We report the whole genome ChIP seq for human TOP2B from MCF7 cells. Using three different peak calling methods, regions of binding were identified in the presence or absence of the nuclear hormone estradiol, as TOP2B has been reported to play a role in ligand-induced transcription. TOP2B peaks were found across the whole genome, 50% of the peaks fell either within a gene or within 5 kb of a transcription start site. TOP2B peaks coincident with gene promoters were less frequently associated with epigenetic features marking active promoters in estradiol treated than in untreated cells. Significantly enriched transcription factor motifs within the DNA sequences underlying the peaks were identified. These included SP1, KLF4, TFAP2A, MYF, REST, CTCF, ESR1 and ESR2. Gene ontology analysis of genes associated with TOP2B peaks found neuronal development terms including axonogenesis and axon guidance were significantly enriched. In the absence of functional TOP2B there are errors in axon guidance in the zebrafish eye. Specific heparin sulphate structures are involved in retinal axon targeting. The glycosaminoglycan biosynthesis–heparin sulphate/heparin pathway is significantly enriched in the TOP2B gene ontology analysis, suggesting changes in this pathway in the absence of TOP2B may cause the axon guidance faults. Summary: Gene ontology enrichment analysis of genes associated with human TOP2B peaks, identified by whole genome ChIP seq used to identify regions of binding, highlighted a number of processes in neuronal development including axonogenesis and axon guidance. The TOPd region . Crystald region . TOP2B dd region . By comping site . Beyond −/−Top2b mice are non-viable. Top2b null embryos exhibit no gross abnormalities in the morphology of major organs during development, but exhibit a defect in neural development where motor axons fail to form contacts with skeletal muscles and sensory axons do not enter the spinal cord, although neurogenesis appears to be normal. Failure to innervate the diaphragm, resulting in an inability to breath is thought to be the cause of perinatal lethality  in −/−Top2b mice compared to wild-type siblings. Top2b was shown to be present in the 5′ and upstream regions of a number of Top2b-sensitive genes, suggesting a direct role of Top2b in transcription of these genes, but not genes that were insensitive to Top2b (Ngfr p75) and up-regulation of this gene has been implicated in cell death of −/−Top2b neurons. A cre-lox −/−Top2b murine model demonstrated that neurons lacking Top2b had defective positioning in the cerebral cortex  mutant isolated in an ENU forward genetic screen is characterised by neurite targeting defects in retinal ganglion cell axons and dendrites and is caused by a mutation in top2b  expression (FAS gene it has been suggested that the DSB serves to activate DNA-PK to then modulate one or more further promoter-bound factor(s) via phosphorylation. It is likely that in at least the case of FAS, TOP2B interacts with DNA-PK since in the absence of DNA-PK TOP2B is not recruited to chromatin  peak calling algorithm   across the genome from untreated and E2-treated cells are shown in a circle diagram in methods) D. TOP2B methods) D. Approxmethods) . In bothmethods) .Fig. 2.2 peaks were analysed for coincidence with histone markers of transcriptional activation  or transcriptional repression (H3K9me3 and H3K27me3) .Next we identified TOP2B peaks that coincided with transcriptional markers. The location of TOP2B3K27me3) , with RN3K27me3) . Estradislightly A. Coincislightly B. There slightly , roughlyFig. S2). For example ∼0.2% of the H3K4Me3 peaks co localised with TOP2B2 peaks in the absence of estradiol and this reduced 4.1 fold with estradiol. 2 peaks relative to the nearest TSS. Upon treatment with estradiol there is a reduction in the number of TOP2B peaks near transcription start sites. In the TOP2B2 set 4.84% (139/2872) of peaks in the untreated data set are within 1 kb either side of a TSS compared to 2.97% (97/3265) of the peaks in the E2-treated data set  and CpG islands associated with TOP2B peaks . Gene ontology enrichment analysis was carried out using GOstats Bioconductor package, using a hypergeometric test . Heparan sulphate is a long unbranched polysaccharide which is covalently attached to various proteins (heparin sulphate proteoglycans) on the cell surface and in the extracellular matrix. A TOP2B peak was observed by all three peak calling methods on the gene encoding heparan sulfate 2-O-sulfotransferase 1 (HS2ST1), shown in 2 sets. Axon guidance is significantly enriched in both E2-treated and untreated cells (FDRs 1.110957e-03 and 7.741285e-05). At