The organization of the five β-type globin genes on chromosome 11 reflects the timing of expression during erythroid cell development, with the embryonic ε-globin gene being located at the 5′ end, followed by the two fetal γ-globin genes, and with the adult β- and δ-globin genes being located at the 3′ end. Here, we functionally characterized a DNase I-hypersensitive site (HS) located 4 kb upstream of the Gγ-globin gene (HBG-4kb HS). in vitro and in the context of cells with a high affinity and specificity. Direct delivery of the HBG-4kb ZF to K562 and primary human erythroid cells caused a reduction in γ-globin gene expression which was associated with decreased binding of transcription factors and active histone marks at and downstream of the HS. The data demonstrate that the HBG-4kb HS is important for fetal globin production and suggest that it may act by opening chromatin in a directional manner.The organization of the five β-type globin genes on chromosome 11 reflects the timing of expression during erythroid cell development, with the embryonic ε-globin gene being located at the 5′ end, followed by the two fetal γ-globin genes, and with the adult β- and δ-globin genes being located at the 3′ end. Here, we functionally characterized a DNase I-hypersensitive site (HS) located 4 kb upstream of the Gγ-globin gene (HBG-4kb HS). This site is occupied by transcription factors USF1, USF2, EGR1, MafK, and NF-E2 in the human erythroleukemia cell line K562 and exhibits histone modifications typical for enhancers. We generated a synthetic zinc finger (ZF) DNA-binding domain targeting the HBG-4kb HS (HBG-4kb ZF). The HBG-4kb ZF interacted with the target site  Enhancers are DNA elements that activate transcription of genes in a position- and orientation-independent manner . Active 4–8–The organization of the β-type globin genes reflects the order by which they are expressed during development, with the embryonic ε-globin gene being located at the 5′ end and the adult genes being located at the 3′ end. High-level expression of the genes is regulated by the LCR . The β-gcis-regulatory DNA elements that are distal to the genes, e.g., 3′ enhancers, or located in the proximal promoter regions  across the human β-globin gene locus that play significant roles in the regulation of γ-globin gene expression  is regulated by positive and negative  regions . The her regions . Several regions . Further regions , 17. Recpression –20. The ression –.Here we interrogated the function of a hitherto uncharacterized DNase I HS located 4 kb upstream of the Gγ-globin gene (HBG-4kb HS). Data from the Encyclopedia of DNA Elements (ENCODE) show that the HBG-4kb HS is associated with histone marks typically found at enhancers , 23. In The analysis of publicly available data from the ENCODE project revealed the presence of a DNase I HS located about 4 kb upstream of the Gγ-globin transcription start site (HBG-4kb HS)  data from the ENCODE project, we found that the HBG-4kb HS interacts with a large number of transcription factors that are either ubiquitously expressed or more restricted to hematopoietic tissues. Among the factors interacting with this element are EGR1, USF1, USF2, NF-E2, and MafK . The assEscherichia coli. We first determined the DNA-binding affinity and specificity in vitro using electrophoretic mobility shift assays (EMSAs). We incubated increasing concentrations of purified recombinant HBG-4kb ZF with a constant amount of fluorescently labeled double-stranded DNA oligonucleotides and resolved the free DNA and the protein-DNA complexes on native polyacrylamide gels (dK) reflects the concentration of protein at which half of the DNA molecules are bound and was determined to be 29.5 nM for the HBG-4kb ZF. Changing only 2 nucleotides in the target sequence  (rs11036496) that is associated with hyperuricemia in African Americans  25). Th. Th25). 26–ide gels . The disTo obtain additional evidence that the HBG-4kb HS functions as an enhancer of γ-globin gene expression, we deleted sequences within the HBG-4kb using the CRISPR/Cas9 technology. We designed a specific guide RNA (gRNA) targeting the center of the HBG-4kb and used lentivirus transduction to deliver the guide RNA together with Cas9 to K562 cells, as outlined in Next, we directly delivered the HBG-4kb ZF to K562 cells and obtained cytoplasmic and nuclear protein fractions at different time points after delivery of the protein. The fractions were then subjected to Western blotting using antibodies specific for CTCF, GAPDH , HBG-4kb ZF , γ-globin, Brg1, and GATA1 . The dat−-associated regulatory DNA element associates with USF1, USF2, EGR1, NF-E2, and MafK, just like the HBG-4kb HS . The binding of the transcription factors at the Gγ-globin gene promoter was relatively low, except for USF2, and delivery of the HBG-4kb ZF reduced the binding of the proteins to this site. Importantly, elevated binding of EGR1, USF1, USF2, NFE2, and MafK at the HBG-4kb HS was reduced after delivery of the HBG-4kb ZF to K562 cells. In contrast, there was no statistically significant reduction in the interaction of these proteins with the CDC27 enhancer after treatment with the HBG-4kb ZF. The data demonstrate that binding of the HBG-4kb ZF interfered with the binding of transcription factors at the HBG-4 kb HS.To examine if the binding of the HBG-4kb ZF interfered with the binding of transcription factors at the HS at kb −4, we performed ChIP experiments in K562 cells 12 h after delivery of the ZF-DBD . The binWe next analyzed Pol II binding and histone modification patterns at six different regions in the globin gene locus in K562 cells (WT) and in K562 cells treated with the HBG-4kb ZF  using thTo examine the function of the HBG-4kb HS in primary human erythroid cells, we analyzed previously published ChIP-seq data from differentiating primary human fetal liver hematopoietic stem and progenitor cells (HSPCs)  20). Th. Th20). cis- and trans-regulatory activities that modulate the developmental stage-specific expression of the globin genes continue to be discovered  . HistoneGSM1480321, GSM2695562, as well as GSM2695565 and aligned to the human genome hg19 by the Bowtie 2 program with default settings  data set of GRO-cap sequencing and ATAC-seq data for WT HBD-1kb and the HBD-1kb knockout (KO) were downloaded from GEO accession numbers settings . A bigwisettings . Snapshosettings , using hsettings . The ChIM1816074 . The snahttp://www.scripps.edu/barbas/zfdesign/zfdesignhome.php) (The HBG-4kb ZF and the negative-control ZF-DBD (control ZF) were designed using the Zinc Finger Tools website (ome.php) . The HBGome.php) , 29. TheE. coli as described previously with minor modifications  at 37°C for 4 h. The cells were lysed using a French press with the high-power mode for 3 cycles. The purification of the ZF-DBDs was done with HisLink resin-packed columns  as previously described . The corresponding oligonucleotide sequences were 5′-CACCGACCAACCTTTTGAGTTGCA-3′ for the forward sequence and 5′-AAACATGCAACTCAAAAGGTTGGTC-3′ for the reverse sequence. Ligation of the gRNA encoding the double-stranded oligonucleotide into the lentiCRISPRv2 vector  was performed as described by Sanjana et al. . DNA from single-cell clones was extracted using a QIAamp DNA minikit  and subjected to PCR using primers spanning the HBG-4kb HS . The PCR products were subjected to Sanger sequencing using the HBG-4kb sequencing primer (5′-GAGGGCCAAGCAAAATACAG-3′). K562 cells were subjected to karyotype analysis by the UF Pathology Core.A guide RNA (gRNA) targeting the HBG-4kb HS was designed using the CRISPR DESIGN tool . A 10-μl volume of each reaction mixture was loaded onto 6% Tris-borate-EDTA gels . The detection and analysis of the fluorescent signals as well as the determination of the dissociation constant (dK) were done as described previously (The purified ZF-DBDs (1 to 100 nmol/liter) were incubated at room temperature for 30 min in a 20-μl reaction mixture mixed with 15 nmol/liter Cy5-labeled double-stranded DNA oligonucleotides, 100 ng herring sperm DNA, and binding buffer . Transfected cells were maintained under Geneticin (Sigma) selection. Human peripheral blood mononuclear cells  were expanded and differentiated into erythroblast cells following the supplier's instructions. The PBMCs (15 × 106 cells) were cultured in StemSpan SFEM II medium (StemCell Technologies), StemSpan erythroid expansion supplement, and 1% (vol/vol) P-S. The cells were incubated in the presence of 5% CO2 at 37°C, and the medium was changed every 2 days.K562 cells were grown in RPMI medium  supplemented with 10% (vol/vol) fetal bovine serum  and 1% (vol/vol) penicillin-streptomycin  in the presence of 5% CO6 cells/ml) were supplied with purified ZF-DBD proteins (from 500 nmol/liter to 2 μmol/liter) for various times. Cells were collected and subjected to Western blotting, chromatin immunoprecipitation (ChIP), and reverse transcription (RT) followed by quantitative PCR (qPCR).Delivery of ZF-DBDs into K562 cells and PBMCs was performed using a direct protein delivery protocol , 29. CelWestern blotting procedures were performed using a previously published protocol . Briefly6 cells were collected and cross-linked with 1% (vol/vol) formaldehyde at room temperature for 10 min. The reaction was quenched with 125 mmol/liter glycine, and the cells were incubated in cell lysis buffer  and nucleus lysis buffer . Chromatin was fragmented to average 300-bp fragments on ice using a Bioruptor disruptor . For each immunoprecipitation reaction, chromatin from 2 × 105 cells was incubated with 2.5 μg antibody plus protein A/G magnetic beads (Pierce) rotating at 4°C overnight. The chromatin-antibody-bead complexes were subjected to washing, eluting, and reverse cross-linking at 68°C overnight as described previously (The chromatin immunoprecipitation (ChIP) protocol used in this study was described previously . Brieflyeviously . The DNATC method. The primer sequences used for RT-qPCR are listed in Table S5.Total RNA was extracted using an RNeasy kit  following the manufacturer's protocol. The RNA was reverse transcribed into cDNA with an IScript cDNA synthesis kit (Bio-Rad). The cDNAs were subjected to qPCR in a 10-μl reaction mix with SYBR green and 1.5 μmol/liter of each forward and reverse primer. Gene expression was analyzed by the normalization of expression to that of GAPDH using the ΔThe experiments were repeated at least three times unless stated otherwise. In experiments involving qPCR, each sample from the three repeats was analyzed by PCR three times. Error bars represent the standard error of the mean (SEM). The statistical analysis was performed with GraphPad Prism software.


