA transcribed by polymerase (pol) III. NDM29 is highly expressed in neuroblastoma cells and is involved in neuroblastoma maturation [NDM29 expression is detected in the brain of AD patients. NDM29-dependent cell maturation induces APP synthesis and results in an increase of Aβ secretion. Moreover, an increase in the production of copies of NDM29 transcripts can be driven by inflammatory stimuli [turation . In addi stimuli .17A is a 159 bp long lncRNA synthesized by RNA pol III that induces an increase of GABA B2 receptor splice variant B, which affects GABA-B function. Thus, 17A impairs GABA-B signaling and might enhance Aβ secretion. In addition, 17A is upregulated in AD compared to its level in control tissues [51A, NAT-Rad18, and GNDFOS might also be involved in AD [ tissues . Other led in AD , 71.(2) Parkinson's Disease. Parkinson's disease (PD) is a common and complex neurodegenerative disease characterized by defects in the body's motor functions . The classical features of PD are associated with Lewy bodies and loss of dopaminergic neurons in the substantia nigra pars compacta in the midbrain. The resultant deficiency of dopamine ultimately induces movement disorder, which is a characteristic of PD [ic of PD .AS Uchl1  is a 1.2 kb lncRNA transcribed from the opposite strand of the ubiquitin carboxy-terminal hydrolase L1 (Uchl1) gene and induces UChl1 translation. AS Uchl1 is expressed in mesencephalic regions, which are degenerated in PD. AS Uchl1 is regulated by Nur11, a major transcription factor functioning in the differentiation and maintenance of dopaminergic neurons. Expressions of AS Uchl1 and UCHL1 have been decreased in PD models in vitro and in vivo [ in vivo .NaPINK1 is transcribed from the antisense direction of the PINK1 gene, which is implicated in PD through an association with unbalanced mitochondrial homeostasis. As naPINK1 is able to stabilize PINK1 splice variant (svPINK1) expression in neurons via a dsRNA-mediated mechanism, naPINK1 might be involved in PD through regulation of the PINK1 locus [K1 locus , 74.(3) Huntington's Disease. Huntington's disease (HD) is a dominantly inherited disease characterized by chorea, psychiatric problems, and dementia. HD is caused by mutation in the huntingtin (HTT) gene. Expansion of the CAG-triplet repeat sequence within the first exon of HTT results in abnormal protein production, which gradually leads to death of brain cells [in cells .TUG1 (taurine upregulated gene 1) was first identified in a screen for genes upregulated by taurine in developing retinal cells [TUG1 increases the phenotypes of apoptosis [TUG1 expression is elevated in HD [TUG1, a direct transcriptional target of p53, combines with enhancer of zeste homolog 2 (EZH2), a component of the PRC2 complex, and epigenetically regulates gene expression. TUG1 and EZH2 bind to the promoter of homeobox B2 (HOXB7) and represses HOXB7 expression [al cells  and, subal cells . Depletipoptosis , and TUGed in HD . Mechanipression .MEG3 is highly expressed in adult human and mouse brains and is differentially expressed in HD patients. MEG3 can associate with the PRC2 complex and is found in the chromatin region in the nucleus, suggesting that MEG3 might be involved in epigenetic regulation in HD [HAR1 (human accelerated region 1), NEAT1 (nuclear paraspeckle assembly transcript 1), and DGCR5  also exhibit altered expression in HD patients, as shown by microarray studies [on in HD , 78. Oth studies , 78.HTTAS (huntingtin antisense) is a natural antisense transcript at the HD CAG repeat. HTTAS is mainly spliced into HTTAS-V1 (exons 1 and 3) and HTTAS-V2 (exons 2 and 3). HTTAS-V1 expression is reduced in human HD frontal cortex, and its overexpression negatively regulates HTT transcription [cription .The immune response is a wide variety of physiological and pathological processes originating from immune system activation. It is triggered by pathogens, antigens, tissues injury, and other noxious stimulations. The innate immune response provides immediate defense against infection and is evolutionarily conserved. The adaptive immune response is highly specific to particular pathogens and provides long-lasting protection. Adaptive immune responses are mainly mediated antibody and cell-mediated immune responses. The inflammatory response is considered an innate immune response. Inflammation functions to eliminate the initial cause of the original insult and to initiate tissue repair, which are regulated by immune mediators including cytokines, chemokines, and soluble inflammatory proteins .THRIL (TNFα- and hnRNP L-related immunoregulatory lincRNA) is an approximately 2 kb lncRNA that changes expression upon activation of innate immune signaling in macrophages. THRIL recruits heterogeneous nuclear ribonucleoprotein L (hnRNP L) to the TNFα promoter and increases the secretion of TNFα, an inflammatory cytokine. An increase in TNFα downregulates THRIL expression via a negative feedback mechanism. Moreover, THRIL is associated with maintaining expression of many innate immunity-associated genes [ed genes .Lnc-DC is exclusively expressed in human conventional dendritic cells and is involved in dendritic cell (DC) differentiation and DC capacity to stimulate T cell activation. Lnc-DC prevents signal transducer and activator of transcription 3 (STAT3) dephosphorylation by SHP1 through a direct association with STAT3. Lnc-DC is known as a specific regulator of DC differentiation and function [function .Lnc-IL7R, which overlaps with the 3′-UTR of interleukin-7 receptor α (IL7R) gene, shows altered expression in response to LPS stimulation. Lnc-IL7R functionally diminishes the LPS-induced inflammatory response and is mechanistically involved in trimethylation of H3K27 at the E-selectin and VCAM-1 promoters. Lnc-IL7R is a regulator of proinflammatory genes via epigenetic modification [fication .LincRNA-EPS is downregulated in response to inflammatory triggers. Gain-of-function and rescue studies have revealed that lincRNA-EPS represses transcriptions of immune response genes by interacting with hnRNP L. LincRNA-EPS has a critical role in restraining lethal inflammatory responses [esponses .Diabetes is a metabolic disease associated with high blood sugar levels. Diabetes can be caused by the pancreas not producing insulin (type 1 diabetes (T1DM)) or by insulin resistance (type 2 diabetes (T2DM)). The majority of type 1 diabetes cases are attributed to a T cell-mediated autoimmune attack, which leads to loss of the insulin-producing beta cells of the islets of Langerhans in the pancreas. It is traditionally termed juvenile diabetes and is partially inherited. T2DM is the most common type of diabetes. Insulin resistance in T2DM might be combined with reduced insulin secretion and defective responsiveness of insulin receptors .RNCR3 is involved in diabetes-induced retinal neurodegeneration [RNRC3 reduces the release of cytokines and results in fewer apoptotic retinal cells and improved visual function. RNCR3 increases in response to high glucose stress in vitro and in vivo and regulates retinal endothelial cell function through the RNCR3/KLF2/miR-185-5p network [neration . Knockdo network .MEG3 is reduced in the retinas of STZ-induced diabetic mice and in endothelial cells under high glucose and oxidative stress. MEG3 knockdown aggravates diabetes-related retinal vessel dysfunction, which is mainly mediated by activation of PI3K/AKT signaling [MEG3 expression is upregulated in hepatocytes through histone acetylation in high-fat diet and ob/ob mice. In addition, MEG3 is involved in hepatic insulin resistance via an increase in FoxO1 expression [ignaling . MEG3 expression .HI-LNC25 was first identified in a transcriptome mapping study of human pancreatic islets and β cells [HI-LNC25 is a β cell-specific lncRNA and an integral component of β cell differentiation and maturation [HI-LNC25 decreases expression of GLIS3 mRNA, which is associated with pancreatic β cell function and mass maintenance [KCNQ1OT1 and HI-LNC45, which were previously genetically associated with T2DM [ β cells . HI-LNC2turation . Depletintenance . KCNQ1OTith T2DM , are sigith T2DM .Muscle development is a multistep process that includes myogenesis, muscle differentiation, and regeneration. Myogenesis is a tightly regulated developmental program to direct myoblasts to form muscle fibers. Myogenic pathways are primarily governed by transcription factors, MyoD, Myf5, myogenin, and MRF4 at the molecular level. Impairment of these processes might be a cause of muscle dysfunction and is an age-related pathological phenomenon .SRA (steroid receptor RNA activator) was initially characterized as an lncRNA functioning to enhance steroid receptor-dependent gene expression [SRA has an unusual property that functions as both SRA RNA and SRAP protein through alternative splicing. The ratio between SRA RNA and SRAP increases during myogenic differentiation, but there is no increase in myotonic dystrophy patients. SRA RNA is an enhancer of myogenic differentiation and myogenic conversion through regulation of MyoD activity [pression . SRA hasactivity .MUNC (MyoD upstream noncoding RNA) is transcribed from the upstream of myogenic differentiation (MyoD), a master transcriptional regulatory factor in muscle differentiation and specifically expressed in skeletal muscle. MUNC depletion reduces myoblast differentiation and impairs muscle regeneration in vivo. MUNC is involved in gene expressions of MyoD, Myogenin, and Myh3 (myosin heavy chain) by acting in trans. MUNC also stimulates the transcription of other genes that are not recognized as MyoD-inducible genes. MUNC is an evolutionarily conserved promyogenic lncRNA that acts directly or indirectly on multiple promoters to increase myogenic gene expression [pression .Linc-RAM (linc RNA activator of myogenesis) is a skeletal