least 20 genes in the KEGG pathway for axon guidance have TOP2B peaks associated with them, these include the netrins, NTNG1 and NTNG2, and the netrin receptor UNC-5; ephrin receptors, EPHA and EPHB, and the membrane bound tyrosine kinase FYN; SLIT3 and its receptor ROBO1 (roundabout axon guidance receptor homolog 1); SEMA3A, SEMA3B and their co-receptor proteins PLXNs and MET. The intracellular effectors include GTPases and their associated proteins and kinases. These include three Rho GTPase regulating proteins ARHGAP13 (SRGAP), ARHGAP26 (FAK), and ARHGEF27 (NGEF), also RHOD, a Rho GTPase and ROCK, a Rho associated protein that acts as a downstream effector of Rho. The kinases included two serine-threonine kinases, PAK that links RHO GTPases to the cytoskeleton and nuclear signalling; and GSK3B, a glycogen synthase kinase subfamily member. Tyrosine kinases included FYN, a membrane bound non-receptor protein tyrosine kinase and FRK, a nuclear tyrosine kinase that negatively regulates proliferation. Other genes involved in axon guidance with TOP2B peaks are AGAP3, GNA1, NCK2, CALN and ABLIM1. Enrichment for the KEGG axon guidance pathway is consistent with TOP2B's previously reported role in axon guidance , glioma (9.174241e-03), and the ErbB signaling pathway (9.770330e-03). A number of signalling proteins are enriched in these three pathways, PKC, PI3K and SHC. PAK is present in focal adhesion and ERBB signalling as well as in axon guidance.Three other KEGG pathways are significant in the TOP2B2 peaks; these include dystrophin (DMD), integrins, and the sarcoglycan complex. Several calcium ion channels involved in cardiomyopathies are associated with TOP2B peaks including the voltage-dependent calcium channel CACNA1C, the sarcoplasmic reticulum calcium release channel RYR1, and the Na+/Ca+ exchanger and calcium anti-porter SLC8A1. The latter is also present in the calcium signalling pathway.In the presence of estradiol, in addition to axon guidance, calcium signalling and three cardiomyopathy pathways are significantly enriched. Calcium signalling pathway (5.857564e-04), arrhythmogenic right ventricular cardiomyopathy (ARVC) (6.770581e-06), dilated cardiomyopathy (4.601454e-04) and hypertrophic cardiomyopathy (HCM) (2.018838e-03). The three cardiomyopathies share a number of components associated with TOP2BTable S2. TOP2B2 peaks from untreated cells are associated with three genes  implicated in both autism and schizophrenia whilst TOP2B2 peaks from E2-treated cells are associated with five genes . CACNA1C is a calcium channel with strong links to autism and schizophrenia , with a higher preference for a downstream rather than an upstream localisation . Peaks within 500 bases of a transcription start site were analysed further, 108 peaks were within 500 bases of a TSS in the untreated set and 57 in the E2-treated set. Of these peaks 100 in the untreated set and 45 in the E2-treated set spanned at least one of the enriched transcription factor motifs. These genes are plotted versus enriched transcription factors in heat maps and are shown in Fig. S6. The shaded blocks in the heat map show that some genes have binding sites for several of the enriched transcription factors. Transcription occurs in transcription factories and it has been suggested that these factories may come in different flavours depending upon which transcription factors are present  in the absence of estradiol is shown in PKM promoter is coincident with a ChIP seq peak for ERα binding and an RNA pol II ChIP seq peak in MCF7 cells in the absence of estradiol, all three of these ChIP binding areas are within a CpG island. Twelve of the 23 enriched transcription factor motifs are found within the TOP2B peak sequence. Five of these motifs are occupied in ENCODE transcription factor ChIP seq analyses; ESR1.1, ESR1.2, PAX5, REST, and two SP1.2 sites. PKM gene expression is activated by SP1 in response to insulin  promoter also overlaps with a CpG island, ERα and RNA pol II ChIP seq peaks. The TOP2B peak on RALGAPA2 has 14 of the enriched transcription factor motifs within its sequence, four of these are occupied in encode data ESR1.1 and ESR1.2 and two SP1.2 sites. RALGAPA2 is a substrate for insulin activated protein kinase AKT and has similarities to the tuberous sclerosis complex  were selected for further analysis. The distribution of TOP2B peaks is shown in 2 peaks fall within 5 kb upstream of an annotated TSS and 48% fall within genes is shown in Fig. S2). Two percent of the TOP2B2 peak set coincided with H3K4Me3 marks in untreated cells and this reduced >4 fold in estradiol treated cells, consistent with reduced TOP2B