The liver is a critical organ of energy metabolism. At least 10% of the liver transcriptome demonstrates rhythmic expression, implying that the circadian clock regulates large programmes of hepatic genes. Here, we review the mechanisms by which this rhythmic regulation is conferred, with a particular focus on the transcription factors whose actions combine to impart liver- and time-specificity to metabolic gene expression. The circadian clock maintains homeostasis in the face of rhythmic environmental changes and as such, regulates critical physiological processes, including inflammation and energy metabolism. Rhythms of hepatic metabolism have been especially well-studied, as interest grows in the potential of circadian medicine to have therapeutic applications for disorders of energy metabolism and storage. Here, we summarise the current understanding of the mechanisms by which programmes of metabolic genes are under circadian regulation in the liver, particularly by core clock transcription factors.In mammals, there is a network of cellular clocks that are coordinated, or synchronized, by the central clock in the suprachiasmatic nucleus of the hypothalamus. The suprachiasmatic nucleus of the hypothalamus functions as the dominant pacemaker tissue, responsive to light via the retinohypothalamic tract. This central clock entrains clocks in peripheral tissues by autonomic and endocrine signals. Entrainment refers to the synchronisation of a rhythm to a fixed time signal, in the absence of entrainment, rhythms are free-running, and as the endogenous circadian clock period is not exactly 24 h, this results in separation of the endogenous circadian phase from that of the external environment through time. Peripheral tissue clocks are sensitive to phase setting by other stimuli (zeitgebers) and are highly responsive to feeding time ,2,3 and Period1/2/3 (PER) and Cryptochrome1/2 (CRY) genes, whose protein products serve to negatively regulate CLOCK and BMAL1 and their transcriptional targets [Per1 and Per2 expression are further modulated by the basic helix-loop-helix factors DEC1 and DEC2, and by the basic leucine zipper transcription factor E4BP4 and its counterbalance transcription factor DBP [At a molecular level, the clock comprises a transcription-translation feedback loop (TTFL) with a periodicity of approximately 24 h . The cir targets ,8. Per1 ctor DBP . Anotherctor DBP . The TTFctor DBP , but rhyctor DBP ,12, thusThis tissue-specificity demonstrates that the core clockwork is not the only determinant of rhythmic gene expression. Gene expression is regulated by transcription factor activity at promoter regions, activity at distal regulatory regions (enhancers) and chromatin architecture ,14. ChroAny of these factors above can confer rhythmic regulation to gene expression. In fact, only 22% of oscillating genes are thought to be rhythmic due to de novo transcription , the remIn terms of conferring tissue-specific rhythmicity, current thinking suggests that patterns of chromatin accessibility, determined by tissue-specific pioneer factors, produce cistromes (the genome-wide binding profiles) of key clock factors distinct to different tissues. These can show surprisingly little overlap. A recent study has found only 6% of BMAL1 binding sites to be common to liver, kidney and heart , whilst Bmal1 [Bmal1−/− with liver-specific Bmal1 reconstitution) only partly restores the rhythms seen in wild-type animals [Approximately 10% of the mouse liver transcriptome is reported to show rhythmic expression (the most recently published liver dataset detected 2029 genes with circadian oscillation ). In perBmal1 , but losBmal1 . Further animals . These f animals , the gut animals  and dist animals , all hav animals , the rolIn the livers of intact animals with normal behaviour, there is a coordinated rhythm of CLOCK:BMAL1 recruitment to the genome during the day, accompanied by histone acetylation and RNA polymerase II recruitment, which triggers a peak phase of transcription in the early part of the night ,24. As tHnf6 deletion leading to lipid accumulation [Following the theory that tissue-specific chromatin accessibility dictates clock protein binding , one coumulation  through Hnf4α knockout [Clock and Bmal1 expression with Hnf4α overexpression and co-occurrence of HNF4α binding at CLOCK:BMAL1 sites [Work demonstrating the synchronising effect of glucocorticoids in the liver has found the HNF4α motif to be associated with synchronised genes and that the synchronising effect was partly lost in liver knockout . This suL1 sites , but thiOver the course of the day-night cycle, the nocturnal mouse undergoes transitions between the inactive and active states and the fasted and fed states. As the key organ of metabolism, the liver can both produce glucose as an immediate energy supply (gluconeogenesis) and produce lipid as a longer-term energy store (de novo lipogenesis) A. It is The CRY proteins in mammals are related to blue-light receptors and photolyases found in plants. They play a key role in both the early and late repressive phases of the circadian cycle, with CRY1 repressing the CLOCK:BMAL1 heterodimer by binding directly to the PAS (Per-Arnt-Sim) domain . DegradaPepck and G6pc, thus suppressing gluconeogenesis further [In mouse liver, activity at the fasting-responsive cyclic adenosine monophosphate (cAMP) response element (CRE), which regulates gluconeogenesis, is lowest at the night-day transition when CRY activity is highest . This ha further .Pepck gene shows time-of-day dependency being highest when CRY levels are lowest. Chromatin immunoprecipitation experiments do suggest that CRY can associate with the Pepck GRE (Glucocorticoid Response Element), in a dexamethasone-dependent manner. Chronic dexamethasone treatment in CRY double-knockout mice significantly impairs glucose tolerance, this effect is not seen in wild-type mice [Separately, CRY has been found to associate with the glucocorticoid receptor C-terminus . It appeype mice . Taken tBmal1 and Rev-erbα transcription [REV-ERBα is a member of the nuclear receptor superfamily. Its endogenous ligand is haem, a cofactor important for enzymes involved in mitochondrial respiration . Haem sycription . When hacription . This iscription . REV-ERBα has properties which distinguish it from other nuclear receptors. It has not, for example, been found to form functional heterodimers with other NR1 family members at the RORE site, unlike RXR and PPAR . Co-reguBmal1 promoter which is proposed to be the mechanism by which REV-ERBα exerts repressive control over Bmal1 transcription [The P-box in the DBD of NR1 receptors recognises the AGGTCA motif, typically found as repeats (direct/inverted/everted) with variable numbers of spacing nucleotides between. REV-ERBA binds WAWNTAGGTCA as a monomer (where WAWNT is an A/T-rich sequence immediately 5’ to AGGTCA) demonstrating activation of transcription in vitro . Later ecription .Rev-erbα knockout, thus it has been proposed that REV-ERBA opposes the formation of functional enhancer-promoter loops at its target genes [Two further mechanisms of REV-ERBα-mediated gene repression have been proposed. One of these suggests that the displacement of ROR at RORE sites is not through competition but through facilitated repression. This model requires the co-activator SRC-2 (GRIP-1) . The SRCet genes .Rev-erbα−/− mice and Fl/FlRorγFl/FlRorα mice injected with AAV-Cre, respectively), suggests that the two proteins might compete for binding at RORE/RevDR2 sites associated with highly rhythmic genes [REV-ERBα is proposed to have roles as a component of the core clock and as a master regulator of metabolism. Over recent years, evidence has suggested that these two roles are mediated by distinct mechanisms. Characterisation of the cistromes of REV-ERBα and REV-ERBβ in mouse liver has revealed overlap at sites associated with core clock genes, suggesting that both contribute to clock regulation  , which is thought to promote gene transcription by recruiting CBP/p300 to the genome. Using mice with a liver-targeted mutation in the REV-ERBα DBD, who concurrently had liver-targeted Rev-erbβ knockdown (AAV-Cre approach), it was hypothesised that REV-ERBα has both DBD-dependent and DBD-independent actions and that these are dissociable [This same study noted that many genes were sociable . The DBDsociable . As withsociable .REV-ERBα is therefore proposed to mediate these DBD-independent metabolic effects by tethering to HNF6 and recruiting the NCOR/HDAC3 co-repressor complex ,34. The Rev-erbα is not as severe as that caused by depletion of HDAC3 or HNF6; this supports a role for Rev-erbβ, and indeed, in Rev-erbα−/− mice who then have hepatic depletion of Rev-erbβ, the hepatosteatosis does become more severe and less NCoR/HDAC3 recruitment to the genome is seen [Rev-erbα−/− mice [Rev-erbα−/− mice driving excessive fat storage and ketogenesis.The metabolic phenotype of mice lacking  is seen . Nonethe−/− mice . Using i−/− mice . They suTogether, these studies build a picture of REV-ERBα/NCoR/HDAC3 repressive activity during the inactive period at sites regulated by HNF6 binding, which serves to downregulate de novo lipogenesis . During the active feeding phase, this repressive activity falls, allowing lipogenesis and lipid accumulation. The absence of these repressive components therefore, produces a phenotype of increased de novo lipogenesis and hepatic lipid accumulation. Furthermore, an in vivo study with a REV-ERBα agonist has demonstrated the loss of fat mass with enhancement of REV-ERBα activity .REV-ERBα is degraded by the proteasome having been ubiquitinated by the F-box protein FBXW7, this ubiquitination is itself triggered by phosphorylation by cyclin-dependent protein kinase 1 (CDK1) . ModulatHepatic energy metabolism is also subject to regulation by rhythmic hormones, notably endogenous glucocorticoids . Acute treatment with dexamethasone (a synthetic glucocorticoid) influences the expression of large programmes of metabolic genes in mouse liver , in keepGlucocorticoid action is context-specific ,70,71 anThis raises the attractive possibility that manipulating the timing of glucocorticoid administration could minimise the undesirable metabolic side effects associated with therapeutic glucocorticoid use. A study of chronic glucocorticoid administration has found that when given at ZT12, glucocorticoids up-regulate lipogenic genes to a lesser extent than at ZT0 . FurtherThe liver plays a critical role in enabling the necessary switch between the fed and the fasted state. As such, gene expression programmes in the liver show a strong time of day dependence. The relative importance of the liver-autonomous circadian clock versus the systemic circadian system in generating these rhythms of gene expression and enzymatic function remains unclear. A number of key circadian transcription factors, including CRY and REVERBa, have been shown to play dominant roles in regulating either carbohydrate or lipid metabolic switches in the liver through time, and both these transcription factors have been targeted by small, drug-like molecules. A further manifestation of the circadian phenotype results from regulation of the hepatic nuclear receptor expressome, with actions identified both for lineage-determining nuclear receptors including HNF4A, and also signal-dependent receptors, such as the glucocorticoid receptor. Taken together, there is now abundant evidence for the importance of the hepatic circadian clock in driving energy metabolism, utilisation and storage and for the actions of systemic diseases, such as obesity, in affecting this process. It seems certain that new approaches to tackle the prevalent disease of over-nutrition in developed economies will require the liver clock to be harnessed for maximal efficacy.