muscle-specific lncRNA that localizes in both cytoplasm and nucleus of myoblast. Depletion of linc-RAM impairs myoblast differentiation and muscle regeneration. Mechanistically, linc-RAM promotes assembly of the MyoD-Baf60c-Brg1 complex and facilitates the recruitment of the SWI/SNF core on target myogenic genes, resulting in transcription of myogenic differentiation genes [on genes .linc-MD1 and lncRNA Dum are also involved in the control of muscle gene expression and muscle regeneration [Other muscle-specific lncRNAs such as neration , 99.Atherosclerosis is the primary cause of heart disease and stroke. It is a chronic disease of the large arteries and is characterized by narrowing or closing of an artery with lipids and fibrous elements. Pathological studies have provided evidence of the critical role of endothelium in mediating inflammation and accumulation of oxidized low-density lipoproteins (LDL) in the intima to recruit monocytes and form macrophage-derived foam cells .SENCR  is an antisense transcript from the first intron of friend leukemia virus integration 1 (FLI1) and is localized in the cytoplasm. SENCR is highly expressed in both smooth muscle and endothelial cells [SENCR impedes migration and proliferation of smooth muscle cells through the regulation of FoxO1 and TRPC6 expression [SENCR is associated with the regulation of endothelial cell differentiation and angiogenic capacity of human umbilical endothelial cells (HUVECs) [al cells . SENCR ipression . In addi(HUVECs) .Recent studies have reported several lncRNAs that are involved in atherosclerosis-related smooth muscle cell, endothelial cell, macrophage, and lipid metabolism regulation, suggesting a potential function of such lncRNAs in atherosclerosis development .Cataract is characterized by the clouding of an eye's lens. Cataract accounts for half the cases of blindness. Lens proteins denature and degrade over time, and this process is accelerated by age and diseases such as diabetes and hypertension. ROS may be mechanistically involved in cataractogenesis. The only treatment for cataract is surgery in the current state of technology .LncRNA-MIAT  is highly expressed in patients with cataracts and is involved in the maintenance of human lens epithelial cells (HLECs) whose dysfunction results in cataract formation. MIAT regulates viability, proliferation, and migration of human HLECs in response to oxidative stress. Mechanistically, MIAT acts as a competing endogenous RNA (ceRNA) and can regulate HLEC function through a feedback loop with AKT and miR-150-5p [R-150-5p .A mechanistic diagram of representative lncRNAs involved in DNA damage and oxidative stress, cellular senescence, and age-related diseases is shown in The RBPs have a pivotal role in mediating posttranscriptional regulation of gene expression by affecting pre-mRNA splicing and maturation as well as mRNA transport, storage, turnover, and translation . RBPs caVEGF, HIF-1α, p53, c-myc, SIRT1, and prothymosin α. The roles of HuR in the regulation of stress response have been extensively reviewed by others [HuR is a member of human antigen (Hu) family and governs turnover and translation of target mRNAs involved in the regulation of cell proliferation, growth, survival, and differentiation in response to various stresses . HuR hasy others , 115. A y others . HuR is y others , 118. Esy others .GADD45α mRNA during DDR [replication protein A (RPA2), thioredoxin (TRX), or HIF-1α [hdm2 and UVE-triggered translation of Apaf-1 in response to UV exposure [p53 mRNA, thereby regulating apoptosis [HIF-1α-mediated gene expression by enhancing translation of HIF-1α in hypoxia [Heterogeneous nuclear ribonucleoproteins (hnRNPs) are nuclear proteins regulating a broad spectrum of RNA metabolism including alternative splicing, translocation, and translation . hnRNP Aring DDR . In respr HIF-1α , 123. hnexposure . After iexposure . hnRNP Hpoptosis . hnRNP I hypoxia .FUS  binds RNA and single- and double-stranded DNA, and it affects multiple steps of DNA/RNA metabolism. FUS has been observed in sites with laser-induced DNA double-strand breaks (DSBs) and regulates DSB repair . FUS alsApaf-1 mRNA, and enhances their translation [T cell-restricted intracellular antigen-1 (TIA-1) is a member of RNA-binding protein involved in alternative pre-mRNA splicing and mRNA translation. TIA-1 is a component of stress granules (SGs) triggered by hypoxia, ischemia, and anoxia, and it has essential roles in regulating mRNAs involved in oxidative stress and DDR through its associations with other SG components . A recennslation .p53 mRNA by protecting it from deadenylation, thereby enhancing the p53-mediated stress response [σ [Wig1  is a transcriptional target gene of p53 and has a zinc-finger domain that binds to double-strand RNA (dsRNA) . Wig1 haresponse . Depletiponse [σ .p21, p16, cyclin A, cyclin B1, c-fos, and SIRT1 [Drosophila as well as to several senescence-associated phenotypes in mouse embryonic fibroblasts (MEF) [HuR is implicated in cellular senescence and the aging process based on its involvement in regulating stability and translation of various target mRNAs including nd SIRT1 . Recent ts (MEF) , 139. Thts (MEF) . Coactivts (MEF) . includes four alternative spliced isoforms  containing two RRMs and regulates mRNA stability and turnover. In addition, AUF1 has been shown to affect proliferation, stress response, immune response, and cellular senescence. AUF1 is differentially regulated during aging and cellular senescence [p21 and p16 in a competitive or cooperative manner with HuR and influences cellular senescence [AU-rich element RNA-binding protein 1 nescence . AUF regnescence , 143. Ponescence .TNFα, COX-2, c-myc, calmodulin 2, small nuclear ribonucleoprotein polypeptide F (SNRPF), and caspase-8 in response to various cellular stresses [TIA-1 and TIAR regulate alternative splicing, SG formation, and translation of various target genes including stresses . It has stresses . TIA-1/Tstresses . Howeverp21 and increases translation of p21 by competing with calreticulin. In senescent cells, increased phosphorylation of CUGBP1 promotes binding to p21 mRNA.CUG triplet repeat, RNA-binding protein 1 (CUGBP1) is a member of the CELF/BRUNOL protein family containing two N-terminal RRMs and regulates pre-mRNA alternative splicing, mRNA editing, and translation . CUGBP1 β (C/EBPβ) expression [C/EBPβ mRNA and enhances its translation, thereby accumulating C/EBPβ-liver-enriched inhibitory protein (C/EBPβ-LIP), a dominant inhibitor of differentiation, in fat cells. Augmented expression of CUGBP1 is responsible for the impairment of adipogenesis in aged-fat tissues. In old liver, CUGBP1 phosphorylation at S302 residue by GSK3β facilitates the association of CUGBP1 with eukaryotic initiation factor 2 (eIF2) and increases translation of HDAC1 and C/EBPβ, which are responsible for epigenetic regulation of gene expression [CUGBP1 has been known to increase with aging in fat tissue and to regulate CCAAT/enhancer-binding protein pression . CUGBP1 pression , 151.E47 mRNA [Tristetraprolin (TTP) is an ARE-binding protein involved in destabilizing target mRNAs, and its expression is upregulated during cellular senescence and aging . TTP is E47 mRNA . SandujaE47 mRNA . E6-AP dp21 mRNA and recruits the RNA-induced silencing complex (RISC) by interacting with Ago2, thereby destabilizing p21 mRNA. Depletion of Wig1 results in a decrease of miR-mediated p21 mRNA decay and promotes cellular senescence via p21 upregulation in various cell types.Wig1 is also implicated in the regulation of cellular senescence. Kim et al. reported that Wig1 prevents cellular senescence by regulating p21 expression . Wig1 biNeuronal cells have their own systems for regulating RNA expression in response to various stimuli via RBPs that are uniquely expressed in neuronal cells. Accumulating evidence indicates that abnormalities in RNA metabolism are a common feature of neurodegeneration , 157. Th(1) TDP-43. TDP-43 was identified as the major component of ubiquitin-positive neuronal inclusion bodies observed in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) patients [patients . TDP-43 patients . Also, Tpatients , 162. Mupatients .(2) FUS. Mutations in the gene coding FUS are found in 5% of familial ALS patients and in rare sporadic cases. Like TDP-43, mutations on FUS gene facilitate delocalization and abnormal aggregation of FUS to cytoplasm and affect the alternative splicing of its target genes [et genes , 165. Alet genes .(3) HuD. HuD  is expressed in the brain and has been implicated in various aspects of RNA metabolism [APP mRNA, β-site APP-cleaving enzyme 1 (BACE1) mRNA, and BACE1 antisense (BACE1-AS) lncRNAs, thereby facilitating the accumulation of the toxic APP cleavage product Aβ [tabolism . HuD funtabolism . HuD hasoduct Aβ .(4) FMRP. Fragile X mental retardation protein (FMRP) is a gene product encoded by fragile X mental retardation 1 (FMR1) and plays essential roles in normal cognitive development and female reproductive function [FXR1 gene lead to fragile X syndrome (FXS), autism, AD, and PD by dysregulating translation of its target genes [APP mRNA translation by recruiting APP mRNA into P-bodies [function . Mutatioet genes . FMRP inP-bodies . FMRP haP-bodies .(5) hnRNPs. hnRNP A1 has essential roles in the regulation of pre-mRNA processing, transport, and translation of mRNAs [RAGE and APP mRNAs [of mRNAs . Loss ofof mRNAs . In addiPP mRNAs , 175.hnRNP A2/B1 affects alternative splicing of ALS-associated D-amino acid oxidase, and ALS mutant (hnRNP A2B1 D290V) dysregulates cellular stress responses , 176. hnAPP mRNA or enhance its translation by competing with FMRP, therefore positively regulates APP expression [hnRNP C has been known to stabilize pression . Borrecapression .Metabolic disease is associated with the risk of developing T2DM, obesity, cardiovascular disease (CVD), and coronary heart disease (CHD) , 179. In(1) HuD. HuD is also found in the islets of the pancreas and mediates RNA quality control of pancreatic β cells [insulin2 mRNA and repressing its translation [ATG5 and INSIG1, respectively [β cells [ β cells . In the  β cells . HuD regnslation . In addiectively , 183. Mo[β cells .