occupancy at active promoters and CpG islands following estradiol treatment . In this first whole genome TOP2B analysis, we show that peaks of occupancy are distributed across the whole genome, and are not located specifically in promoter regions.To determine whether TOP2 recruitment is a common feature of estradiol-regulated genes we carried out ChIP-SEQ with anti-TOP2B antibodies in human MCF7 cells before and after estradiol treatment (10 nM) for 30 min to visualise the distribution of TOP2B in the human genome . Peaks oreatment ; Fig. S22 data sets in Table S2. Twenty one percent of the non-expressed genes were associated with TOP2B peaks whilst 32% of expressed genes were associated with TOP2B peaks. So expressed genes were more frequently associated with TOP2B peaks than non-expressed genes (32% and 21% respectively) (Table S3).When expressed genes were divided into those that changed in response to estradiol and those that did not, 30% of the genes that did not change in response to estradiol had TOP2B peaks and 34% of those that changed had TOP2B peaks. Interestingly of the transcripts that change in level following exposure to estradiol more transcripts go down than up, consistent with estradiol being able to both repress and activate gene expression (Table S4).Published GroSEQ data for MCF7 cells treated with 100 nM estradiol for 10, 40 and 160 min was utilised to determine which genes are expressed in these cells and which change in response to estradiol . InterseFig. S3) including protein tyrosine kinase activity, ion channel activity, GTPase regulator activity and glutamate receptor activity. Consistent with these molecular functions, cellular response to stimulus, regulation of signal transduction and glutamate receptor signalling were highly enriched in the biological processes. The enriched cellular components were consistent with the previously described role in neuronal development  lacks functional top2b and displays neurite targeting defects in the retinal ganglion. Specific heparin sulphate structures are involved in axon guidance of retinal ganglion cells  was dissolved in the above medium at a stock concentration of 20 µg/ml and for E2 treatment was added to cells to a final concentration of 10 nM for 30 min.MCF7 cells were cultured in Minimum Essential Medium Eagle (MEME)  supplemented with 10% (v/v) foetal bovine serum , 1% (v/v) penicillin and streptomycin  and 1% (v/v) glutamine (200 mM) , 1% NEAA  in a 37°C incubator with 5% COFig. S1.Rabbit polyclonal antisera 3535 and 30400 were raised in-house  to recomChIP was performed using the kits and the protocol from Millipore . Briefly, MCF7 cells were grown to ∼80% confluency and treated with 17β-estradiol  (Sigma-Aldrich). E2-treated and untreated (control) cells were fixed with formaldehyde (1% (v/v), Sigma-Aldrich), neutralised with glycine, washed with 1× PBS and trypsinised . Cells were collected and lysed with cell lysis buffer containing protease inhibitors, collected and resuspended in nuclear lysis buffer containing protease inhibitors. Chromatin was sonicated to between 500-1000 bp, 14×15 s cycles, and 5% was removed as ‘input’. 3.6 µg anti-TOP2B (3535), 7 µg anti-TOP2B (30400), or 1 µg anti-GFP  or 5 µg anti-AcH3  were added to protein A magnetic beads . Chromatin was diluted, incubated with the beads and rotated for 4.5 h at 4°C. Immunoprecipitated protein-DNA complexes were washed sequentially in Low Salt Buffer, High Salt Buffer, LiCl Buffer and TE Buffer and then resuspended in ChIP Elution Buffer+Proteinase K, rotated at 65°C for 2 h followed by incubation at 95°C for 10 min. DNA was then purified using spin columns .Table S1. qPCR parameters were −95°C for 5 min (1 cycle), 95°C for 25 s, then 60°C for 25 s, then 72°C for 10 s (40 cycles), 72°C for 6 min (1 cycle). Results were quantified as % input with GFP and AcH3% input values being the negative and positive controls respectively.qPCR was performed using iQ SYBR green supermix , primers including GAPDH control primers are shown in the Libraries were generated with chromatin immunoprecipitated from MCF7 cells±E2 (10 nM for 30 min). Multiple immunoprecipitates were pooled for each antibody  to obtain between 400 and 800 ng DNA, at a concentration of 6-21 ng/µl for library preparation for Illumina ChIP Sequencing on an Illumina Genome Analyser II. Each library was sequenced 3 or 4 times for input libraries and for TOP2B libraries. The data from the 3535 and 30400 libraries was merged to give >10 million uniquely aligned reads. Genome Alignment, the raw sequencing reads were aligned to the human reference genome (hg18) using BWA (v0.5.8c)  and aligP<0.01.Gene ontology and KEGG pathway enrichment analysis was carried out using GOstats (v 2.24.0)  BioconduP<0.05. A distance measure for binary attributes and Ward's method was used for clustering of genes and transcription factor motifs associated with TOP2B peaks. All heatmaps were generated with the gplots v 2.12.1 BioConductor package.Vertebrate transcription factor motifs were obtained in MEME format from the Jaspar database (2014 release) . FIMO v, was use