EGR1 gene is also known to be transcribed in early G1 phase. However, the regulation of EGR1 transcription in early G1 phase is not clarified well. Here we found that CCCTC-binding factor (CTCF), a chromatin binding protein, is required to transcribe EGR1 gene at the onset of early G1 phase. We found that CTCF mediated the formation of higher-order chromatin structures among CTCF binding sites located in the EGR1 locus. Disruption of the CTCF-dependent higher-order chromatin structure using nuclease-dead Cas9 (dCas9)-mediated interference reduced the EGR1 transcription in early G1 phase. Collectively, we propose that CTCF has functional roles for the temporal expression of EGR1 in early G1 phase through regulation of higher-order chromatin structure organization.Early growth response 1 (EGR1) is a transcription factor and regulates cellular processes such as proliferation, differentiation, and apoptosis. The expression of EGR1 is rapidly induced in response to several stimuli, and it activates the expression of downstream target genes involved in signaling cascades.  Most IEGs encode transcription factors involved in initiation of signaling cascades by modulating transcription of the target genes. EGR1 belongs to IEGs and is transcribed rapidly and transiently in response to various kinds of stimuli2. EGR1 functions as both an activator and a repressor for transcriptional regulation of numerous genes, including EGF, TGF-β, and Bax, in concert with several cofactors4 to control cell growth, tumorigenesis5, and apoptosis6. Analyses of the EGR1 promoter revealed that a number of cis-elements are located within 720 bp upstream of the EGR1 transcription start site (TSS)7. It has been proposed that several serum response elements (SREs) located at approximately 300 bp upstream of the EGR1 TSS have a crucial role for the expression of EGR17. Serum response factor (SRF) binds to SREs by interacting with TCF co-regulator proteins in a phosphorylation-dependent manner8. It has been reported that the expression of EGR1 was induced in early G1 phase9. Because SRF-TCF complex is activated in early G1 phase by growth factors to induce genes involved in G1 progression10, EGR1 is thought to be regulated by SRF-TCF complex in early G1 phase. From functional analyses of CTCF in the EGR1 expression, CTCF was thought to function as a negative regulator during mouse myeloid cell differentiation or in LPS-stimulated macrophages11. CTCF binding motif is located at approximately 1.2 kb upstream of the EGR1 TSS11. CTCF is a DNA binding protein possessing C2H2 zinc finger motifs and was originally found as a repressor of c-myc12 and lysozyme13. CTCF is a highly conserved multifunctional protein in vertebrates. CTCF functions not only as a transcription factor with a variety of binding partners14, but also a regulator of higher-order chromatin structure through the formation of chromatin loops that define the boundary between transcriptionally active and inactive chromatin15. Recently we reported that CTCF is highly phospholylated in mitosis and the phosphorylation of CTCF impairs its DNA binding activity16. In parallel with the reformation of nucleus at telophase, dephosphorylation of CTCF and its recruitment on chromatin might occur, although a phosphatase(s) involved in this process is Unknown. Higher order chromatin structures, including structures possibly regulated by CTCF, are disrupted in mitosis and reformed in G1 phase17. Regulation of gene expressions through higher-order chromatin structures are observed through whole cell cycles. In particular, early G1 phase may be a time in which profound reformation of higher-order chromatin structure and transcription restart simultaneously proceed. Thus, CTCF might be involved in the expression of early G1 genes, such as EGR1. However, the precise function of CTCF in the EGR1 expression in early G1 phase is not well known.Immediate early genes (IEGs) are a set of cellular genes, and transcription of mRNAs from which is rapidly induced by both extracellular and intracellular signals through several factors which does not requires EGR1 in early G1 phase. Chromatin Immunoprecipitation (ChIP) and Chromosome Conformation Capture (3 C) analyses indicated that CTCF-mediated higher-order chromatin structure is formed among the promoter and the upstream and the downstream CTCF-binding sites of the EGR1 gene locus after mitotic exit. dCas9-mediated interference of the formation of higher-order chromatin structure in early G1 phase also reduced EGR1 transcription. Collectively, these results suggest that CTCF is important for the temporal transcription regulation of EGR1 through its function in the organization of higher-order chromatin structure.Here, we have shown that CTCF is required for the transcription of EGR1 in early G1 phase, we examined the effect of CTCF knockdown (KD) on the EGR1 transcription level in early G1 phase. In CTCF KD cells, using plasmids expressing shRNA against CTCF (shCTCF#1 and #2), the expression level of the CTCF proteins was less than 25% of that in the control cells  of CTCF in the EGR1 transcription in early G1 phase, we used a luciferase reporter system to analyze the effect of CTCF bound to the promoter on EGR1 transcription in early G1 phase. A DNA fragment corresponding to the promoter region between −720 bp and + 120 bp of the EGR1 TSS was amplified by PCR and cloned into a luciferase reporter plasmid (pEGR1 pro-720/+120 Luc). We also constructed a luciferase reporter plasmid containing a DNA fragment between −1358 bp and + 120 bp of the EGR1 TSS (pEGR1 pro-1358/+120 Luc) containing a promoter-proximal CTCF binding site located at the nucleotide position between −1227 bp and −1208 bp of the EGR1 TSS. pEGR1 pro-1358/+120ΔCTCF BS Luc has mutated nucleotide sequences of the promoter-proximal CTCF binding site  and downstream (38 kb from the TSS) sites in the EGR1 gene locus in early G1 phase. Both the upstream and downstream CTCF binding sites showed similar increment patterns of CTCF binding as seen in the promoter-proximal CTCF binding site in early G1 phase  and a specific target DNA sequence, resulting in the induction of a double-strand DNA break22. Because dCas9 has two alanine substitutions at amino acid positions 10 and 840 , dCas9 has gRNA-dependent DNA binding activity but not nuclease activity. Thus, the dCas9-gRNA complex is recruited to a specific chromatin site without DNA damages23. To bring about interference of CTCF at each CTCF binding site, we designed gRNA sequences covering each of the CTCF binding sites. Cells were transfected with a plasmid construct expressing both 3× FLAG tagged dCas9 and gRNA. The gRNA targets were either a control nonspecific sequence (non-homologous with any CTCF binding sites in the EGR1 locus), the upstream, downstream, or promoter proximal CTCF binding site in the EGR1 locus , while the downstream CTCF binding site of the EGR1 locus is on the reverse strand (the template strand of EGR1). Because, it is reported that two CTCF binding sites located in convergent orientation interacts more often than that of located in the same orientation24. The 3 C interaction of the upstream-promoter region seems weaker compared with others  of the EGR1 expression which is controlled independently of CTCF-mediated higher-order chromatin structures. The gRNA against the promoter proximal CTCF binding site reduced the expression of EGR1 in early G1 phase, suggesting that there is a functional significance of the promoter proximal CTCF binding site on the EGR1 expression, even though it can not be reconstituted in transiently transfected plasmid lacking additional CTCF binding sites. These results suggest that higher order interactions between the promoter proximal CTCF binding site and other CTCF binding sites are important for its proper function. It has been reported that nucleosomes surrounding a CTCF binding site are well positioned25. Hence, we can not exclude the possibility that the local chromatin structure around the promoter proximal CTCF binding site might be important for its function in the EGR1 locus because nucleosome positioning on transiently transfected DNA is usually different from that of native chromatin26. It has been reported that in mouse erythroblast cells, chromatin loop structure is generally disrupted in mitosis and increased sharply until early G1 phase (60–90 min post nocodazole release)27. Reformation of chromatin loop structures occur independently of its gene expression patterns during cell cycle, although the functional relationship between chromatin loop structures and its direct contribution to a gene expression is not clear27. Our findings suggest that the formation of higher order chromatin structures in the EGR1 locus in early G1 phase may contribute to the expression of the EGR1 pre-mRNA in early G1 phase. Enhancers regulate transcription of their target genes through the binding of transcription factors in a distance-independent manner28. It has been suggested that distal enhancers and their target promoters are closely positioned in three dimensional space through higher order chromatin structures to establish a microenvironment to regulate efficient gene expressions29. The enhancer database predicts a possibility of the presence of cell type specific putative enhancer elements in the human EGR1 gene locus30. These putative enhancer elements may contribute to an optimal induction of EGR1 in early G1 phase mediated by CTCF.EGR1 regulates several cellular processes such as cell proliferation and apoptosis as a transcription factortor Fig. . CTCF isase Figs  and 6. Tase Figs  and 5A,Cite Figs  and 3B acus Fig.  did not EGR1 but also other early G1 genes9 have CTCF binding sites in their gene loci . After 20 h post transfection, puromycin was added in the medium at the concentration of 2 μg/ml, and cells were incubated further for 20 h. After selection in the presence of puromycin, mitotic cells synchronized with nocodazole were collected. Briefly, cells were treated with 2.5 mM thymidine (Sigma) in the culture medium for 16 h. Thymidine blocked cells were released into a fresh culture medium and incubated for 7 h, so that cells stayed in late S and G2 phases. Then, cells were treated with nocodazole (Sigma) at a final concentration of 165 nM and further incubated for 6 h. Mitotic cells were collected by gentle shaking of cell culture dishes. For release of cells from mitosis, nocodazole-arrested cells were reattached to poly-L-lysine coated culture dishes, then washed with PBS, and cultured for indicated periods.HeLa S3 cells were maintained at 37 °C in DMEM supplemented with 10% FBS. Cells