(2) CUGBP1. CUGBP1 has been reported to regulate insulin resistance and alternative splicing of the insulin [ insulin . In addi insulin . CUGBP1  insulin , 187.(3) RBFOX2. RBFOX2  is a RNA-binding protein and a homolog of C. elegans Fox-1 and regulates alternative splicing by directly binding to the consensus (U)GCAUG motif in the target pre-mRNAs [re-mRNAs . A recenre-mRNAs . DN-RBFO(4) IGF2BP2/IMP2. Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2/IMP2) belongs to IGF2 mRNA-binding protein (IMP) family and is known to regulate IGF2 translation by interacting with the 5′-UTR of IGF2 mRNA [GF2 mRNA . Genome-GF2 mRNA , 192. DaGF2 mRNA .Representative RBPs involved in DNA damage and oxidative stress, cellular senescence, and age-related diseases are shown in Increasing evidence indicates that ncRNAs and RBPs are essential regulators of various cellular processes, and dysregulation of these RNA regulators is implicated in the pathogenesis of several diseases including neurodegenerative diseases, metabolic diseases, and cancer. In this review, we tried to discuss the regulatory lncRNAs and RBPs that are involved in stress response, cellular senescence, and the pathogenesis of age-related diseases including neurodegenerative diseases, metabolic diseases, immune response, and muscle dysfunction Tables  and 2. WThe results of studies undertaken to uncover the roles of lncRNAs and RBPs during stress response, cellular senescence, and the pathogenesis of age-related diseases are prompting several questions for immediate consideration. For example, what are the molecular targets of lncRNAs and RBPs? What signaling pathways control the expression and function of lncRNAs and RBPs during the stress response or in the pathogenesis of age-related diseases? How do they contribute to the stress response and cellular senescence? Do lncRNAs and RBPs interplay in order to fine tune RNA metabolism? How are RNA regulators including lncRNAs, miRNAs, and RBPs differentially expressed in age-related diseases? As we begin to consider these questions, the importance of functional networking between RBPs and ncRNAs is coming to the forefront –199.A deeper and more comprehensive knowledge of the fine mechanisms involving lncRNAs and RBPs in the regulation of RNA metabolism is warranted because regulatory lncRNAs and RBPs are promising novel targets for intervention in physiopathologies with underlying deficiencies in stress response, cellular senescence, and the aging process.


Set1 and Jhd2 regulate the methylation state of histone H3 lysine-4 (H3K4me) through their opposing methyltransferase and demethylase activities in the budding yeast Saccharomyces cerevisiae. H3K4me associates with actively transcribed genes and, like both SET1 and JHD2 themselves, is known to regulate gene expression diversely. It remains unclear, however, if Set1 and Jhd2 act solely through H3K4me. Relevantly, Set1 methylates lysine residues in the kinetochore protein Dam1 while genetic studies of the S. pombe SET1 ortholog suggest the existence of non-H3K4 Set1 targets relevant to gene regulation. We interrogated genetic interactions of JHD2 and SET1 with essential genes involved in varied aspects of the transcription cycle. Our findings implicate JHD2 in genetic inhibition of the histone chaperone complexes Spt16-Pob3 (FACT) and Spt6-Spn1. This targeted screen also revealed that JHD2 inhibits the Nrd1-Nab3-Sen1 (NNS) transcription termination complex. We find that while Jhd2’s impact on these transcription regulatory complexes likely acts via H3K4me, Set1 governs the roles of FACT and NNS through opposing H3K4-dependent and -independent functions. We also identify diametrically opposing consequences for mutation of H3K4 to alanine or arginine, illuminating that caution must be taken in interpreting histone mutation studies. Unlike FACT and NNS, detailed genetic studies suggest an H3K4me-centric mode of Spt6-Spn1 regulation by JHD2 and SET1. Chromatin immunoprecipitation and transcript quantification experiments show that Jhd2 opposes the positioning of a Spt6-deposited nucleosome near the transcription start site of SER3, a Spt6-Spn1 regulated gene, leading to hyper-induction of SER3. In addition to confirming and extending an emerging role for Jhd2 in the control of nucleosome occupancy near transcription start sites, our findings suggest some of the chromatin regulatory functions of Set1 are independent of H3K4 methylation. Saccharomyces cerevisiae, H3K4me levels are controlled by the opposing functions of two highly conserved enzymes belonging to the Trithorax/MLL and JARID1 families: the Set1 methyltransferase and the Jhd2 demethylase  have surprisingly limited phenotypic impact in otherwise wild-type (WT) mitotic cells grown in the presence of glucose, confounding the study of this important chromatin regulatory protein using yeast (Methylation of histone H3 lysine-4 (H3K4me) represents one of the most comprehensively studied chromatin modifications. Like most histone lysine methylations, H3K4 exhibits mono, di, and tri-methylated states  that have distinctive regulatory outputs reflecting the recruitment of effector proteins  and overlapping with transcription termination sites (TTSs). Associated with these H3K4me defects, JHD2 also represses the accumulation of noncoding intergenic transcripts genome-wide  and causes defects in mRNA 3′ untranslated region length for some genes , and NNS (Nab3-Nrd1-Sen1)  and grown overnight at room temperature (23°). Each strain was diluted to an OD600 = 0.4, serially diluted five times and spotted on synthetic complete media (YNB media (Multicell Wisent) containing 5 g/L of ammonium sulfate and either 2% glucose or 2% galactose.Standard yeast genetic methods were used for construction of all strains. Yeast strains and plasmids used in this study are listed in 600 between 1-2 were lysed by vortexing with acid washed glass beads in SUMEB buffer as described previously  and then immediately quenched with glycine at a final concentration of 125 mM. Cells were pelleted and washed 2 times with ice cold PBS. Cell pellets were snap frozen in 2 mL screwcap tubes using liquid nitrogen and stored at -80°. Frozen pellets were resuspended in lysis buffer  without thawing and topped off with glass beads. Cells were lysed through multiple rounds of beadbeating and then sonicated to shear the chromatin to fragment sizes of approximately 200 to 500 bp. Cross-linked chromatin fragments were immunoprecipitated with antibody overnight. Protein A-Sepharose beads were then added to the samples, and samples were incubated for 90 min. The immunoprecipitated complexes were washed with lysis buffer, lysis buffer containing 500 mM NaCl, wash buffer , and TE . Next, the immunoprecipitated chromatin was eluted from beads with elution buffer  and then eluted with TE/0.67% SDS . The two elutions were combined. Whole cell extract samples were topped off with TE/1% SDS . Formaldehyde cross-linking was reversed by incubating the eluates at 65° overnight. DNA from the eluates was treated with RNAse A and purified with a QIAquick PCR purification kit (Qiagen). Immunoprecipitated fractions (IP) and whole-cell extracts (Input) containing DNA were analyzed by real-time PCR as described previously . Each strain was diluted to an ODThe authors state that all data necessary for confirming the conclusions presented in the article are represented fully within the article. Strains are available upon request.jhd2∆. Because Jhd2 reverses a histone modification associated with active transcription, we evaluated temperature sensitive (TS) alleles of essential genes related to transcriptional control as interacting candidates of jhd2∆. We used strains sourced from a library created and curated previously . Deletion of JHD2 also modestly suppressed sen1-1 . As shown, incubation at high temperature was sufficient to eliminate growth of nab3-42, confirming that jhd2∆ did not provide bypass suppression and that Jhd2 therefore acted to inhibit nab3-42 function at semi-permissive temperatures , only temperature sensitive alleles of genes encoding Spt6-Spn1 and FACT subunits exhibited genetic interactions with jhd2∆ . Furthermore, and consistent with the NAB3 results, sen1-1 was also suppressed by both jhd2∆ and set1∆, again with the triple mutants resembling sen1-1 set1∆ . While none of our experiments overlap with those of Terzi et al., their findings would not have predicted ours nor vice versa. In particular, our finding that set1∆ suppressed the nab3-11 growth defect is somewhat discordant with Terzi et al.’s finding of an aggravating consequence of set1∆ on nab3-11 for transcriptional termination. Among the many possible explanations for this discrepancy, a simple view is that the essential functions of NAB3 may not be directly reflected by termination defects described by Terzi et al.As 42 set1∆ . Terzi epob3-7 and spt16-ts as exhibiting alleviating genetic interactions with jhd2∆ . The differences in SET1 genetic interaction phenotypes observed for SPT16 and POB3 mutants suggest complexities in nucleosome interactions, or that their encoded proteins may exhibit separable functions apart from the FACT complex.Our targeted screen also identified the FACT subunit encoding alleles th jhd2∆  and 1C.  synergy . In contfunction . We confset1∆ and jhd2∆ can rescue certain temperature sensitive growth defects, we employed strains that express H3K4 amino acid substitution mutants from a chromosomal locus in place