Saccharomyces cerevisiae share a conserved −8 to +1 sequence with +1+2 AA, AG or AT initiation sequence, which dictates the efficiency of transcription initiation by the mitochondrial RNA polymerase Rpo41 and its initiation factor Mtf1. We used 2-aminopurine fluorescence to monitor promoter melting and measured the kcat/Km of 2-mer synthesis to quantify initiation efficiency with systematic changes of the +1+2 base pairs to matched and mismatched pairs. We show that AA promoters are most efficient, followed by AG and then AT promoters, and the differences in their efficiencies stem specifically from differential melting of +1+2 region without affecting melting of the upstream −4 to −1 region. Inefficient +1+2 melting increases the initial NTPs Kms of the AG and AT promoters relative to AA or singly mispaired promoters. The 16–100-fold higher catalytic efficiency of AA initiation sequence relative to AG and AT, respectively, is partly due to Rpo41-Mtf1 interactions with the +1+2 non-template adenines that generate a stable pre-transcribing complex. We propose a model where the +2 base pair regulates the efficiency of initial transcription by controlling multiple steps including downstream promoter opening, +1+2 NTPs binding, and the rate of 2-mer synthesis.Mitochondrial promoters of  Saccharomyces cerevisiae mitochondrial genes are transcribed by nuclear-encoded mitochondrial RNA polymerase protein, Rpo41, which is homologous to the single subunit T7 RNA polymerase (RNAP). Unlike T7 RNAP that transcribes without requiring any initiation factors, Rpo41 requires the initiation factor Mtf1 to transcribe specifically from duplex promoters .Recombinant Rpo41 and Mtf1 proteins were expressed and purified as reported previously  using suSynthetic oligonucleotides were purchased from IDT and short 20-mer ssDNAs were HPLC purified and longer 45-mer and 57-mer were purified by urea-denatured polyacrylamide gel electrophoresis and electroelution. The molar concentration of purified DNA was determined from its extinction coefficient and absorbance at 260 nm. The double-stranded (ds) DNA were prepared by annealing complementary non-template and template strands in 1.1 to 1 ratio.in vitro transcription assays and resolving the RNA products using denaturing polyacrylamide gel electrophoresis  and formamide dye , heated to 95°C for 2 min and analyzed on 24% sequencing gel containing 4 M urea. The gel was exposed to a phosphor screen overnight and scanned on a Typhoon 9410 PhosphorImager instrument (Amersham Biosciences). The free ATP and RNA bands were quantified using ImageQuant and molar amounts of RNA synthesized were calculated according to Equation  in the case of AA promoters, or 5–3000 μM each of +1 ATP (+ [γ-32P] ATP) and +2 GTP or +2 UTP for AG and AU promoters, respectively. The 2-mer RNA was resolved from the free NTP on a 24% sequencing gel containing 4 M urea and quantified using ImageQuant. The rates of 2-mer RNA synthesis were calculated according to Equation . A pre-incubated complex of 2 μM Rpo41, 2.5 μM Mtf1 and 2.5 μM U25D32 15S rRNA promoter loaded in one syringe was rapidly mixed with equal volume of 500 μM ATP from the second syringe for various time intervals, and quenched with EDTA from the third syringe. The 2-mer RNA was resolved from free ATP on a 24% sequencing gel containing 4 M urea.Steady-state fluorescence measurements were carried out at 25°C on a Fluoro-Max-2 spectrofluorometer  in buffer containing 50 mM Tris-acetate, pH 7.5, 100 mM potassium glutamate and 10 mM magnesium acetate. Fluorescence spectra of 200 nM 2-AP incorporated duplex promoters were collected from 350 to 420 nm (6-nm bandwidth) with excitation at 315 nm (2-nm bandwidth) after sequential addition of 400 nM Rpo41, 400 nM Mtf1 and 1 mM of initiating +1+2 NTPs. After subtracting contributions from buffer and proteins in the presence of unmodified DNA, the corrected 2-AP fluorescence intensities between 360 and 380 nm were integrated for comparison.in vitro transcription profiles of the U25D32 promoters. Purified Rpo41 plus Mtf1 is incubated with the promoter DNA and 500 μM ATP + [γ-32P]ATP, UTP, GTP and 3’dCTP are added to monitor RNA synthesis at 25°C from 1 to 8 min