were transfected with pU6-puro-shCTCF or pU6-puro-shEGFPCTCF, corresponding to the sequence between nucleotide positions 868 and 888 (NCBI Reference Sequence: NM_006565.3), 5′-CACCGTGTCTAAAGAGGGTCTTGTGGTGTGCTGTCCCGCAAGGCCCTCTTTAGACACTTTTTT-3′ and 5′-GCATAAAAAAGTGTCTAAAGAGGGCCTTGCGGGACAGCACACCACAAGACCCTCTTTAGACAC-3′ oligonucleotides were phosphorylated, annealed each other, and cloned into pU6-puro32 that had been digested with BspM I. Oligonucleotides corresponding to the sequence between nucleotide positions 2,819 and 2,839 (NM_006565.3), 5′-CACCGTAAATTATGGAGTGTTCTGATTGATATCCGTCAGAACATTCCATGATTTGCTTTTTT-3′ and 5′-GCATAAAAAAGCAAATCATGGAATGTTCTGACGGATATCAATCAGAACACTCCATAATTTAC-3′ were used to generate pU6-puro-shCTCF#2 plasmid. The human EGR1 promoter was amplified by PCR using the genomic DNA extracted from HeLa S3 cells as template and cloned into PGV-B (TOYOBO). The primers 5′-TCCTCCCCCGCACTCCCGGTTC-3′ and 5′-GGGAACACTGAGAAGCGTGC-3′ (EGR1+120 reverse primer) were used for amplification of the EGR1 promoter region harboring nucleotide positions from –720 to+120 relative to the TSS to make pEGR1 pro-720/+120 Luc. The primers 5′-GCTCAGTTCGTGCTCACTGC-3′ and EGR1+120 reverse primer were used for amplification of the EGR1 promoter region corresponding to nucleotide positions from –1358 to +120 to make pEGR1 pro-1358/+120 Luc. A CTCF binding site (CTCF BS) corresponding to nucleotide positions from –1227 to –1208 was changed from GGAACCTCCAGGGGGCAGCA to GGAAGTTATAAGCTTCAGCA to make pEGR1 pro-1358/+120ΔCTCF BS Luc. The sequencing information of nucleotide regions on pEGR1 pro-1358/+120ΔCTCF BS Luc corresponding to the CTCF binding site are shown in Fig. EGR1 upstream CTCF binding site (5′-CACCGAGGCAGCTGCGCCACCTAGT-3′ and 5′-AAACACTAGGTGGCGCAGCTGCCTC-3′) and downstream CTCF binding site (5′-CACCGTTGTGCTGGTGACCACAAGG-3′ and 5′-AAACCCTTGTGGTCACCAGCACAAC-3′), and promoter proximal CTCF binding site (5′-CACCGAGTGGAGAGGGAACCTCCAG-3′ and 5′-AAACCTGGAGGTTCCCTCTCCACTC-3′) were phosphorylated, annealed, and ligated into Bbs I digested pX330-dCas9. To make a control template for 3 C assay, the DNA fragments corresponding to the Igf2 gene locus were amplified by PCR and cloned into pBluescriptII SK + (pBS). The primers 5′-GGAGCTCACTCTAGTCTCCAAGGCTATT-3′ and 5′-CTCACCTGTCTCCATAAAGCATGTGTCC-3′ were used for amplification of the Igf2 upstream region harboring nucleotide positions 11 kb upstream relative to the TSS to make pBS-Igf2-upstream. The primers 5′-CCTAGGATTGGAAGGACCCCATCATCTG-3′ and 5′-CTGCAAGGTCAGGGGACATTGTTCAGGG-3′ were used for amplification of the Igf2 downstream region harboring nucleotide positions 102 kb downstream relative to the TSS to make pBS-Igf2-downstream. 5′-ends of PCR products were phosphorylated by T4 Polynucleotide Kinase and ligated into pBS digested with EcoR V.To generate pU6-puro-shCTCF#1 plasmid, which expresses 21 nucleotides-long hairpin-type shRNA against human 33, anti-α-tublin , anti-EGR1 , anti-FLAG  antibodies and rabbit normal IgG (Millipore).Antibodies used in this study were anti-CTCF Toal cell lysats were prepared by lysing cells in sample loading buffer . Proteins were separated by 7.5% SDS-PAGE and subjected to western blot analysis. The signal was detected by Chemi-Lumi One L  and ImageQuant LAS4000 .EGR1 pre-mRNA, 5′-CTGTGCATCTACACCGACAACTC-3′ and 5′-CTCGGAGGAGCAGATATGTCAGA-3′ for CCND1 pre-mRNA, 5′-GCAATGAAGGTGAAGGCCGGCG-3′ and 5′-TAACACGTGCGCTCGTGCTCCACCTC-3′ for 28 S rRNA.Total RNA was prepared from cells using RNeasy mini kit (Qiagen) and RNase-free DNase I (Qiagen), and subjected to reverse transcription using ReverTra Ace reverse transcriptase (TOYOBO) with random 9 mer primers. Synthesized cDNA was used as template for quantitative real-time PCR (qRT-PCR) using FastStart SYBR Green Master (Roche) with specific primer sets, 5′-CAGCCCTACGAGCACCTGAC-3′ and 5′-ACTCCTGCGGTGAAGGACAG-3′ for 3 and 10 mM DTT. The primers used here were as follows: 5′-AAACGGTGCCATATCCAGGCTG-3′ and 5′-TCCAGGGAGCGGAGGATGAG-3′ for EGR1 gene 15 kb upstream region, 5′-GCTCAGTTCGTGCTCACTGC-3′ and 5′-GTTCCCTCTCCACTCCCAGC-3′ for EGR1 gene 1.2 kb upstream region, 5′-CAGGCACCTCTTAATGCTTGTC-3′ and 5′-GCCAGGGAAAGTTTGTGCTG-3′ for EGR1 gene 38 kb downstream region. To perform a specific PCR amplification from the promoter proximal CTCF binding site corresponding to the transfected reporter plasmid, the primers used here were as follows: 5′-CCAAACTCATCAATGTATCTTATGG-3′ and 5′-GGCTTGGCGGCTCGGTGCTG-3′. The former primer corresponds to the unique region on the plasmid. The latter primer corresponds to the promoter proximal CTCF binding site in EGR1 gene. The quantitative PCR (qPCR) was performed using FastStart SYBR Green Master and Thermal Cycler Dice Real Time System (TaKaRa). The results were analyzed using Thermal Cycler Dice Real Time System Software Ver. 5.11. (TaKaRa).Cells were fixed by direct addition of formaldehyde to culture dishes at the final concentration of 0.5% and incubated at room temperature for 10 min. Cross-linking was terminated by the addition of glycine. Cells were washed with 125 mM glycine/PBS and collected by scraping. ChIP assays were carried out according to the protocol from Chromatin Immunoprecipitation Assay Kit  with minor modifications. Elution buffer contains 1% SDS, 100 mM NaHCO34. Cells were fixed by direct addition of formaldehyde to culture dishes at the final concentration of 1% and incubated at room temperature for 10 min. Cross-linking was terminated by the addition of glycine. Cells were washed with 125 mM glycine/PBS and collected by scraping. Cells were resuspended in Dpn II digestion buffer containing 0.3% SDS and incubated at 37 °C for 8 h. Triton X-100 was added at the concentration of 1.8%, and the samples were incubated at 37 °C for 1.5 h to sequester SDS. DNAs in samples were digested at 37 °C for 16 h with restriction enzyme Dpn II (New England Biolabs). The restriction enzyme was inactivated by the addition of SDS at the concentration of 1.6% and heating at 65 °C for 20 min. The samples were diluted by ligation buffer , to adjust concentration of DNA to 2.5 ng genomic DNA/μl each. Triton X-100 was added to 1% and incubated at 37 °C for 1 h to sequester SDS. T4 DNA ligase (TOYOBO), DTT, and ATP were added and incubated at 16 °C for 20 h followed by 30 min at room temperature. The samples were heated at 65 °C for 6 h to reverse cross-linking and incubated with Proteinase K  at 55 °C for 1 h. DNAs were purified from the samples by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation. A typical size distribution of genome DNA of uncut genome, Dpn II digested genome without ligation, and ligated genome DNA are shown in Fig. EGR1 locus on human chromosome 5, pBS-Igf2-upstream, and pBS-Igf2-downstream. BAC DNA and plasmid DNA are mixed in equimolar amounts and digested with Dpn II. Digested DNA was randomly ligated by T4 DNA ligase. The random ligated DNA was purified and used as a standard template for semi-quantitative PCR. PCR was performed with primers shown in Table 35.3C assays were performed essentially as described previouslyEGR1 pro-720/+120 Luc, pEGR1 pro-1358/+120 Luc, pEGR1 pro-1358/+120ΔCTCF BS Luc plasmids together with pRL-SV40 (Promega) plasmid. Renilla luciferase expression plasmid pRL-SV40 was used as a normalizer. Transient transfection of plasmid DNA for reporter gene assay was performed using GeneJuice (Novagen). Cells were collected at 0, 1, 2, and 3 h post release from nocodazole treatment as described previously. The cells were lysed with lysis buffer . The cell lysates were mixed with a firefly luciferase substrate (Promega) or rennilla luciferase substrate (Promega), and each luciferase activity was measured by Lumat LB9506 (BERTHOLD). Each value was normalized by the renilla luciferase activity.HeLa S3 cells were transfected with pHeLa S3 cells were transfected with shEGFP or shCTCF#1 expression plasmid and cell cycle were synchronized as described above. Nocodazole arrested cells were collected and reattached to a poly-L-lysine coated 12-well plate. The plate was put in a chamber of ARRAYSCAN XTI (Thermo Fisher Scientific) and maintainted at 37 °C in 5% CO2. Then, cells were washed with PBS, and time-lapse images were collected by ARRAYSCAN XTI.Supplementary Figures


NR5A2 regulates reproduction in sheep. In this study, we amplified the promoter sequence of NR5A2 and determined that its core promoter region ranged from -721 nt to -281 nt. A T > G polymorphism at -700 nt was detected in the core promoter region. Association analysis found that the litter sizes of Hu ewes at their second and average parities with genotype GG  were significantly higher than those of ewes with genotype TG  (p < 0.05) and TT  (p < 0.05). The litter size of Hu ewes at their third parity with genotype GG (2.10 ± 0.10) was significantly higher than that of ewes with genotype TT (1.56 ± 0.12) (p < 0.05). A luciferase assay showed that the -700G allele increased the luciferase activity relative to the -700T allele. Furthermore, the -700T > G polymorphism created a novel binding site for metal-regulatory transcription factor 1 (MTF-1). A competitive electrophoretic mobility shift assay confirmed that MTF-1 specifically bound with the G-type promoter of NR5A2. An overexpression experiment demonstrated that MTF-1 was involved in the alteration of NR5A2 transcription activity and further increased NR5A2 gene mRNA expression. Our findings revealed that the -700T > G polymorphism promoted NR5A2 expression due to the positive effects on NR5A2 gene transcription activity by MTF-1 and thereby increased fecundity in Hu sheep.Nuclear receptor subfamily 5 group A member 2 (NR5A2), also referred to as LRH-1 or FTF, is an orphan nuclear hormone receptor that is involved in regulating embryonic development, ovarian granulosa cell differentiation, gonadal sex differentiation, and steroidogenesis in mammals. However, little is known about how  Drosophila (Nuclear receptor subfamily 5 group A member 2 (NR5A2), also referred to as liver receptor homolog-1 (LRH-1) or fetoprotein transcription factor (FTF) in osophila , is an oosophila , and is osophila , ovarianosophila , gonadalosophila , and steosophila .NR5A2 gene were associated with preterm birth in humans  from Helen Sheep Industry Co., Ltd., Jiangsu Province, China. DNA was extracted using a standard phenol–chloroform extraction protocol . Five he2, and 0.5 μl of 10 nmol/L upstream and downstream primers (A PCR was conducted using a 20-μl mixture containing 1 μl of reverse transcription (RT) product or 60 ng of DNA, 2 U of Taq DNA polymerase , 2 μl of 10× PCR buffer, 0.5 mmol/L dNTP, 2.5 mmol/L MgCl primers Table 1.NR5A2 promoter sequences were screened for SNPs using a DNA pooling sequencing assay with primers P4 including core promoter region of NR5A2 gene . The core promoter was predicted using Promoter 2.0 (http://jaspar.binf.ku.dk/).The oter 2.0  and tranNR5A2 promoter deletion fragments  were amplified and cloned in the NheI–XhoI  site of a pGL3-basic vector to create pGL3-281, pGL3-721, and pGL3-1373 products. The MTF1 CDS sequence of Hu sheep was amplified and cloned in the XhoI–XbaI  site of a pcDNA3.1(+)  vector to obtain the overexpression vector pcDNA3.1-MTF-1. Plasmids were extracted with an Endo-free Plasmid Mini Kit .To identify the core promoter region, three The -700GG promoter region containing an MTF1-binding site was amplified and cloned into pGL3-basic to obtain pGL3-G, and the -700TT promoter region was amplified by point mutation primer P5 Table 1 Pyrobest DNA Polymerase) was used to PCR amplification, then DNA was purified, next was Blunting kination and ligation reaction to corresponding plasmids.Point mutation amplification was followed according to the manufacturer’s instruction. In brief, firstly, a pair of primers (P5) was designed which was adjacent at 5′-ends and opposite-direction at 3′-ends to introduce mutation site (T). High-fidelity polymerase  were cultured in Dulbecco’s modified Eagle’s medium  and supplemented with 10% fetal bovine serum  and 100 U/ml penicillin/streptomycin . 