of wild type H3 . Unexpectedly, we found that H3K4A and H3K4R modified pob3-7 and nab3-11 mutants in opposing fashion. H3K4A suppressed pob3-7 and nab3-11 temperature sensitivity . Suppression by H3K4A and set1∆ did not display any synergy. H3K4R caused the opposite effect of H3K4A, exacerbating the temperature sensitive growth defect of nab3-11 and pob3-7 . We note that this latter result is consistent with previous findings suggesting a positive impact for H3K4me3 on NNS function  encodes a conserved yet poorly characterized protein required for growth on glucose at low temperatures and is involved in secretory protein maturation and amino acid metabolism. . The csf1∆ enhancement by JHD2GAL- was abrogated by mutation of histidine-427 to alanine (H427A), which renders Jhd2 catalytically inactive .To shed light on the confounding differences in H3K4A and H3K4R phenotypes, we performed additional studies exploiting an annotated synthetic lethal interaction between csf1∆, set1∆, and H3K4A or H3K4R mutants and interrogated them using tetrad dissections. In addition to confirming csf1∆ set1∆ synthetic lethality, we found that H3K4R exhibited synthetic lethality with csf1∆ . Thus, the suppression of NNS and FACT subunits by set1∆ in H3K4R mutants suggests that some activity of Set1 acted in opposition to NNS and FACT independent of H3K4me . Based on these results as well as previous findings from Terzi et al., we propose counterbalancing regulation of NNS and FACT by Set1 and Jhd2, with H3K4me promoting their functions and an unknown inhibitory impact conferred by Set1 in a H3K4 independent manner . As enzymatically dead alleles of SET1 lead to a depletion of Set1 protein levels, this was to be expected . Similar results were obtained with nab3-11 (data not shown). These results, while complex, suggest that the H3K4-independent inhibition of pob3-7 by Set1 may involve both enzymatic and non-enzymatic roles of this protein . Importantly, this suppression was lost in the H427A and PHD domain mutants . Our findings support the conclusion that suppression of Spt6-Spn1 alleles by jhd2∆ was due to the loss of H3K4 demethylation.To validate that Spt6-Spn1, we genetically interrogated COMPASS complex subunits known to specifically perturb H3K4me3 or H3K4me3 and -me2. The effects of bre2∆ and spp1∆ on H3K4me have been extensively described. Deletion of BRE2 results in a complete loss of H3K4me3 and a significant decrease of H3K4me2 but no change in H3K4me1, while spp1∆ results in a substantial decrease of H3K4me3 with no detectable consequence on bulk H3K4me2 or -me1 . Collectively, our data support the conclusion that JHD2 and SET1 opposingly governed the function of Spt6-Spn1 through H3K4me3 .Reflecting its role in the recycling of nucleosomes in the wake of elongating PolII, e of 23° . At the haperone . This reJhd2 and Spt6-Spn1, we measured chromatin-associated histone H3 in WT, jhd2∆, spt6-14, and spt6-14 jhd2∆ cells at the SRG1-SER3 locus using chromatin immunoprecipitation with an anti-H3 antibody followed by real time PCR quantification of the immunoprecipitated DNA (ChIP-qPCR). In order to evaluate nucleosome positions at high resolution across SRG1-SER3, we used 33 closely spaced PCR amplicons to measure immunoprecipitated DNA , confirming that their deposition was Spt6 dependent . This JHD2-repressed nucleosome was positioned ∼75 base pairs upstream of the SER3 TSS, and positioned ∼75 base pairs downstream of this TSS we observed an additional H3 ChIP peak in jhd2∆, though this second peak was not statistically significant . To determine if it instead impacted SER3 induction, we subjected WT and jhd2∆ cells to serine starvation and compared their relative SER3 mRNA abundance using quantitative PCR amplification of reverse transcribed RNA (RT-qPCR). We found that SER3 was maximally expressed 1 hr after serine withdrawal, and that SER3 mRNA accumulated to ∼1.7 increased levels in jhd2∆ at this timepoint  controls transcription at silent mating loci, telomeric regions, and retrotransposon (Tf) elements, as well as the clustering of diverse genomic regions encoding Tf elements. Interestingly, spSet1 accomplishes these sundry functions in a manner dependent on differing domains of Set1 that do, or do not, have consequences for H3K4me. The two key regulatory roles of spSet1 appear dependent on spSet1’s enzymatic activity and on its RNA binding RRM2 domain, a domain found in both fission and budding yeast Set1 (Set1 RRM2 domain (Set1 we identify here. Our genetic results, however, also suggest that enzymatic activity of Set1 directed toward a non-H3K4 substrate has regulatory significance, as may be the case for S. pombe Set1 (Set1 and other histone methyltransferases.An important feature of our findings relates to functions of www.g3journal.org/lookup/suppl/doi:10.1534/g3.118.200151/-/DC1.Supplemental material is available online at Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.


DSCAM-AS1 lncRNA gene. This represents a paradigm of apoERα activity since its expression is largely unaffected by estrogenic treatment, despite the fact that E2 increases ERα binding on DSCAM-AS1 promoter. We validated the enrichment of apoERα, p300, GATA3, FoxM1 and CTCF at both DSCAM-AS1 TSS and at its associated SE by ChIP-qPCR. Furthermore, by analyzing MCF-7 ChIA-PET data and by 3C assays, we confirmed long range chromatin interaction between the SE and the DSCAM-AS1 TSS. Interestingly, CTCF and p300 binding showed an enrichment in hormone-depleted medium and in the presence of ERα, elucidating the dynamics of the estrogen-independent regulation of DSCAM-AS1 expression. The analysis of this lncRNA provides a paradigm of transcriptional regulation of a luminal specific apoERα regulated lncRNA.Estrogen receptor-α (ERα) is a ligand-inducible protein which mediates estrogenic hormones signaling and defines the luminal BC phenotype. Recently, we demonstrated that even in absence of ligands ERα (apoERα) binds chromatin sites where it regulates transcription of several protein-coding and lncRNA genes. Noteworthy, apoERα-regulated lncRNAs marginally overlap estrogen-induced transcripts, thus representing a new signature of luminal BC genes. By the analysis of H3K27ac enrichment in hormone-deprived MCF-7 cells, we defined a set of Super Enhancers (SEs) occupied by apoERα, including one mapped in proximity of the  Estrogen Receptor α (ERα) is the key transcriptional factor of the luminal breast cancer subtype, which comprises three quarter of breast cancer cases , by mediERα regulates biological processes in breast cancer cells by ligand-dependent (estrogen-ERα complex) or ligand-independent action (apoERα) . Whereascis regulatory elements, defined as large clusters of putative enhancers in close genomic proximity which drive gene expression to define cell lineage identity [cis-regulation of coding and non-coding genes expression [Recently, Super-Enhancers (SEs) emerged as a novel class of distal identity . SEs tenidentity . SEs repidentity ,12. Decipression . In MCF-pression . This stDSCAM-AS1 is the most abundantly expressed lncRNA in BC cell lines and tissue samples. Expression of DSCAM-AS1 has a very peculiar dependence over ERα: the increased ERα binding at DSCAM-AS1 promoter upon estradiol stimulus does not lead to a concordant up-regulation of gene transcription over at least 24 h indicating a necessary and sufficient apoERα action for the regulation of DSCAM-AS1 transcription [DSCAM-AS1 promoter is high in tumor tissues from patients unresponsive to Tamoxifen treatment and that DSCAM-AS1 expression is upregulated in Tamoxifen-resistant cellular models [A full collection of lncRNAs has emerged during the last decade, stressing on their importance as regulators of cellular development and differentiation. They are expressed at lower levels than protein-coding transcripts, but compared to the latter, their pattern of expression is remarkably cell type-specific ,16. Indecription . It has r models , contrarDSCAM-AS1 associated SE which is stabilized in a hormone-independent manner and whose regulation depends on the presence of apoERα. It represents a paradigmatic example of how luminal lncRNAs are regulated by specific SEs activated through unliganded ERα, which is extremely important to understand luminal breast tumor growth and progression, in view of the fact that common endocrine treatments today (AIs) deplete the organism of estrogenic hormones.In the present study, we extended the analysis of apoERα-regulated transcriptome in MCF-7, characterized apoERα binding at SEs (SE-aERBSs) and defined apoERα-regulated lncRNAs that are associated and regulated by SE activation. In particular, we focused our attention on ESR1, the most significant down-regulated protein coding genes were CALCR, ARL1, and TFF1 while KLF6, ZNF469, and TP53INP1 were the most significant up-regulated protein coding genes  for 96 h and transfected with control siRNA (siCTR) or against ERα (siERα). Differential expression analysis revealed a set of 2487 differentially expressed (DE) coding and non-coding genes of which 1376 were down-regulated and 1110 up-regulated upon ERα silencing . Excluding genes a. We ide lncRNAs b.mitotic nuclear division (GO:0007067), and cell cycle phase transition (GO:0044770) as the most enriched processes (neuron projection guidance (GO:0097485), axon guidance (GO:0007411), and epithelium development (GO:0060429) while down-regulated genes were enriched for cell cycle- and DNA replication-related processes , rocesses . Interesrocesses c.To correlate apoERα genomic Binding Sites (aERBS) with gene regulation, we took advantage from our ERα ChIP-seq experiment performed in hormone-deprived MCF-7 cells . As prevESR1, GATA3, and FOXA1. Furthermore, lncRNA genes were also associated with this SEs set, including MALAT1 and DSCAM-AS1.To verify