reaction times. Inclusion of 3’dCTP prevents non-templated RNA extension and a means to assess correct transcription start site. The RNA products are resolved on 24% sequencing gel to visualize the complete transcription profile  promoter at 0.03±0.002 min−1  of 2-mer synthesis, which takes into consideration both the binding efficiency of the initial NTPs and the rate of 2-mer synthesis. The kcat and Km kinetic parameters are typically determined from the hyperbolic increase of steady-state 2-mer synthesis rates with increasing +1+2 NTPs concentrations. In order for the steady-state kcat/Km to accurately represent the efficiency of transcription, it is important that observed rate is not limited by 2-mer RNA dissociation. We verified this with the AA and AG promoters by conducting presteady-state experiments . The lack of presteady-state burst of 2-mer synthesis indicates that 2-mer dissociation is faster than its synthesis rate. Hence, production of 2-mer is limited either by the chemical step or steps before synthesis such as promoter melting. Previous studies have shown that the 20 bp 15S rRNA promoter binds one molecule of Rpo41-Mtf1 to form a complex with Kd of 0.2 nM  with the consensus sequence of the 15s rRNA promoter from −12 to +8 Figure , and carf 0.2 nM , and sinKm of Rpo41-Mtf1 in the literature, measured from steady-state rates of run-off RNA synthesis as a function of initial NTPs, are much lower than what we report here  of transcription initiation from various yeast mitochondrial promoters.Note that the initial NTPs ort here –20. Monikcat and Km values . We assure that the observed rate from the 3 min assay (0.075 μM s−1 at 250 μM ATP) is consistent with the observed rate from the time course (0.081 μM s−1 at 250 μM ATP)  to AG (40 M−1 s−1) . Therefore, we measured 2-mer synthesis after 3 min reaction time as a function of increasing +1+2 NTPs concentration to estimate the Km. However, on comparing rates from 2-mer  to determine the kinetic parameters. It should be noted that the rate of dissociation of 2–8 mer RNA can be lower than that of 2-mer RNA, which might result in a decrease in the overall 2–8 mer catalytic rate and subsequently an apparent decrease in NTPs r Figure  versus 2r Figure  assays aKm of the initiating NTPs with AG and AT promoters is due to inefficient promoter opening, we introduced mismatches at +2, while keeping the +1 A:T constant. The identity of the +2 non-template and template bases can influence the efficiency of promoter opening/stabilization; therefore, we changed the +2 base pair to all combinations of mismatches , so now the +2 mismatched promoter has almost the same NTPs Km as the strong AA promoter (Table kcat of 2-mer synthesis (0.8 s−1 for +2 A/C versus 0.3 s−1 for +2 G:C and 0.5 s−1 for +2 A:T). Thus, introducing an A/C mismatch at +2 increases the catalytic efficiency by 26-fold. Changing the +2 G to other bases to create T/C and C/C mismatches increases the catalytic efficiency of 2-mer synthesis by 10–18-fold  with +2 A:T versus G:C . Thus, the identity of +2 base pair appears to dictate the efficiency of transcription initiation by affecting promoter opening/stabilization and 2-mer synthesis.kcat/Km of 6 M−1 s−1 relative to the +2 A:T promoter  is slightly better than the consensus +2 A:T promoter (630 M−1 s−1). Changing the +2T to G or C, to create +2 G/A or C/A mismatches, respectively, also lowers the NTPs Km, but the kcat is not increased relative to the +2 T:A promoter  or [α-32P] ATP (for the rest) and monitor synthesis of 2-mer to 8-mer on the sequencing gel  have low activity. Thus, both +1 C:G and T:A promoters are ∼15-fold less active than +1 A:T  that lacks the 6-amino group of adenine, but contains 2-amino group to form a canonical base pair with thymine, and di-AP that contains 2- and 6-amino groups Figure . The 2-m1 Figure  and C, a1 Figure . InteresT Figure . SimilarT Figure . The rat1 Figure . The res−1, Figure −1) and −2+1 (0.8 min−1)  versus 2-AP:T  of 2-mer synthesis by Rpo41 on the bubble promoter with +2 A:T (141 M−1 s−1) is only 2-fold higher than +2 G:C (67 M−1 s−1)  over G:C (40 M−1 s−1) on the duplex promoter (Table kcat/Km of +2 A:T promoter (kcat/Km of 141 M−1 s−1 for Rpo41 versus 630 M−1s−1 for Rpo41-Mtf1) rather than lowered kcat/Km of +2 G:C (kcat/Km of 67 M−1 s−1 for Rpo41 versus 40 M−1 s−1 for Rpo41-Mtf1). It should be noted that catalytic efficiencies of Rpo41 and Rpo41-Mtf1 have been tested on different DNA substrates, and hence their absolute values are not directly comparable; however, the specificity or the ratio of the catalytic efficiencies of AA and AG for duplex (16-fold) versus bubble (2-fold) should