293T cell lines were thawed and cultured under humidified air containing 5% CO2 at 37°C and subcultured in 24-well plates at 1×105 cells/dish for transfection to measure the luciferase activity.Granulosa cells of Hu sheep were separated from the ovaries following the methods described by Luciferase reporter plasmids were transfected into 293T cell lines using Lipofectamine 3000  according to the manufacturer’s instructions. Briefly, 293T cells were seeded to be 70–90% confluent at 24-well plates for transfection. Then, Lipofectamine 3000 reagent diluted in Opti-MEM medium was added and mixed. A master mix of different constructs was prepared by diluting constructs in Opti-MEM medium, adding P300 reagent and mixing well. Diluted constructs were added to each tube of diluted Lipofectamine 3000 reagent (1:1 ratio) and incubated for 25 min. Finally, the constructs–lipid complex was added to cells. Forty-eight hours after transfection, the cells were harvested and treated for measuring the luciferase activity; all remaining steps were performed following the methods described by Nuclear extracts were prepared from ovine ovaries using a cytoplasmic and nuclear extraction kit  according to the manufacturer’s instructions. Synthetic oligonucleotides 5′-labeled with biotin  were annealed to generate double-stranded oligonucleotides as probes Table 1.http://www.ualberta.ca/∼fyeh/fyeh). Linkage analyses among different loci were performed with Haploview 4.2 .The allele frequencies, heterozygosity, and polymorphism information content were calculated using PopGene v1.31 software . However, the difference in luciferase activity was not statistically significant between pGL3-721 and pGL3-1373 (p > 0.05), which revealed that the location of the core promoter region of the ovine NR5A2 gene ranged from -721 to -281 bp  and TT . The litter size of Hu ewes at their third parity with genotype GG (2.10 ± 0.10) was significantly higher than that of ewes with genotype TT , and there was no significant difference between the litter sizes of ewes with genotypes GG (2.10 ± 0.10) and TG  (NR5A2 gene was involved in regulating the litter size of Hu sheep.SNPs in the  > 0.05) Table 2.NR5A2 gene, constructs of pGL3-T and pGL3-G were transfected into 293T cells, and the results showed that luciferase activity of plasmid pGL3-G was significantly higher than that of plasmid pGL3-T in 293T cells (p < 0.05) (To investigate whether the -700T/G polymorphism affected promoter activity of the  < 0.05) Figure 2We predicted that the -700T/G polymorphism would create a novel binding site for transcription factor MTF-1. A competitive EMSA showed that when nuclear extracts of ewes’ ovaries were incubated with a bio-probe, there was a significant DNA–protein complex, and when nuclear extracts were incubated with a bio-probe and mut-probe at the same time, the DNA–protein complex was still present. However, when nuclear extracts were incubated with a bio-probe and cold probe at the same time, DNA–protein complex significantly decreased with the cold-probe concentration increased. These results suggest that MTF-1 binds specifically to sequences with -700GG Figure 3NR5A2 gene expression, the plasmid pcDNA3.1-MTF-1 was transfected in sheep granulosa cells. The qRT-PCR results showed that the MTF-1 expression levels were higher in sheep granulosa cells transfected with plasmid pcDNA3.1-MTF-1 (p < 0.01) (NR5A2 gene increased after pcDNA3.1-MTF-1 transfection (NR5A2 gene expression in the ovarian granulosa cells of Hu sheep.To determine whether MTF-1 was involved in regulating  < 0.01) Figure 4pcDNA3.1-MTF-1 was co-transfected with pGL3-T or pGL3-G into 293T cells. The promoter activity assay showed that the overexpression of MTF-1 significantly enhanced the luciferase activity of the -700G promoter genotype (p < 0.01), but the -700T promoter genotype did not have a significant effect on luciferase activity (p > 0.05) (NR5A2 was associated with ovulation and litter size in Hu sheep (NR5A2 gene. Polymorphisms of NR5A2 are associated with preterm birth in humans (p < 0.05) and TT (p < 0.05). Moreover, ewes with genotype GG had a significantly greater litter size at the third parity than those with genotype TT (p < 0.05). By combining all three polymorphisms  of the NR5A2 gene, we found that ewes with the GG/TC/TC haplotype have higher litter sizes in the second and third parities than those with other haplotypes  may create a new binding site of transcription factor ARNT, which could enhance HOMER1 promoter activity and expression. Promoter haplotypes of the ABCB1 gene encoding the P-glycoprotein differentially affects its promoter activity by altering the transcription factor binding .YL is responsible for the design and the writing of the whole article. JZa is responsible for SNP screening. YQ is responsible for samples collection. CM is responsible for real-time PCR. HW is responsible for cell assay. SC is responsible for EMSA assay. JZo is responsible for the modification of the whole article.The funding is from the National Natural Science Foundation of China, and the number is 31501934. The title of this funding is the Molecular Mechanisms of -700T/G Mutation Regulating the Transcription of NR5A2 and High Fecundity in Hu Sheep. This paper is completely consistent with this funding, and this funding includes the publication fees. Another funding is Agricultural Science and Technology Innovation of Jiangsu Province, the number is CX(2018)3004.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pyruvate Kinase M2 (PKM2) mediates metabolic reshuffling and is ubiquitously upregulated in several cancer types. The non-metabolic function of PKM2 as key nuclear kinase and modulator of gene expression is instrumental in cancer progression and tumorigenesis. Here, we attempt to discern the non-canonical function of PKM2 as an epigenetic modulator and the underlying implication of this activity. Using 5’-FAM labelled reconstituted mononucleosome we have shown that PKM2 interacts with the complex through Histone H3 and possibly obstruct the access to DNA binding factors. Subsequently, the interaction negatively impacts the ATP dependent remodeling activity of Chromodomain Helicase DNA binding protein-7 (Chd7). Chd7 remodeling activity is required to ameliorate DNA damage and is crucial to genome stability. Our study shows that PKM2 blocks the Chd7 mediated sliding of nucleosome. It can be conjectured that stalling Chd7 may lead to impaired DNA damage and increased genomic instability. We propose a mechanism in which PKM2 negatively regulate nucleosome repositioning in chromatin and may exacerbate cancer by altering the nucleosome architecture. This research is imperative to our understanding of how altered cancer metabolism can potentially modulate the gene expression and sustain incessant proliferation by tweaking the chromatin topography. Metabolism and gene expression are stringently regulated physiological process that are concurrently modulated by a wide array of enzymes and regulatory protein. The concerted control mechanism exerted by the milieu of governing factors lead to dynamic regulation of the cellular machinery and contribute to incessant cell proliferation and tumorigenesis . Metabol−1 , 150 mM NaCl, 3 mM EDTA, and 0.05% NP-40 surfactant. The association kinetics for PKM2 was monitored for 400 s, followed by dissociation kinetics for 300 s. Different concentrations of Histone H3 (100 nM- 1000 nM) diluted in running buffer were injected across the sensor surface. SPR signals and resultant sensogram for PKM2-Histone H3 interactions were analyzed. Resultant sensogram was analyzed by subtracting change in the signal from activated/blocked control panel from that obtained as a consequence of binding of histone H3 to PKM2. At last the surface was regenerated with 75 mM NaOH. All the data were recorded at room temperature . Autolab SPR Kinetic Evaluation software was used for data analysis.2, 5% sucrose, 0.1mg/ml of BSA, and 1 mM DTT.500 nM of PKM2 was incubated with 100 nM of FAM-labeled nucleosome on ice overnight. Buffer composition was as 20mM HEPES buffer, pH 7.6, 50mM KCl, 5mM MgClVarying concentration of ExoIII enzyme was added to the 10ul of reaction mixture. After incubation at 23 °C reaction was terminated with 2X stop buffer containing 20mM EDTA and 2% SDS and addition of ~20μg of glycogen. DNA was extracted using phenol–chloroform–isoamyl alcohol (25:24:1). The samples were subjected to denaturing electrophoresis in a 7% polyacrylamide—7 M urea gel in 1X TBE buffer. The gel was scanned by using Typhoon Phosphorimager FLA9000 .Nucleosome sliding experiment was performed using an electrophoretic mobility shift assay (EMSA)-based method as described previously –47 with GCG⌃C has been inserted at dyad position 73 after each 208 bp and is usually obscured in nucleosome. Remodeling results in exposed restriction site and subsequent digestion of the DNA. To summarize 5nM of the pJ201 (34 × 601) chromatinized plasmid was incubated with 50 nM of Chd7 remodeler and 2.5mM of ATP with or without PKM2 at 23°C for 120 mins. Buffer used was 20mM HEPES, pH 7.6, 50mM KCl, 5 mM MgCl2, 1 mM EDTA, 1mM DTT, 0.1 mg/ml of BSA. Post incubation restriction digestion was