whether the apoERα binding involved in DE gene regulation occurs within SEs in MCF-7 cells, we have applied a bioinformatics strategy that is summarized in ESR1, SPDEF, ZNF217, and RARA  . To identify which of these regions is involved in DSCAM-AS1 regulation, we used public ERα Chromatin Interaction Analysis with Paired-End-Tag sequencing (ChIA-PET) data of MCF-7 grown in full medium condition (GSE39495) [DSCAM-AS1 promoter interacts directly with two SE-aERBSs, E6 and E8, in particular E6 was characterized by the higher number of interactions (EnhT). Known long-range interactions were also confirmed using the TFF1 gene as positive control for the analysis.In the SE mapped upstream of the SE39495) . We obseractions . OverlapDSCAM-AS1 locus, we took advantage of public MCF-7 ChIP-Seq data obtained in HD condition. Using the WashU genome browser, we integrated these data with the RNA-seq and ChIP-seq experiments of HD MCF-7 transfected with siERα or control siRNA  occupancy. In addition, the SE-aERBSs (particularly E5 and E6) were characterized by the enrichment of Forkhead Box A1 (FoxA1), Forkhead box protein M1 (FoxM1), histone acetyltransferase p300 and GATA Binding Protein 3 (GATA3), confirming the active status of these regulatory regions. Interestingly, the RNAPII ChIP-Seq signal at the DSCAM-AS1 locus decreases rapidly in the region downstream DSCAM-AS1, where a CTCF binding sites is characterized by a ChIP-Seq enrichment of components of the cohesin complex RAD21 and STAG1 (GSE25021) [DSCAM-AS1 locus and the SE-aERBSs, ERα and CTCF ChIA-PET data were visualized. This data confirms a consistent interaction between the DSCAM-AS1 promoter and E5, E6, and E8 SE-aERBSs.Then, to explore the genomic and epigenomic context of the nine SE-aERBSs of the ol siRNA a. As expSE25021) . To furtDSCAM-AS1 expression, we focused our analysis on E5 and E6 SE-aERBSs characterized by the highest apoERα signal and co-factor co-occupancy. By ChIP-qPCR we first evaluated ERα enrichment in MCF-7 cells maintained in HD medium and transfected with siERα or control siRNA. Knockdown of ERα was monitored by western blot, observing a reduced level of the protein  or vehicle for 45 min, by ChIP-qPCR. We observed a significant enrichment of ERα binding upon E2 treatment at the promoter   analysis. Interestingly, we detected E6-promoter interaction in vehicle-treated cells, whereas no interactions between the E5 site and DSCAM-AS1 promoter were observed  is enriched at ata from  in whichll lines .DSCAM-AS1 expression and the activity of the associated SE, we analyzed public ERα and H3K27ac ChIP-Seq data obtained in Long-Term Estrogen Deprived (LTED) models and Tamoxifen resistant model from [DSCAM-AS1 SE occurs as well in wild-type as in Tamoxifen resistant and in LTED models. Only in a LTED model, which is resistant also to Tamoxifen, is the binding absent, despite the high H3K27ac ChIP-Seq signal still measured in this region. Noteworthy, the highest ERα ChIP-Seq signal at E6 SE-aERBS was observed in the LTED model.To further explore the relationship between del from . As repoDSCAM-AS1 promoter and gene locus combines with a low but evident ERα ChIP-Seq signal at both E6 and DSCAM-AS1 promoter , which is a critical issue for Breast Cancer (BC) patients given adjuvant treatment with Aromatase Inhibitors (AIs). We performed a deep triplicate RNA-Seq in hormone-depleted MCF-7 cells upon ERα silencing and, specifically, we were interested to understand whether Super Enhancers (SEs) were involved in sustaining the basal expression of a group of luminal-specific lncRNAs. Our study indicates that a set of apoERα dependent lncRNAs lie close to SEs, suggesting a strict dependence on these regions. Focusing on a representative lncRNA gene, The identification of SEs helped us to understand the peculiar transcriptional program regulating cell-type specific gene expression. Recently, several evidences demonstrated that cell-specific enhancers play an important role in tumorigenesis . Indeed,DSCAM-AS1, the prediction appeared correct and could be confirmed by our ChIA-PET network model.One important limitation of our results is represented by the fact that the possible targets of SE were identified only based on the distance, i.e., we attributed to each SE the closest apoERα-regulated lncRNA gene. While on the basis of the reported frequency of enhancer-promoter interaction this is justified, the ENCODE project and other studies have reported that in a number of cases enhancers interact with promoters jumping over several genes . ClearlyDSCAM-AS1 is particularly interesting due to its abundance and close association with unliganded ERα activity. DSCAM-AS1 loss impairs indeed BC cells proliferation and its expression is somehow implicated in Tamoxifen-resistance [DSCAM-AS1 expression in BC cells.The expression of the carcinoma-specific sistance ,22. Heresistance  by validsistance ,34, suggRARA) [RARA gene has a very important developmental function in epithelial mammary cells. One important novel finding in our data is represented by the state of activation of the aERBS within the major SE upstream the DSCAM-AS1 gene. We evaluated p300 enrichment confirming the active status of these regulatory regions. In particular, the transcriptional coactivator p300 possesses intrinsic acetyltransferase activity and it was shown to play a critical role in transcriptional regulation [DSCAM-AS1 enhancer is conserved rather in hormone-deprived medium than upon estrogen treatment, indicating that this SE is not an early estrogen target site. However, the dynamic distribution of p300 in BC cells in the presence or absence of estrogenic hormones and its effect on the prognosis of BC are poorly understood [The presence of apoERα bound to an intronic element, which can increase quantitatively in response to estrogen but not transcriptionally, is not novel: a similar situation was reported for an intronic site in the retinoic acid receptor alpha 1 gene (RARA)  suggestigulation . Consistgulation ,38. InteDSCAM-AS1 gene expression seems to be based on long range interaction between apoERα-bound enhancer and the gene promoter as previously described for some estrogen-dependent ERα target genes such as TFF1 and GREB1 [DSCAM-AS1 regulation. In fact, both ERα binding and 3C analysis show very low activity of E5, also reflecting public ChIA-PET data of MCF-7 cells grown in full medium, i.e., under low hormone concentrations [Our analysis confirms the model where apoERα collaborates with other TFs to maintain the luminal epithelial enhancer landscape . These cnd GREB1 . Notewortrations .DSCAM-AS1 promoter-enhancer interaction before and after estrogen stimulation and we could detect at least the E6-promoter interaction, further demonstrating that this enhancer is active even in the absence of estrogen. However, it should be emphasized that this is a preliminary and qualitative result, which should be confirmed by further experiments to quantify the frequency of DSCAM-AS1 promoter-enhancer interaction in different experimental conditions. This data will allow understanding if this interaction is more favored or stabilized in absence of hormones, as we hypothesize.Therefore, we attempted DSCAM-AS1 and a long-range interaction was observed between a site close to DSCAM-AS1 3’ end and a site farther upstream the SE, we asked whether CTCF binding could justify the peculiar mode of action of the DSCAM-AS1 enhancer/promoter regulatory system. Noteworthy, we found that CTCF occupancy upstream DSCAM-AS1 decreases after ERα silencing in HD medium, whereas it decreases significantly downstream the DSCAM-AS1 gene region after estradiol stimulation, suggesting that estrogen, or its absence, may induce a redistribution of long-range interactions. Taken together, these results suggest a non-trivial regulatory model at this site, which may involve channeling SE action on the DSCAM-AS1 promoter in cancer cells overexpressing ERα due to strict domain definition by CTCF. We also supposed that the reduction of CTCF signal upon estradiol stimulation indicate that these enhancers might regulate distal genes surmounting the protein insulator. Interestingly, the analysis of public RNA-Seq data MCF-7 transfected with siCTCF and treated with E2 for 3 h show a reduction of DSCAM-AS1 level in siCTCF-treated cells , the DSCAM-AS1 SE is completely active, while only the DSCAM-AS1 promoter region is active in ZR-75.1 (characterized by a lower ERα expression). Furthermore, the LTED cell model system, which is characterized by an elevated ERα expression [Finally, analysis of the pression , were alpression , limitedDSCAM-AS1 SE, but we clearly observed that DSCAM-AS1 locus is active in four out six patients non-responsive to AI treatments. Since patients with a good prognosis present no H3K4me3 signal at DSCAM-AS-SE, its activated-status is correlated with a negative prognosis, suggesting it as epigenetic-predictor of AI-treatment response. As far as the other common endocrine treatment is concerned, i.e., Tamoxifen, Niknafs and coworkers reported that Tamoxifen-resistant patients display ERα binding to DSCAM-AS1 promoter, as opposed to Tamoxifen responders [DSCAM-AS1 SE, confirming that ERα binding is limited to non-responder patients  and in the presence of antagonistic ERα ligands (Tamoxifen).Analysis of ERα and H3K4me3 ChIP-Seq data clearly show extensive heterogeneity among patients. Using these data, we were not able to confirm a strong ERα binding at sponders . Using tsponders , we addipatients . AltogetDSCAM-AS1 genes was included in the gene classifier of drug responsiveness proposed by Jansen and colleagues [Moreover, lleagues .DSCAM-AS1 expression in tissues of BC patients to demonstrate it possible role as hormone-responsiveness marker. At the same time, several other lncRNA loci are being studied to understand unliganded ERα action in luminal breast cancer.In our lab, we are actively exploring l-glutamine, 50 U/mL penicillin and 50 μg/mL streptomycin. T-47D cells were routinely grown in RPMI medium supplemented with 10% heat-inactivated FBS, 2 mM l-glutamine, 50 U/mL penicillin and 50 μg/mL streptomycin. Hormone-deprived medium (HD) was obtained from phenol red-free DMEM supplemented with 5% charcoal-dextran-treated serum. MCF-7 cells were grown at least 4 days in HD medium before 17β-estradiol treatment (E2). 