be comparable. Thus, the increased preference for AA in the presence of Mtf1 is either because of direct interactions of Rpo41-Mtf1 with the +1+2 template/non-template region that regulate melting of +2 A:T bp, or by an indirect mechanism, where Rpo41-Mtf1 melts the −4 to −2 region efficiently to create a competent open complex, which is not achieved by Rpo41 alone even when the −4 to −2 is pre-melted.Using the −4 to −2 bubble promoter where the non-template sequence is changed, we show that Rpo41 by itself is not very specific and synthesizes 2-mer on +2 A:T, +2 G:C and +2 C:G, but not +2 T:A  Figure  and E. Oer Table . The higTaken together, these results indicate that differential promoter efficiencies are achieved by selectively augmenting the catalytic initiation efficiencies of promoters with +2 A:T, and both Rpo41 and Mtf1 contribute to specificity for the +2 A:T in the promoter sequence and/or specificity for initiation with +2 ATP.We have investigated the biochemical basis of the higher transcription initiation efficiency of the yeast mitochondrial RNA polymerase Rpo41-Mtf1 complex on promoters that initiate with the +1+2 AA sequence. The mitochondrial promoters of the yeast have a consensus −8 to +1 sequence, but initiate with +1+2 AA, AG or AT coding sequence. By studying the complete transcription profile of five representative promoters, we show that AA promoters produce the highest amount of run-off product, followed by +2 G:C, and least efficient is +2 T:A. Although, +1+2 AA promoters are highly efficient, they produce ∼5–10-fold more abortive products than the AG promoters. One explanation for the lower amount of abortive products on AG promoters is the stability of short initial RNA products bound to the RNAP–DNA complex. This is further exemplified by the difference in the abortive products of the two AA promoters, where Cox1 with a +5 G:C base pair synthesizes ∼3-fold less abortive products than the 15S rRNA promoter with no such G:C base pair. Consistent with this, we observe accumulation of 2-mer to 4-mer abortive products on Cox1 promoter, after which they significantly reduce.kcat/mK of 2-mer synthesis on short 20 bp promoters that have been successfully used previously to study the initial DNA binding, bending and melting steps , followed by GTP (0.2–1.3 mM), and lowest for CTP (<0.01–0.24) and UTP (0.01–0.13)  ,23. In athe cell ,24–27; wopen complex and (iii) Rpo41-Mtf1-DNA-+1+2NTPs pre-transcribing complex  Rpo41-DNA complex, (ii) Rpo41-Mtf1-DNA x Figure , and thex Figure , Mtf1  and Rpo41 by itself bends the promoter by 52° and shows transient visitations to the severely bent DNA conformation (Previous studies have shown that Rpo41 has tight interactions with the promoter (ormation ,11. Our ormation ,12. Togeormation ,10, thesOur results indicate that initiating NTPs are required to stably unstack the −1 template base and effectively melt/stabilize the +1+2 region to generate the ‘pre-transcribing complex’, where the template bases are optimally aligned in Rpo41's active site to facilitate correct start-site selection and 2-mer synthesis. In addition to multiple interactions throughout the −4 to +2 region, specific interactions with the 6-amino groups of +1 and +2 non-template adenines play an important role in stabilization of pre-transcribing complex and efficient transcription from the AA promoter.Unlike the extensive non-template interactions in the yeast mitochondrial initiation complex, the homologous T7 RNAP does not have any significant interactions with the melted non-template strand during initiation ,31, indiInteractions with +2 base are observed in the bacterial initiation complex, where the core RNAP β subunit flips the +2 non-template G base and buries it in a β-pocket . MutatinDespite great diversity in promoter architecture, RNAP assembly and mechanism of transcription regulation, the yeast mt transcription system seems to share common RNAP–DNA interactions with the bacteriophage, prokaryotic and eukaryotic systems. Almost all single- and multi-subunit RNAPs initiate with purines, hinting at a conserved mechanism of start-site selection. In this paper, we have established the biochemical basis of preference for initiating purines in yeast mitochondria. We envision that the sequence-specific information will facilitate designing of optimal DNA substrates for capturing structural snapshots of the thus-so-far missing initiation complexes of yeast mitochondria. In addition to providing structural and mechanistic insights, this study has potential applications in developing commercial tools for custom RNA synthesis.Supplementary Data are available at NAR Online.