performed using 1 U of HhaI enzyme for a time period of 30 min. The reaction was allowed for the defined time and stopped using Glycogen stop buffer . Samples were loaded on to 1.4% agarose gels. Electrophoresis was carried at 120V for 2hrs and gel was stained with ethidium bromide [To study the effect of PKM2 on nucleosome accessibility at chromatin level restriction digestion of pre-assembled nucleosome array of plasmid was carried out using HhaI enzyme. HhaI digestion site  bromide .kd value of very stable interactions is difficult and at the same time might be misleading under mild or physiological conditions. The complex decay in such cases is extremely slow and kd value should be only a qualitative measure and not quantitative. We can conclude from the aforementioned binding parameters that immobilized PKM2 interacts with Histone H3 and there is a gradual increase in the response unit with increase in Histone H3 concentration. A kinetic equilibrium is obtained represented by plateau in the association phase of the plot. Affinity Constant (KD) value for the binding kinetics is 6.39E-06M (Affinity error 3.20E-07) and calculated from the equilibrium equation. The obtained value is mean of the experiment repeated in triplicate. However the rate of complex decay is remarkably low as we do not observe any significant dissociation of the PKM2-Histone H3 complex suggesting that some other factor might be needed to break the association between the two biomolecules. This dissociation phase kinetics is interesting and needs to be further investigated. Fluorescence spectroscopy experiment using FITC labelled PKM2 and Histone H3 further validate the interaction between the two proteins. Interestingly we observe a decrease in the fluorescence intensity of labelled PKM2 when titrated with increasing concentration of Histone H3. With subsequent increase in the Histone H3 concentration in the PKM2 microenvironment the fluorescence is gradually quenched and a steady depreciation in the FITC fluorescence intensity is recorded which finally becomes stabilized and no further change in the intensity corresponding to higher concentrations of Histone H3 is observed 2 tetramer and two units H2A–H2B dimers onto the Widom 601 nucleosome positioning sequence DNA is critical for the nucleosome assembly. Microscale reconstitution by employing Salt gradient dialysis with 1:1.5 DNA-to-octamer ratio as previously determined and subsequent titrations by varying the molar ratio PKM2 envince that nucleosome reconstitution can be possibly altered by PKM2 interaction. As can be inferred from native PAGE electrophoresis at a 1:2.4 PKM2-to-octamer ratio PKM2 interacts with the nucleosome and forms a complex of higher molecular weight , Lane 2 observed . The DisExonuclease III possess the phosphomonoesterase activity and catalyzes the 3' exonucleolytic digestion of the duplex DNA. The activity is hindered by the DNA-histone interaction and at regulated low levels of ExoIII, digestion of the nucleosome monomers yield a compact core of 145 bp which defines the boundary of the nucleosome. On extensive ExoIII digestion the nucleosome core is further chopped to yield discrete bands that are integer multiples of 10 bases signifying sequential disruption of the DNA–histone contacts at every turn of the helix with the advance of ExoIII . We usedAs evident from the restriction digestion assay PKM2 interacts with nucleosome and reduces the accessibility to the complex. We were interested in exploring the possible outcomes of this finding and how it might contribute to altered gene expression. Chd7 is a member of the SNF2-protein superfamily and is reported to play a role in DDR (DNA Damage Repair). To cite as per the reports any aberrant expression or somatic mutations of Chd7 often in many cases may lead to cancer. Chd7 exhibits an intrinsic ATP-dependent nucleosome remodeling activity by sliding the histone octamer from one end of the DNA fragment to the centered position . This suSince DNA exist as chromatin in the nucleus and DNA-directed processes have majorly evolved with the chromatin  we furthTo dissect the impact of PKM2 on nucleosome positioning in a chromatin array and investigate if it could hinder Chd7 driven remodeling we used 34-mer nucleosome array for our study. The pJ201 (34 × 601) construct has restriction site for HhaI at the dyad position. The 34-mer reconstituted chromatin when digested by HhaI do not show any digested fingerprint , lane1 sHere, we report that PKM2 potentially acts as a modulator of chromatin remodeling activity by directly interacting with chromatin and thus contribute to oncogenesis. We highlight a rather unaddressed role of PKM2 that may be pivotal in exacerbating genomic integrity and trigger cell proliferation. The metabolic functions of PKM2 as key catalytic enzyme of glycolytic cycle and regulator of the energy flux in a cell is well established. However, the non-metabolic functions of PKM2 and its various aspects need to be discerned. The oligomeric switch as a consequence of oncogenic signal mediated post-translational modification subsequently facilitate nuclear translocation. Using fluorescence labelled NCP (nucleosome core particle) and 34-mer chromatin array we have demonstrated direct interaction of PKM2 with the nucleosome complex. We examined the possible interaction of PKM2 through nucleosome reconstitution. Gel mobility shift assay result of the reconstituted complex validate the potential binding of PKM2 to nucleosome. It can be inferred that PKM2 may have a role to play in nucleosome assembly and thereby dictate a likely control over gene expression. However the implications of this interaction is immense and needs to be further explored. Intuitively the nature of this interaction was further verified by establishing an evident association of PKM2 with Histone H3. Employing native PAGE gel electrophoresis we confirmed that PKM2 binds with Histone H3 and there is a possible interaction with Histone H4 (as seen in the native PAGE) which was although not as promising as Histone H3. The possibility of interaction with Histone H4 is a subject of study and further interpretation.Our finding was consistent as confirmed by SPR and fluorescence spectrometry experiments. This study was further extended to the validation of 34-mer chromatin array emphasizing the veracity of this interaction to the chromatin level. The results further prove the possibility of wider involvement of PKM2 in the chromatin structural organization and the downstream implication it may exert on gene regulation.Nucleosome restrict the accessibility of transcription factors and impede transcription and other chromatin transactions. This can be attributed to the 14 contact points between DNA and the histone octamer. However, protein complexes like chromatin remodelers can dynamically reposition and slide nucleosome to alter gene expression suggesting multi-layered regulation To dissect the impact of PKM2 on nucleosome accessibility we performed Exonuclease III digestion of PKM2-Nucleosome complex. Constrained digestion of nucleosome at low concentration of enzyme yield a nucleosome core with 145 bp wrapped around it which is further cleaved with the increase in enzyme units. However, in case of PKM2 bound nucleosome it is remarkable to observe from the lack of cleaved bands on the denaturing gel that digestion cannot proceed and the enzyme activity is significantly compromised. Results suggest that PKM2 probably impose a barrier to the remodeling enzymes, insulate nucleosome and obstruct access.As previously noted, in presence of nucleosome, transcription machinery face a roadblock and the highly coordinated process of gene regulation is impaired. Several chromatin remodelers relieve this transcriptional blockade by facilitating nucleosome sliding. CHD family of remodelers harness the energy generated from ATP hydrolysis to alter the chromatin structure. The interplay between the repair machinery and the remodeler is crucial to gain an access to DNA double-strand breaks (DSB) and prevent genomic instability . Chd7 isNucleosome mapping reactions with HhaI using 34-mer pre-assembled nucleosome array imply that PKM2 impair remodeling in chromatin without altering the structure. In contrast to chromatin digestion in the presence of the ΔChd7 which was on account of the exposed HhaI site at the dyad position we fail to observe any digestion in presence of PKM2. This was evidently on account of inhibition of nucleosome sliding.To summarize, we have shown that PKM2 can directly interact with the nucleosome through Histone H3 and obscure the chromatin from undergoing epigenetic modifications catalyzed by remodeling complexes. The basis of this prediction was evident from the suspended remodeling activity of Chd7 in the presence of PKM2 which can possibly lead to dysfunctional DDR machinery and genomic instability . As per In a cancer cell metabolic overhaul often commensurate with dysfunctional metabolic enzymes that synergistically impact the epigenetic blueprint and promote altered gene expression. In this research article we report a so far unaddressed role of PKM2 as a modulator of chromatin remodeling activity. Our study show that PKM2 can directly interact with nucleosome through Histone H3 and inhibit Chd7 mediated remodeling. At present non-metabolic functions of PKM2 is an intensely researched domain and its role in cancer progression and tumorigenesis is widely being acknowledged. Besides acting as a key nuclear kinase it is known to regulate the expression of several oncogenes. Here, we attempt to answer how PKM2 can directly alter the expression of genes by hindering nucleosome positioning and inhibiting remodeling. Exonuclease III digestion of Pyruvate Kinase M2- NCP complex suggest that PKM2 obscure nucleosome and create a roadblock for the Chd7 remodeler and thereby impede nucleosome sliding. Chd7 is implicated in a number of developmental disorder and is reported to play a role in cancer as well. As an important factor for DNA Damage Repair machinery any impairment in its functions can have detrimental implications. It can be inferred that PKM2 by negatively modulating Chd7 catalyzed chromatin remodeling may probably exacerbate genomic instability and in turn contribute to cancer. Through this study we aim to highlight the impact of PKM2 on chromatin architecture and its impact on the epigenetic landscape of the cell.S1 Fig(A) Interaction of Chd7 with PKM2 with increasing molar concentration of PKM2. (B) Interaction of PKM2 with Chd7 with increasing molar concentration of Chd7.(PDF)Click here for additional data file.