17β-estradiol  was added at a final concentration of 10–8 M.MCF-7 and ZR-75.1 cells were routinely grown in DMEM medium supplemented with 10% heat-inactivated FBS, 2 mM MCF-7 cells were grown in HD medium (for two days in this conditioned medium) before being transfected with siRNAs using Lipofectamine2000 , according to the reverse transfection protocol. Stealth RNAi™ siRNAs from Invitrogen were used to target ERα mRNA ; stealth RNAi™ siRNA Negative Control medium GC was used as a control (siCTR). Any experiment was carried out 48 h after siRNA transfection in order to achieve the maximum efficiency of RNA interference.MCF-7 cells were grown for 2 days in HD medium before siRNAs transfection (siCTR or siERa). RNA was harvested after 48 h from transfection by RNA isolation kit . RNA quality check (RNA integrity number (RIN) > 8) was achieved with Fragment Analyzer  and quantified with Qubit . Two μg of RNA were polyA+ selected and RNA-Seq libraries were constructed using Illumina TruSeq RNA sample prep kit . Paired-end (PE) cluster generation was performed using cBot on Flow Cell v3 . Sequencing of libraries was performed on the HiSeq sequencing system .Raw and processed RNA-Seq data were deposited at GSE108693.TopHat v2 [featureCounts algorithm [DESeq2 package [Raw RNA-Seq reads were aligned using opHat v2 . Gencodelgorithm  and read package . NormaliDESeq2 in default settings. A gene was considered as differentially expressed if associated with an adjusted p-value < 0.001. To identify genes differentially expressed in the time-course E2-treatment experiment, maSigPro R package [p-value computed using the p.vector function of the package. A gene was considered as differentially expressed if associated with an adjusted p-value < 0.05. Gene to SEs association was performed by computing the distance between gene TSSs and each SE center. Only genes mapped within 100 kbp from a SE region were considered for the analysis.Differentially expression between siCTR and siERα-treated MCF-7 was performed using  package  was applGene set functional analysis was performed using Enrichr algorithm .local option.Analysis of GRO-Seq datasets from GSE45822 was performed using Bowtie v2.1.0  in defauhttps://genome.ucsc.edu/cgi-bin/hgLiftOver).A H3K27ac ChIP-Seq dataset performed on 48-h hormone-deprived MCF-7 cells treated with vehicle GSM986079) was used for the identification of SE regions. Raw ChIP-Seq reads were aligned using Bowtie 2.0 algorithm in default settings . Signifi079 was uThe network model of Gene-aERBSs chromatin interaction was generated using the ChIA-PET data from GSE39495. Each pair of interacting genomic regions was overlapped with the aERBSs and gene promoter coordinates. A promoter was defined as a region of −2 kbp and +500 bp around the gene TSS. Each aERBS was equally extended by 1 kbp in each size. The average number of interacting region between each aERBS-gene, gene-gene, or aERBS-aERBS pair was computed and represented as link width between the connected nodes. Gene were color-coded according to their expression log2FC in the siERα RNA-Seq experiment while aERBS nodes were colored according to their epigenetic classification defined in .DSCAM-AS1 locus and upstream enhancer regions was performed using the Washu Genome Browser [The analysis of  Browser . ChIP-Se Browser . Each ChData from ENCODE long-range chromatin interaction experiments were analyzed by considering the ERα and CTCF ChIA-PET experiments performed in complete-medium grown MCF-7. The processed data of these experiments were provided by the public track hub “long-range chromatin interaction experiments” of the Washu Genome Browser.Whole-cell lysate was harvested from organic-phenol phase after RNA purification in Purezol™ reagent . 70 μg of protein extracts were denatured by boiling in 6x Loading Dye, fractionated by SDS-PAGE electrophoresis in 6% polyacrylamide gel and finally transferred on PVDF membrane (Millipore). Antibodies used were designed against ERα , CTCF , GATA3 , p300 , FOXM1  and Hsp90  proteins. Proteins quantitation was performed with Volume Analysis Tool from Quantity One™ software, version 4.6.6 (Bio-Rad).RNA was isolated from MCF-7 cells using Purezol™ reagent (Bio-Rad). All total isolated RNAs were subjected to DNase treatment to remove contaminating genomic DNA . First strand cDNA synthesis from 250 ng of total RNA template was performed with SensiFAST cDNA Synthesis Kit , followed by SYBR-green qRT-PCR amplification . Real-time PCR primers for human 18S, ERα, GREB1 RNAs were purchased from Qiagen . Custom expression-primer pairs are reported in MCF-7 cells were grown for 2 days in HD medium before siRNAs transfection or 45 min of E2 treatment. ChIP experiments were then performed as follows: cells were cross-linked by addition of formaldehyde at 1% final concentration  and incubated 10 min at 37 °C. Cross-linking was stopped by addition of glycine solution to a final concentration of 0.125 M for 5 min on a shaker at room temperature. Cross-linked cells were then washed twice with ice-cold PBS supplemented with complete protease inhibitors cocktail and collect by scraping . Cell pellets were subjected to lysis on ice for 10 min with Lysis Buffer 1  supplemented with complete protease inhibitors cocktail. Subsequently, nuclei pellets, obtained by a 5 min spin cycle at 4 °C (4000 rpm) were exposed once again to 10 min lysis in Lysis Buffer 2  supplemented with complete protease inhibitors cocktail. Total extracted chromatin was sonicated to an average size of 250–500 bp by using an immersion sonicator device. The desired fragments size was checked on 1.2% agarose-gel and quantified by Nanodrop spectrophotometer at 280 nm, in order to use 50 μg of chromatin per IP. 10 μL (1%) of chromatin extracts was recovered as input normalization-control for each experimental condition. Chromatin extracts were diluted with IP-buffer  and incubated overnight with the specific antibody or IgG at 4° C on a rotating platform. Protein A and G sepharose-beads  were pre-coated with IP buffer supplemented with 5% BSA, in order to reduce nonspecific antibody binding. Upon 2 h of Protein A or G sepharose-beads incubation , samples were washed sequentially for 5 min, on a rotating platform with 1 mL of three different Washing Buffer  and twice with TE buffer . After complexes elution at RT with elution buffer , DNA fragments were de-crosslinked at 65 °C overnight with NaCl 5 M and by 1 h of proteinase K treatment. DNA purification was achieved with Phenol:Chloroform:IAA  according to the manufacturer’s instructions. Quantitative Real-time PCR was carried out on ChIP-enriched DNA using SYBR Green Master Mix. ChIP enrichment was normalized on input samples  and expressed as fold-enrichment of specific binding over the control nonspecific IgG binding. Antibodies against human ERα , p300 , CTCF , GATA3 , FOXM1  and normal rabbit IgG  were used in this assay. Custom ChIP primer pairs are reported in P2RY2 gene-enhancer interaction were used as a technique positive control [DSCAM-AS1 promoter-enhancer interaction. Primers are reported in MCF-7 cells were grown for 2 days in HD medium before 45 min of E2 treatment. 3C procedure was performed essentially as described by  with som control  and cust


Bacteria have evolved sophisticated mechanisms for regulation of metabolic pathways. Such regulatory circuits ensure that anabolic pathways remain repressed unless final products are in short supply and that catabolic enzymes are not produced in absence of their substrates. The precisely tuned gene activity underlying such circuits is in the purview of transcription factors that may bind pathway intermediates, which in turn modulate transcription factor function and therefore gene expression. This review focuses on the role of ligand-responsive MarR family transcription factors in controlling expression of genes encoding metabolic enzymes and the mechanisms by which such control is exerted. Prospects for exploiting these transcription factors for optimization of gene expression for metabolic engineering and for the development of biosensors are considered. In many cases, ligand- and DNA-binding functions reside in the same protein, either within a single domain as seen in members of the multiple antibiotic resistance regulator (MarR) protein family A change in the conformation or flexibility of the transcription factor is elicited upon ligand binding, and this results in altered interaction with cognate DNA and therefore a change in gene expression . Such alThe ability to sense and metabolize specific nutrients confers a fitness advantage, as it avoids costly synthesis of unnecessary enzymes and allows utilization of resources that competing species may not be able to catabolize. In addition, genes encoding enzymes that participate in a specific pathway are often encoded together in an operon, a genome organization that allows concerted regulation of all genes. While metabolic pathways are often conserved, regulatory networks vary between species. This review highlights examples of ligand-responsive MarR family transcription factors that respond to specific pathway intermediates to effect differential gene expression; a particular focus is on MarRs that respond to lignin-derived aromatics, as high resolution structural analyses have been reported that