Cellular differentiation programs are controlled, to a large extent, by the combinatorial functioning of specific transcription factors. Cortical projection neurons constitute the major excitatory neuron population within the cortex and mediate long distance communication between the cortex and other brain regions. Our understanding of effector transcription factors and their downstream transcriptional programs that direct the differentiation process of cortical projection neurons is far from complete.In this study, we carried out a ChIP-Seq (chromatin-immunoprecipitation and sequencing) analysis of NEUROD2, an effector transcription factor expressed in lineages of cortical projection neurons during the peak of cortical excitatory neurogenesis. Our results suggest that during cortical development NEUROD2 targets key genes that are required for Reelin signaling, a major pathway that regulates the migration of neurons from germinal zones to their final layers of residence within the cortex. We also find that NEUROD2 binds to a large set of genes with functions in layer-specific differentiation and in axonal pathfinding of cortical projection neurons.in vivo NEUROD2 target genes offers mechanistic insight into signaling pathways that regulate neuronal migration and axon guidance and identifies genes that are likely to be required for proper cortical development.Our analysis of The online version of this article (doi:10.1186/s12864-015-1882-9) contains supplementary material, which is available to authorized users. The neocortex is a six-layered structure composed of myriad excitatory and inhibitory neuron types. Developmental programs, controlled by multiple cell-intrinsic and –extrinsic cues, establish unique patterns of connectivity for the diverse neuronal subtypes located in different layers. While the granular neurons of layer IV form networks of local connections within cerebral hemispheres, projection neurons of layers II/III, V and VI mediate long distance communications both within and outside the cortex.Developmentally, excitatory neurons are born from dividing progenitors located in distinct germinal domains situated at ventricular and subventricular zones of the dorsal telencephalon. Once born, they migrate to superficial cortical layers, with each wave of migrating neurons surpassing predecessors, resulting in six distinct cortical layers generated in an inside-out manner . ConsequNeurog1 and Neurog2 proneural basic helix loop helix (bHLH) transcription factors commit neural progenitors to cortical excitatory neuron identity .210, representing 10 % of input chromatin) was subtracted from the Ct value of the input sample to generate the adjusted input Ct. Following formula was used to calculate the input normalized chromatin immunoprecipitated DNA amount: 100 × 2(Adjusted input Ct – ChIP Ct). ChIP DNA was prepared using NEUROD2 antibody 2 and negative control ChIP DNA by using GFP antibody 2 for all qPCR experiments  and chromatin immunoprecipitated DNA (ChIP DNA) were each dissolved in 300 μl water and 1 μl of each was used in a single qPCR reaction. The numerical value 3.32 .An shRNA targeting Neurod2 was cloned into pSUPER-neo-GFP backbone vector as described in oligoengine.com. Oligos used for cloning are provided in Additional file Ten nanogram total RNA was used to prepare cDNA from Neurod2 knockdown and non-targeting samples . mRNA levels were quantified by qPCR analysis  with primers listed in Additional file Bowtie for Illumina (version 1.1.2) [rmdup (version 1.0.0). Reads that mapped onto multiple locations on the genome were removed with Filter SAM (version 1.0.0). Peaks locations were identified with MACS  [−5 p-value cutoff and an MFOLD high confidence enrichment ratio of 20 were used as input parameters. Results from ChIP performed using GFP antibodies were used as ChIP-Seq control files for MACS analysis. GFP ChIP reads generated with a rabbit antibody (Santa Cruz cat. sc-8334) were used as input controls for NEUROD2 ChIP reads generated with rabbit antibodies (Abcam cat. ab104430 and ab109406). GFP ChIP reads generated with a mouse antibody  were used as input controls for NEUROD2 ChIP reads generated with a mouse antibody (Abcam cat. ab168932). The antibody information is presented in Additional file Peak calling was performed using tools available on Galaxy Project . Tool names and versions are indicated below. Single end 50 bp raw sequences in fastq format were mapped onto the mouse genome (mm10 build) with n 1.1.2) , 18. Twon 1.0.1) . 50 nt tMACS outputs from ChIP-Seq data using different NEUROD2 antibodies were combined to yield a single set of high-confidence peak locations. To this end, we first eliminated isolated instances where a MACS peak from an antibody has no overlap with those derived from the other two antibodies. Remaining overlapping peaks were next merged to yield a single set of 32,367 peak regions which, by construction, are confirmed by at least two experiments. The midpoint of each peak was used as the peak location.The statistical analysis of peak locations was done on the genome assembly GRCm38/mm10 with Ensembl annotations for coding and noncoding regions CDS, 5′UTRs, 3′UTRs, and introns , 55. In n for a given region type with the corresponding mean σ, both of which were obtained from a uniformly random reassignment of the observed intragenic peaks to transcript regions genome-wide. We measured the binding preference in terms of the deviation of the observed peak count from the random expectation, where the deviation was expressed in multiples of the standard deviation, i.e., Binding preference of NEUROD2 to different types of intragenic regions was investigated by comparing the observed peak count 6 bps apart and constructing the corresponding peak-TSS distance histogram.Affinity of NEUROD2 for binding specifically in the proximity of transcription start sites (TSSs) was investigated by considering all peak/TSS pairs located up to 10NEUROD2 consensus sequence was determined by using MEME ChIP Suite using 32,367 NEUROD2 peak sequences as input. Accession numbers for data downloaded from Encode Project (encodeproject.org) were: For E14.5 whole brain ChIP using antibodies against, H3K4me1 (ENCSR000CCZ), H3K4me3 (ENCSR000CDA) and CTCF (ENCSR000CEH) . All datClosestGene method as described in [ClosestGene target score which was the sum of the scores of all associated peaks. As a background control 32,367 randomly distributed peaks along the mouse genome were assigned to specific genes using the same method.Individual peaks were assigned to genes using the ribed in . BrieflyAll mice were bred and housed at Koç University Animal Research Laboratory. Timed pregnant BALB/c strain mice at E14.5 stage were sacrificed by cervical dislocation, and embryos were retrieved into ice-cold 1x HBSS for cortical dissection. All animal experiments were done in accordance with guidelines provided by the Koç University, The Turkish Ministries of Food, Agriculture and Live Stock, Forestry and Water Management and the European Union. Ethics approval was obtained from The Koç University Institutional Animal Care and Use Committee (no. 2013–1).