Transcription factors are involved in a large number of human diseases such as cancers for which they account for about 20% of all oncogenes identified so far. For long time, with the exception of ligand-inducible nuclear receptors, transcription factors were considered as “undruggable” targets. Advances knowledge of these transcription factors, in terms of structure, function  and the dynamics of their mode of binding to DNA has changed this postulate and paved the way for new therapies targeted against transcription factors. Here, we discuss various ways to target transcription factors in cancer models: by modulating their expression or degradation, by blocking protein/protein interactions, by targeting the transcription factor itself to prevent its DNA binding either through a binding pocket or at the DNA-interacting site, some of these inhibitors being currently used or evaluated for cancer treatment. Such different targeting of transcription factors by small molecules is facilitated by modern chemistry developing a wide variety of original molecules designed to specifically abort transcription factor and by an increased knowledge of their pathological implication through the use of new technologies in order to make it possible to improve therapeutic control of transcription factor oncogenic functions. DOT1L cZ004777) ,46. ConsZ004777) ,49,50 anZ004777) . -The macrocyclic peptidomimetic MCP-1 ,-The thienopyrimidine MI-2-2  and its -50 for interation to menin of 390 nM indoles 1 and its N-substituted hydrazides 9b, 9c and 9e  and the hydronaphthoquinones S01, S05 and S47 [g]indole skeleton in compounds 1, 9b, 9c and 9e series induces a 5- to 10-fold decrease of Nrf2/Keap1 PPI inhibitory constant Ki and 10-fold increase of the compound metabolic stability as evidenced using human liver microsomes, compound 9e being the most stable compound -thiophene dioxide, dibenzylidenecyclohexanone derivatives and naphthalene-5,8-dione-1-sulfonamide  was developed and showed interesting preclinical evaluations in DU145 prostate cancer xenograft model -2-oxo-5-phenylpyrrol-1-yl] benzoic acid  Consequently, InS3-54 treatment of human breast and lung cancer cell lines results in apoptosis but also in reduced cell migration and invasion, in correlation with reduced expression of the STAT3 downstream target MMP-9. InS3-54 also inhibits STAT3 binding to cyclinD1 (CCND1) promoters in ChIP experiments and reduces tumor growth and metastasis -bis(methylene)]-bis2+ against the interaction of PUrine-rich box-1 (PU.1)/SFFV Proviral Integration Site-1 (SPI1), an ETS-family member transcription factor, to its minimal cognate ETS-family-core binding site 5′-GGAA/T-3′ + is able to disrupt SP1 and TFIID DNA binding through its interaction with the major groove [2]3+([Rh(phen)2(phi)], + pyrene, 50 ≈ 40 μM in order to inhibit CREB binding to CRE-containing DNA but is however specific as demonstrated with other transcription factors  which interaction with their respective cognate sequences could not at all be disrupted at the highest evaluated concentration of drug (80 µM) -1,3-dimethylthiourea] that evidenced TBP/DNA binding inhibition activity (NOactivity . The lac ligands . The nat ligands  that eff ligands . Tanshin ligands . Tanshin ligands .Today most developed compounds for transcription factor/DNA binding inhibition are mithramycins (MTMs). Indeed, even if their specificity for DNA sequences and their affinity to those sequences are not the best one from all minor groove binding compounds presented here, those compound and derivatives entered clinical trials. MTMs represent a family of natural antibiotics isolated from different Streptomyces species such as MTM-A  or synthMTMs were first as a protein/DNA inhibitor of the SP1/3 family of transcription factors ,385 to cMithramycin was also evidenced as an inhibitor of the EWS-FLI1 fusion transcription factor activity but without evidencing if it comes directly from inhibition of EWS-FLI1/DNA binding or indirectly from inhibition of SP1/DNA binding . ConversBecause it controls genes associated with many pathways as evidenced above, the cellular consequences of MTMs treatment are also diverse and correspond to various ways to target the different hallmarks of cancer as defined by Hanahan and Weinberg : anti-prFinally, chromomycin A3  is anothCancer is a consequence of multiple deregulated processes first defined as the “Hallmarks of Cancer” by Hanahan and Weinberg two decades ago, implicating six main processes, now revised more recently to ten different processes ,411. BecAmong the various strategies presented in this review , all present advantages and disadvantages that are moreover to see in light of the nature and function of each transcription factor. About the nature of the target transcription factor, the reactivation of mutated p53 is an interesting model to highlight how the complexity of cancer biology interferes with drug discovery. Indeed, mut-p53 cannot be considered as a unique entity since many different point mutations (single or multiple) affect this transcription factor function leading to cancer. As a matter of fact, hotspot and minor mutations could occur in the DNA binding domain of mut-p53 protein affecting only its conformation  or together with p53 interaction with DNA  . Mut-p53Furthermore, transcription factors activate or repress wide repertories of downstream target genes which differ depending on cancerous or normal cell type. Identifying an oncogenic signature from normal function repertories of genes driven by this transcription factor is not always easy due to some redundancy and the difficulty to establish the threshold of expression associated with the oncogenic process. GGAAGT-3′ followed by an additional T base , corresponding statistically to ¼ of the total of ERG cognate sequence on the genome. Such specificity may be limitated (considering that only a potion of ERG-driven genes could be deregulated) or may offer the opportunity to modulate more precisely ERG-driven genes assuming that they are associated with the oncogenic process. Identifying such set of deregulated genes depending not only from the presence of an ERG cognate sequence but also of the surrounding bases through bioinformatics tools would be helpful for future development of such type of compound.Moreover, for DNA ligands, DNA recognition context is important as illustrated with DB1255. Indeed, DB1255 recognizes only the ERG binding site 5′-In a general manner, a better knowledge of the precise modalities of transcription factor/DNA or co-factor binding would take benefit of global analysis using high throughout analysis and next-generation sequencing  of these transcription factors and of their different co-factors in the context of each tumor cell type, particularly from comparison with normal cell context and would be helpful for future design of transcription factor inhibitors. Futhermore, transcription factors could interact with some epigenetic modifiers and their cooperation regulates gene expression pathways that are responsible for the cellular oncogenic phenotype . A betteMoreover, the development of specific transcription factor inhibitors against cancer would have to take into account compensation phenomena between different transcription factors that may be very close to each other in the same family. Finally, the dynamics of transcription factor/DNA or co-factor interaction process have to be further elucidated to open new opportunities to develop inhibitors that would block transcription factor 3D orientation in an active or an inactive structure. Altogether, if the inhibition of transcription factor to treat cancer is already a part of the current anti-tumor pharmacopoeia, it would surely be improved in the future and opened to other pathologies such as genetic or inflammatory diseases, diabetes, Parkinson and Alzheimer diseases.


Telomeres are unique nucleoprotein structures. Found at the edge of each chromosome, their main purpose is to mask DNA ends from the DNA-repair machinery by formation of protective loops. Through life and cell divisions, telomeres shorten and bring cells closer to either cell proliferation crisis or senescence. Beyond this mitotic clock role attributed to the need for telomere to be maintained over a critical length, the very tip of our DNA has been shown to impact transcription by position effect. TPE and a long-reach counterpart, TPE-OLD, are mechanisms recently described in human biology. Still in infancy, the mechanism of action of these processes and their respective genome wide impact remain to be resolved. In this review, we will discuss recent findings on telomere dynamics, TPE, TPE-OLD, and lessons learnt from model organisms. Drosophila melanogaster fruit flies, they observed that mutations never occurred at the end of the DNA molecule suggesting the presence of a protective cap restricted to these regions. Since then, explorations and hypothesis on their complete function never ceased within the scientific community and beyond .Unlike circular DNA found in prokaryotes, eukaryotes share minisatellite sequences at the end of linear chromosomes named telomeres. The importance of this particular structure was first demonstrated in the late 1930s by McClintock and Muller [Tetrahymena ciliate model, which possess hundreds of mini telomeres, with the identification of its nucleotide composition [Major findings on the organization of telomeres were obtained in the position  and the position ,4,5.Thanks to the increased sensibility of techniques, the human telomeric sequence composed of a 5′-TTAGGG-3′ repeated motif was later identified  followedThe genetic information of eukaryotes is assembled in a linear genome. Hence, the first major role of DNA ends is to protect informative genetic sequences from erosion. Free DNA extremities are exposed to exonuclease and at risk for non-homologous ends joining between DNA molecules . These rFrom cancer research to aging studies, investigations on the regulation of the very tip of chromosomes led to paradoxical observations: (i) telomerase, the enzyme responsible for telomere elongation is not produced in somatic cells, (ii) most cancer telomerase-positive cells display rather short telomeres as telomerase upregulation is considered a late event during malignant transformation; (iii) stem cells harbors long telomeres and express telomerase; (iv) short telomeres are associated with aging and increased cancer risk, or the complete opposite; (v) telomere maintenance involves epigenetic mechanisms and chromatin changes. Altogether, the wealth of studies available in the literature raises questions on the relevance, feasibility, and caveats that one encounters when questioning telomeres dynamics. In the end, does telomere length and size matter?In this review, we will discuss the past decades of telomere research by crossing lessons on telomere length regulation and impact in Human. We will first focus on telomere length dynamics before developing on consequences at the epigenetic level and possible implication on human health through position effect mechanisms. As previously mentioned, DNA extremities are lost through cell divisions. This mechanical DNA erosion is the consequence of incomplete duplication of the lagging strand  during synthesis. Indeed, during replication, the DNA polymerase fails to generate a completed 3′ end in the absence of RNA primer ,24. TeloSingle stranded DNA or free double stranded extremities are toxic for the cell. The shelterin complex protects telomeric DNA to prevent recognition as damaged DNA . Defect For a long time, telomere erosion has been associated with the proliferative state of tissues . However, a recent cross study exploiting samples from 87 well-characterized individuals did not sustain this dogma . Indeed,Telomere attrition is involved both in normal and pathological aging whereas re-activation of telomerase is able to revert aging in mice  or in viWhere telomere shortening can be seen as a tumor-suppressor pathway through replicative senescence , the line between tumor-suppressing and tumor-promoting is thin. At senescence, cell proliferation is inhibited. In the presence of cancer-initiating changes, senescence can be bypassed and cell lifespan extended, a stage called crisis. During crisis, signals to undergo replicative senescence or continue cell divisions are in balance with chromosome end-to-end fusions, accumulation of chromosome abnormalities and massive apoptosis . To acquhTERT gene is 42 kb long and located in the subtelomeric region of the short arm of chromosome 5 (5p13.3). TERT promoter is unconventional with a complex regulation dynamics still under investigations [TERT promoter mutations, including recurrent mutations identified in 19% of cancers [Telomerase is the specific enzyme that allows such elongation, both by providing the template and catalytic unit. It is composed of the hTERT (human telomerase reverse transcriptase) catalytic subunit and the hTR (human telomerase RNA), a RNA component serving as a primer and template for DNA synthesis. The igations ,42. Reacigations . In the  cancers .Telomeres can also be maintained in cancer cells through the telomerase-independent ALT pathway. Human ALT tumor cells exhibit specific characteristics including heterogeneous telomere length, presence of extrachromosomal telomeric repeats (ECTR), C-circles, ALT-associated promyelocytic leukemia (PML) bodies (APBs) and elevated frequency of telomere sister chromatid exchanges (TSCEs) . ALT is Zscan4 gene. Its activation is associated with rapid telomere extension by telomere recombination [As in cancer cells, telomeres need to be finely regulated in stem cells, a cell type where telomerase is constitutively expressed albeit to a lower extent. In pluripotent stem cells (PSCs), in addition to being essential for the maintenance of telomere length, telomerase is