illustrate distinct mechanisms by which binding of structurally related ligands may alter association with cognate DNA. Advances towards optimizing or (re)designing MarR protein function for metabolic engineering or generation of biosensors are discussed.2Escherichia coli Multiple Antibiotic Resistance Regulator, MarR family transcription factors are ubiquitous in the bacterial kingdom Helicobacter pylori does not uncover any MarR homologs whereas the human pathogen Staphylococcus aureus, which can infect multiple ecological niches within the host environment, encodes 18 Named for the E. coli MarR MarR proteins exist as obligate homodimers in which each monomer contributes a winged helix-turn-helix (wHTH) DNA-binding motif. Recognition helices from each DNA-binding motif bind palindromic DNA sequences in consecutive major grooves, with residues in the wing contacting the adjacent minor grooves A [12]. D3Bacteria continuously monitor their environment and cellular metabolic state and modify their gene expression patterns in response to perceived cues. The ensuing changes in gene activity are the result of complex transcriptional networks that may include global regulators that control a large number of transcription units  as well as more specific transcription factors that are dedicated to particular genes 3.1Mineralization of lignin-derived aromatic compounds by soil bacteria is a key step in the terrestrial carbon cycle, and several bacterial species have been characterized that can utilize such compounds as carbon and energy sources Acinetobacter baylyi ADP1, which controls expression of the hca operon that encodes proteins involved in catabolism of the p-hydroxycinnamic acid derivatives caffeate, p-coumarate, and ferulate. Hydroxycinnamates are constituents of lignin and other plant components, and they are precursors in the synthesis of flavonoids. For A. baylyi ADP1 HcaR, the inducing metabolites were reported to be the hydroxycinnamate-CoA thioesters Neisseria NadR, which controls expression of an adhesin that mediates binding to human cells Pseudomonas aeruginosa MexR, which regulates expression of genes encoding the MexAB-OprM efflux pump, suggested that a flexible DNA-binding state is reached only transiently and that ligand-binding shifts the conformational ensemble towards a less flexible conformation that cannot bind DNA One example is the MarR protein HcaR from Rhodococcus jostii RHA1 can also grow on p-hydroxycinnamate derivatives such as p-coumarate. Catabolism requires expression of the cou genes, which are under control of CouR. CouR acts as a repressor, and gene expression is induced on binding of the inducer p-coumaroyl-CoA, but not p-coumarate. Structural analysis revealed binding of two ligand molecules per protein dimer, with no significant structural differences on ligand binding compared to apo-CouR. However, while the phenolic ligand moieties occupy equivalent hydrophobic pockets between DNA-binding and dimerization regions of the protein, the CoA parts extend down and are predicted to interfere sterically with DNA binding as well as impose a charge repulsion  represses multiple genes linked to the citric acid cycle, including the gene encoding aconitase cis-aconitate. Citrate and trans-aconitate levels may increase when the iron-sulfur cluster required for enzymatic activity is damaged by reactive oxygen species, causing accumulation of the substrate citrate and release of the intermediate cis-aconitate, which is then converted to the more stable trans isomer Metabolic pathways are frequently regulated in response to stress, as exemplified by the wide-spread repression of gene activity during the bacterial stringent response, when genes associated with growth are repressed in favor of genes linked to survival 4A thorough understanding of transcriptional regulatory networks responsible for optimizing cellular metabolism is essential, not only from the perspective of bacterial physiology but for optimization of such networks for industrial applications or for generation of biosensors. The interest in metabolic engineering in which existing metabolic fluxes are redirected to target pathways or heterologous pathways are expressed is fueled by prospects for commercial generation of value-added compounds such as biofuels and polymer precursors and by switching to sustainable “green” production. Metabolic engineering of bacteria to produce desirable products is challenging, however, as linked reactions may be adversely affected by channeling intermediates towards a specific pathway, resulting in a failure to maintain metabolic balance and leading to a reduced yield of the desired compound 4.1Hydrolysis of plant biomass is actively considered for generation of renewable energy such as biofuels and for production of aromatic precursors in plastics manufacturing 4.2Synthetic biology, the (re)design and implementation of novel biological devices or circuits, aims to create systems with predictable function. Transcription factors play a major role in biosensor design, since they can be implemented in synthetic circuits controlling gene expression in response to specific ligands. Such synthetic circuits may be designed to control downstream metabolic functions in response to changes in the environment or an imbalance of cellular metabolites. Such circuits have been successfully designed based on bacterial transcription factors with specificity for cellular metabolites. However, redesign of transcription factors to respond to new effector molecules remains a challenge, as changes in the ligand-binding residues required to accommodate novel scaffolds may disrupt allosteric communication with the DNA-binding domain Deinococcus radiodurans HucR. In D. radiodurans, the gene encoding uricase is divergently oriented from hucR; HucR binds urate with low micromolar affinity, a binding event that leads to loss of DNA binding and upregulation of the divergently oriented genes. HucR binds with high affinity and specificity to a single DNA site in the intergenic region spanning genes encoding uricase and HucR Synthetic sensor–effector gene networks may be designed to correct metabolic defects and restore metabolic homeostasis. One successful application of a ligand-responsive transcription factor for this purpose was aimed at treating tumor lysis syndrome or gouty arthritis by sensing and reducing levels of urate in the bloodstream Apergillus flavus uricase, and microencapsulated cells engineered to express this prosthetic gene network successfully reduced serum levels of urate in mice A synthetic mammalian circuit designed to maintain urate homeostasis in the bloodstream was created in which detection of urate by HucR results in derepression of a gene encoding Agrobacterium fabrum encodes a homolog of HucR, named PecS, which binds urate and xanthine with equivalent low-micromolar affinity, and both ligands effectively attenuate the specific binding of PecS to its cognate DNA site HucR binds urate in preference to xanthine, a preference ascribed to conformational changes induced only on binding of a negatively charged ligand 5As exemplified above, MarR family transcription factors frequently bind phenolic compounds. This property makes them ideally suited for regulation of genes encoding enzymes involved, for example, in degradation of lignin-derived aromatics as well as man-made environmental pollutants marR gene promoters, identification of specific ligands may be a challenge. Expanding the inventory of MarR proteins with known DNA- and ligand-specificities promises not only to further understanding of bacterial metabolism, but also to develop a larger repertoire of biosensors with clinical potential.The ability to sense metabolites as a readout of cellular metabolic state makes MarR proteins well suited for the development of biosensors. So far, such detection has relied on the native ligand-specificity, as illustrated by the successful implementation of HucR in several urate-sensing devices. Because these transcription factors are very sensitive to changes that alter the dynamics of the dimer interface, redesign of ligand-specificity may be confounded by resulting changes in communication between ligand-binding pockets and the DNA-binding wHTH motif. While identification of cognate DNA sites for MarR proteins is frequently facile due to the presence of palindromic sequences in


SAGA and ATAC are two distinct chromatin modifying co‐activator complexes with distinct enzymatic activities involved in RNA polymerase II (Pol II) transcription regulation. To investigate the mobility of co‐activator complexes and general transcription factors in live‐cell nuclei, we performed imaging experiments based on photobleaching. SAGA and ATAC, but also two general transcription factors (TFIID and TFIIB), were highly dynamic, exhibiting mainly transient associations with chromatin, contrary to Pol II, which formed more stable chromatin interactions. Fluorescence correlation spectroscopy analyses revealed that the mobile pool of the two co‐activators, as well as that of TFIID and TFIIB, can be subdivided into “fast” (free) and “slow” (chromatin‐interacting) populations. Inhibiting transcription elongation decreased H3K4 trimethylation and reduced the “slow” population of SAGA, ATAC, TFIIB and TFIID. In addition, inhibiting histone H3K4 trimethylation also reduced the “slow” populations of SAGA and ATAC. Thus, our results demonstrate that in the nuclei of live cells the equilibrium between fast and slow population of SAGA or ATAC complexes is regulated by active transcription via changes in the abundance of H3K4me3 on chromatin. Importantly, the apparent diffusion coefficient provides an objective reference value to compare mobility properties between different factors.Through the application of various microscopy‐based techniques . Importantly, the slow fraction of SAGA and ATAC is reduced upon inhibition of transcription elongation or histone H3K4 trimethylation suggesting that association of SAGA and ATAC with chromatin in live cells is largely mediated by changes in the levels of transcription and the associated H3K4me3 histone mark.To obtain information on the mobility of human SAGA/ATAC co‐activator complexes at the subsecond range, and to investigate the presence of their immobile fractions, we performed FRAP and FLIP. Our measurements indicate that, in contrast to RPB1 (Pol II subunit), SAGA and ATAC subunits are highly dynamic and exhibit only transient interactions with chromatin with no detectable immobile fractions. Similar mobility was also found for the general Pol II transcription factors, TAF5 (TFIID subunit) and TFIIB. To characterize the diffusion of TFIIB, TFIID, SAGA, and ATAC complexes in the nuclei of living cells with high sensitivity, we performed live‐cell FCS experiments to study those factors. Our FCS analyses show that two distinct populations dominate the diffusion of coactivators (SAGA and ATAC) and GTFs (TFIIB and TFIID): a “fast” population (having the approximate size of the studied complexes) and a “slow”, probably chromatin‐interacting population  known to have stable interactions with chromatin  might influence the FRAP measurements, we complemented our live‐cell analyses by performing FLIP. Importantly, the exposure time in FLIP is 500 ms, 10 times longer than that used for FRAP (50 ms), allowing the selection of cells that express the given GFP‐tagged factors at even lower levels than in FRAP, with a higher S/N ratio.et al, A comparison between representative eGFP‐NLS‐, GCN5‐ and RPB1‐expressing cells subjected to FLIP is shown in Fig et al, To test the above live‐cell imaging findings with an independent biochemical approach, we performed subcellular fractionation of human cells and verified the relative abundance of endogenous SAGA, ATAC and Pol II, subunits in the different cellular and nuclear compartments Fig . In agreet al, Our FRAP and FLIP analyses strongly suggest that TFIIB, TFIID, SAGA and ATAC complexes have different dynamics compared to Pol II. However, it remained unclear to what extent, if any, active transcription may affect the dynamics of the complexes. To answer this question, we applied FRAP and FLIP measurements in cells treated with flavopiridol (FVP). FVP is a potent cyclin‐dependent kinase (CDK) inhibitor, which inhibits Pol II transcription elongation through the positive transcription elongation factor b  was lower than 200  observation volumes, where the total number of molecules . As expected, the average autocorrelation curve fits well with a one‐component free diffusion model Fig A. The si2/s Fig C, as pre 2/s Fig C, in gooa priori about the number of diffusing species and (ii) the low reduced chi square values obtained for all factors with the two‐population fit  and a slow one . These apparent Dfast and Dslow measures of individual SAGA or ATAC subunits showed comparable distributions to each other, as well as to the studied GTFs  by comparing each factor's apparent Dfast and Dslow values with DeGFP . Thus, these subpopulations of SAGA and ATAC appear to move freely in the nuclear environment, with little to no association with chromatin. We noted that the fast population of the ATAC subunit ZZZ3 had a lower than expected MWA (0.32 MDa instead of ~0.7 MDa). For ZZZ3, we also observed a significant overlap between the distributions of diffusion coefficients of its fast population with that of eGFP . We hypothesized that such interactions may affect chromatin association and thus influence the mobility, or proportion of the chromatin‐associated drug Fig A. As expslow, chromatin‐associated population of the mutant protein  proteins that are stably bound  to chromatin, as identified by other imaging (e.g. FRAP/FLIP kinetics of RPB1), biochemical or ChIP approaches. The estimated apparent diffusion constant of the slow component (ranging from 0.56 to 1.26 μm2/s) is higher than what has been observed for transcription activators with high affinity for specific binding site motifs on the DNA sequence [e.g. Dslow of eGFP‐RAR‐α  is 0.05–0.10 μm2/s  is reduced and/or exhibits reduced association with chromatin (i.e. SPT20). These results suggest that the chromatin status  when transcription is blocked is less favourable for the recruitment of the two complexes. In addition, we demonstrated that although FRAP/FLIP did not detect significant changes in the mobility of TAF5 and TFIIB, the analysis of their diffusion properties by FCS revealed that these GTFs exhibit reduced interactions with chromatin upon inhibition of transcription elongation. Interestingly, our live‐cell observations suggest that inhibition of Pol II elongation creates a chromatin environment which may increase the mobility of GTFs and coactivator complexes.Our FRAP/FLIP experiments performed in normal conditions demonstrated that SAGA and ATAC mobility and recruitment is independent of interactions with elongating RNA polymerase II. However, since the two complexes have multiple subunits with chromatin‐interacting domains, we also investigated whether the chromatin state defined by active transcription affects the dynamics of the complexes. FRAP and FLIP analysis after FVP‐induced transcription elongation inhibition did not detect any significant changes for SAGA or ATAC subunits. On the contrary, FCS measurements performed under the same conditions revealed that indeed the chromatin‐interacting fraction of both complexes  of transcribed and poised genes when analysed by ChIP‐seq ; mouse monoclonal anti‐alpha‐Tubulin ; rabbit polyclonal anti‐ASH2L ; rabbit polyclonal anti‐Lamin A ; mouse monoclonal anti‐RPB1 CTD . Evaluation of transcription inhibition was tested by Western blot (WB) assays on whole cell extracts of U2OS cells using an antibody against the CTD of RPB1 (1PB 7C2). For imaging experiments , drug was added ~2 h before imaging. Each treated sample was then used for 1–2 h. To ensure homogenous distribution of the drug, the required amount was first diluted in 500 μl of medium, prewarmed at 37°C, which was vortexed and finally added on cells growing on Ibidi μ (35 mm) high glass bottom dishes.2 using the Tokai Hit INUBG2E‐TIZ stage top incubator. Roper iLas2 FRAP unit was used to bleach a circular nuclear ROI of 6 pixels radius, corresponding to ~21.9 μm2. The selected ROI was monitored every 50 ms. After 5 s (100 prebleach collected frames), the circular ROI was quickly bleached at maximum laser power with a double pulse of 50 ms. Subsequently, recovery of fluorescence was monitored with the same frequency for a total of 100–200 s. MetaMorph (Molecular Devices) image analysis software was used to process the raw images and obtain the average intensity of three ROIs, which were used for further normalization: the bleached ROI, the whole nucleus ROI and a ROI corresponding to background signal. EasyFRAP software  high glass bottom dishes in phenol red free medium. FRAP was performed using a Nikon Ti‐E inverted microscope  equipped with a CSU X1 Yokogawa confocal spinning disc and a 60 × CFI Plan Apo VC oil NA 1.40 (Nikon) objective. Fluorescence signal was detected with a Photometrics Evolve 512 EMCCD camera. During all FRAP measurements, cell was maintained at 37°C in 5% CO2, was bleached every 3 s at maximum laser power by a double bleach pulse for a minimum total period of 4 min. Like in FRAP, raw images were quantified by MetaMorph software. Average fluorescence values were extracted from three ROIs required for FLIP data normalization: the first corresponds to the whole nucleus, the second corresponds to a neighbouring cell that was always included to the field of view , and a third region corresponds to background signal. Normalization of FLIP data was done according to the formulas described in Nissim‐Rafinia and Meshorer . The algorithm provides as an output the distribution of the obtained apparent diffusion constants for a specific condition. To test the robustness of the algorithm, we used free eGFP diffusing in aqueous solution, for which we obtained a single peak distribution centred around 92 μm2/s, a value which is in agreement with previous studies  above 200 in the observation volume of a given cell. The remaining cells, for which N < 200 in the observation volume, were re‐analysed with the Maximum Entropy model to obtain the diffusion constant distributions.The two‐population fit was further used to test whether the measured parameters were showing any correlation with the expression level of cells used for our measurements. Particularly, to work in conditions where the diffusion time is independent of the expression level in the cells of interest, a threshold was used to filter out autocorrelation curves giving a number of molecules , it is possible to determine the apparent molecular mass (MW) of the labelled protein by using the following relation: DeGFP and D are the diffusion constants of the free eGFP and the eGFP‐tagged protein, respectively.In the frame of the Stokes‐Einstein formalism, the diffusion constant of a sphere is related to its hydrodynamic radius according to: Additional NV and ES performed the molecular laboratory work, NV, MK and PD performed imaging experiments. NV, PD and LT designed the study; NV, MK, PK and PD analysed data. All authors contributed text and figure panels to the manuscript. NV, PD, YM and LT wrote the manuscript. All authors gave final approval for publication.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Review Process FileClick here for additional data file.Source Data for Figure 2Click here for additional data file.Source Data for Figure 3Click here for additional data file.Source Data for Figure 7Click here for additional data file.