required for long-term self-renewal and pluripotency . Use of bination . Human ebination . These dAdult stem cells are long-lived and actively cycling stem cells. The importance of telomerase in adult stem cells compartment is directly observable in patients suffering from a number of syndromes associated with dysfunctional telomeres that will be discussed below.It is now widely admitted that telomere length is variable from one individual to another and even variable between each chromosome and their respective arms within one cell. This observation raises questions on telomere length heritability for the 92 telomere ends. Are they partially or totally inherited? Does the age of the parents  matter? Similarly, does the parental overall telomere length matter? If answers can be grasped from a few studies including some focusing on twins, work on these fascinating questions remains to be done.Using Fluorescence In-Situ Hybridization (FISH), a group had previously shown some differences in telomeres length between homologous chromosomes , suggestMore recently, parental contribution to inherited telomere length has been proposed. A meta-analysis on nearly 20,000 participants revealed strong correlation between paternal age at conception (PAC) and offspring telomere length. Sperm telomere length increases with age in humans. As a result older fathers’ offspring inherit longer telomeres . In a moThese different studies show heterogeneity in the length of telomeres between individuals, either because of genetic factors inherited from the parents, age of the father at conception or because of environmental factors specific to each person/generation with a theoretically minimal impact for the later . In thiAs mentioned previously, critical telomere length and sensitivity to DNA damage signaling has been restrained to a small subset of chromosome ends ,32. Thoscis  [trans, through TRF2 interactions  [cis and trans interactions at play in their respective territories.For a long time, chromosome organization within the cell was thought to be random. In mammalian cells, the exact opposite is now well established ,60. Chroa, BAF1) ,64,65 an, LAP2a) ,67. Sinc, LAP2a) . Indeed,, LAP2a) . HoweverChromosome folding and subnuclear localization are also influenced in response to gene replication . ConsideIn a human model of immortalized cells,  there was no change in the global replication profile of telomeres throughout the immortalization process . This co2AG3)n motif, mostly in link with the possibility of development of anti-oncogenic therapies targeting telomerase. Thus, an integrative vision of telomeres within its 3D environment remains to be established to further understand the consequences of nuclear localization on higher-order telomere organization and consequences on transcription or DNA repair of chromosome ends , a long-reach counterpart, TPE-OLD (Telomere Position Effect Over Long Distances) and TERRA (Telomeric Repeat-containing RNA) have been recently described in Human ,76,77. S2AG3)n motifs [Mammalian chromosome ends are enriched in constitutive heterochromatin marks such as di- and trimethylation of H3K9, trimethylation of H4K20 and recruit heterochromatin protein 1 (HP1) isoforms in a telomere length-dependent manner . Thanks n motifs ,80. The n motifs , telomern motifs . ReciproSaccharomyces cerevisiae [Telomere length also alters expression of genes located in subtelomeric regions through TPE. This mechanism first revealed in Drosophila was extensively described in revisiae . By placrevisiae  , first described in 1930 in nogaster . This sinogaster .The capacity of heterochromatin marks to spread along chromosomes suggested the existence of similar mechanisms at the level of telomeric heterochromatin and lead to the identification of TPE. The first in vivo evidence came from a replication timing assay on chromosome 22 with a heterozygous microdeletion of 130 kb at the end of the 22q arm . The micNot all subtelomeric genes are susceptible to TPE suggesting the existence of subtelomeric sequences able to counteract TPE by acting as boundaries. Insulators and boundaries are DNA element able to alter gene expression by preventing activation or inhibitory effects due to the chromatin environment . The firISG15 (Interferon Stimulated Gene 15 kDa) as the first TPE gene up-regulated upon telomere shortening. These findings were consistent with yeast TPE mechanisms. In this specie, most subtelomeric genes are involved in metabolism and stress response and are silenced in optimal growth conditions [ISG15 gene product is involved in the innate immunity response pathway suggesting a conservative role of TPE, as a stress-response and adaptation mechanism [Indeed, in human, evidence for the impact of telomere length was verified in young and old cells (with respectively short and long telomeres) by transcriptomic analysis . This wonditions . These gnditions  or FLO inditions , nutrienechanism .Interestingly, 15 genes are present between this gene and the telomere of the short arm of chromosome 1. None of these genes showed telomere length-dependent regulation, a contradiction with the TPE rule, advocating for higher complexity in the regulation of this mechanism.trans-acting changes occurring elsewhere in response to telomere shortening  transgene flanked by LoxP sites were made [TERT can be excised at different time points to generate isogenic clones in which the only significant difference is the length of telomeres after TERT removal .The major difficulty when looking at telomere length-dependent mechanism is the capacity to impute gene expression changes to telomeres only. To overcome this limitation, reversibly immortalized cell lines expressing a ere made . After cThis system was applied first to primary myoblasts and fibroblasts. Comparing myoblasts with long and short telomeres, 144 telomere length-sensitive genes were identified, amongst the 1423 subtelomeric genes analyzed. These genes were located as far as 10 Mb from the telomere. Analysis of long-distance interactions revealed the existence of loops between TPE-sensitive gene loci and long telomeres disrupted upon telomere shortening. This looping mechanism was dubbed TPE-OLD for TPE over long distance .To test whether changes in the higher-order organization of TPE-OLD genes correlate with epigenetics modifications, ChIP assay were used and revealed enrichment in H3K9me3 marks and, in the TRF2 shelterin component at the promoter of TPE-OLD genes indicating a possible cooperation between chromatin marks and telomeric factors in telomere-dependent gene silencing. TRF2 is specifically enriched at TPE-OLD sensitive genes, decreased upon telomere shortening and absent at the promoter of genes located within the loops. This suggests a gene-specific effect more than a global spreading of telomeric chromatin and heterochromatin marks.Recently, a separate group explored telomere length influence through ChIP-Seq assays . While cTPE-OLD has been involved in other mechanisms not linked to telomere attrition but as an active mechanism involved in the regulation of genes during physiological responses , cancer For years, the longstanding belief was that telomeres are transcriptionally silent. Recent evidences show that telomeres are transcribed by RNA polymerase II to give rise to a class of long non-coding RNA containing telomeric repeats called TERRA (Telomeric Repeat-containing RNA) ,103. Thecis or trans and suggested to interact with telomerase hence leading to different activities when occurring at telomeres or short/damaged telomeres [Telomeric RNAs are also implicated in R-loops formation which are RNA:DNA hybrids in which TERRA act either in elomeres ,110,111.elomeres .Telomeropathies usually refer to diseases caused by defects in factors involved in the telomere maintenance machinery such as Dyskeratosis congenital, Revesz, Coat Plus or Hoyeraal Hreidarsson syndromes ,114,115.Chromosomal translocation of region encompassing whole gene loci and thus implicating modification of the chromatin environment in gene expression modulation were observed in numerous genetic diseases. In the context of these so called “position effect mutations” modification in gene expression can be the consequence of two mechanisms. First, the chromosomal abnormality can separate the coding sequence from its regulating enhancers or silencers. Second, in case of translocations, the gene can be delocalized to a region harboring a different chromatin state. In both cases, a gene normally repressed can be activated and vice versa . In the Non-physiological modification of telomere and chromosome ends integrity has also been associated to a number of diseases. Some patients diagnosed with various malformations, mental impairment, and growth retardation display formation of a ring chromosome. Such structures can occur on any autosomes and are formed by deletion on the short and long arms of the chromosomes followed by fusion at the breakage point. Consequences of ring chromosomes depend on different parameters: the chromosome implicated, the number of genes deleted, and the stability of the ring chromosomes. Most ring chromosomes are associated with loss of genetic material but in some rare cases, only telomeric regions are lost resulting in fusion of residual telomeres from both ends, forming a single telomere in the ring. This mechanism has been observed in ring 14, 17, and 20 syndromes ,122,123.DUX4 activation.One of the best characterized human genetic diseases implicating TPE is Facio-Scapulo-Humeral Dystrophy (FSHD) . A partihTERT transgene [DUX4 expression was upregulated upon telomere shortening in FSHD cells, with a progressive effect as telomeres shorten, long before replicative senescence. TPE also extended to the upstream FRG2 gene, located 70 kb more centromeric to DUX4, but was less prominent and not observed for FRG1 located 90 kb upstream of FRG2. These observations uncovered FSHD as the first disease linked to classical TPE [FRG1 and SORBS2 locus were also identified in myoblasts from FSHD1 patients [SORBS2 upregulation [SORBS2 gene is silenced in normal cells by TPE-OLD mechanisms while this telomere-dependent mechanism is impaired in FSHD myoblasts with cooperation between the telomere and the repetitive macrosatellite array [In this disease, the impact of telomere shortening was investigated in immortalized myoblasts expressing a floxable ransgene ,90. In mical TPE . Using Hpatients . Interesgulation . This sute array ,126.DNMT3B (ICF1), ZBTB24 (ICF2), CDCA7 (ICF3), and HELLS (ICF4) [Another example of rare a disease involving telomeric changes is the immunodeficiency, centromeric region instability, and facial anomalies syndrome (ICF). ICF arises due to mutation in four different genes, S (ICF4) ,129,130.S (ICF4) ,132, theS (ICF4) . This agLMNA gene encoding A-type Lamins [Furthermore, in agreement for a role of telomere subnuclear positioning in telomere regulation, telomeric defects are also observed in premature aging syndrome such as Hutchinson Gilford progeria linked to mutation in the e Lamins . In thise Lamins ,136,137.e Lamins .Telomere length is thus at the corner of different biological mechanisms but also associated to a broad range of diseases, from rare genetic syndromes to common ones. As we previously described, telomere shortening occurs normally during aging. This information has been broadly disseminated in the general population with an increasing number of newspapers mentioning the links between aging and telomeres length. As a consequence, numerous private companies offer telomere length measurement with the promise to “help you stay younger longer”, not mentioning cures or food supplement protecting telomeres.A study published in 2009 aimed at establishing correlations between telomere length, causes of death and years of healthy life (YHL) by applying different statistical analysis on large population cohorts to prove correlation between these three factors . The onlIf one can criticize the statistical tests power, we now know the extreme variability of telomere length among individuals. Such studies validated telomere length as a good biomarker for healthy aging but interpretation must be taken with caution given the number of additional factors to be considered.Topics on aging never get old and telomere biology is no exception. Telomere epidemiology studies already revealed that we are not “telomerically” equal and discoveries on telomere length regulation, heritability, dynamics and association with aging, raised interest on the topic to a general audience, from journalists to insurance companies. This peculiar part of our DNA has been at the center of interest for the past 80 years. In this context, it falls under the responsibility of the scientific community to convey a balanced message regarding the implication of telomere homeostasis in diseases and wellness, considering that numerous additional factors must be considered. Indeed, in human, the clear-cut influence of telomeres, beyond the well-described effect of critical shortening, remains unclear and record on telomere shortening rate at specific telomere-ends are missing, leaving the former observation to potential biased conclusions. Moreover, the recent identification of long non-coding RNA (TERRAs) produced by telomeres and the long-distance impacts of telomeres on gene regulation represents a new challenge. Thanks to the rise of high-resolution methods  and accessibility to affordable deep sequencing platforms , future research will help deciphering subtelomere complexity, likely uncover novel pathways linked to telomere biology and opens new research opportunities to understand the genome wide impact of telomere in physiological and pathological situations or adaptation to environmental cues. In this context, if all agree that telomere matters, questions on their influence, weakness and strength linger.