Several nuclear localised intergenic long noncoding RNAs (lncRNAs) have been ascribed regulatory roles in transcriptional control and their number is growing rapidly. Initially, these transcripts were shown to function locally, near their sites of synthesis, by regulating the expression of neighbouring genes. More recently, lncRNAs have been demonstrated to interact with chromatin at several thousand different locations across multiple chromosomes and to modulate large-scale gene expression programs. Although the molecular mechanisms involved in targeting lncRNAs to distal binding sites remain poorly understood, the spatial organisation of the genome may have a role in specifying lncRNA function. Recent advances indicate that intergenic lncRNAs may exert more widespread effects on gene regulation than previously anticipated. The mammalian genome contains large numbers of noncoding RNA (ncRNA) loci that interdigitate between, within, and among protein-coding genes on either strand. To date, more than 10 000 mammalian intergenic lncRNAs [>200 nucleotides (nt)] see  have beeSome intergenic lncRNAs function as transcriptional regulators that can act locally, near their sites of synthesis, to regulate the expression of nearby genes, or distally to regulate gene expression across multiple chromosomes . Here, wIntergenic lncRNAs have been divided, on the basis of chromatin marks at their promoters, into two broad categories: those emanating from enhancer regions or those transcribed from promoter-like lncRNA loci in vitro and to induce looping interactions between their enhancer elements and the promoters of nearby target genes MyoD1 core enhancer region, both MyoD1 chromatin accessibility and RNA polymerase II (PolII) occupancy were reduced and MyoD1 expression was decreased It is currently unknown whether eRNAs or elncRNAs are commonly simply a by-product of or an actual cause of enhancer action on neighbouring protein-coding genes. However, a small but growing number of eRNAs and elncRNAs have been shown to function at their site of synthesis in a RNA-dependent manner to regulate positively the expression of neighbouring protein coding genes on the same chromosome fbp1+ gene activation in yeast hGH) HS1 enhancer was shown to be stimulated by lncRNA transcription that initiates immediately downstream of HS1 and is noncontiguous with the hGH target promoter hGH expression. When the sequence of this lncRNA was replaced by that of an unrelated bacteriophage RNA, the enhancing effect of the natural transcript was recapitulated. Taken together, these studies show that enhancer-associated lncRNAs can also act locally, near their site of synthesis, using either RNA-dependent or -independent mechanisms to increase the transcriptional activity of chromosomally proximal protein coding genes.lncRNA loci have also been ascribed RNA-independent functions in gene activation, for example that arise from transcription through loci affecting local chromatin accessibility, as described during cis-acting lncRNA modes of action on the same chromosome, and in an allele-specific manner. However, trans-acting lncRNA mechanisms could also operate to control nearby gene expression. The lncRNA Jpx, for example, is transcribed from the active X chromosome and can upregulate expression of the adjacent Xist gene on the other future inactive X allele in trans during the process of X chromosome inactivation Evf-2, in addition, modulates the activity of an enhancer element present within its locus by binding distal-less homeobox 2 (DLX2) and methyl CpG binding protein 2 (MECP2) and inhibiting DNA methylation of this enhancer to control expression of the genomically adjacent Dlx6 gene trans because Evf-2 and DLX2 cooperate to increase the activity of the Dlx6 enhancer when they are co-expressed from transiently transfected plasmids in a reporter assay, and Evf-2 inhibits DNA methylation when expressed ectopically from a transgene in a mouse model. In general, further mechanistic studies will be needed to assess the relative contributions of cis- and trans-acting lncRNA mechanisms controlling local gene expression.Local changes in gene expression are presumed to be mediated by TFF1 and NRIP1 eRNA containing loci, located 27 Mb apart on chromosome 21, are brought into close spatial proximity by long-range DNA looping interactions. This looping is induced by E2 and appears to be dependent on the NRIP1 eRNA. Therefore, a subset of eRNAs may have so far uncharacterised distal regulatory roles.Many experiments thus far have focussed on the possible mechanisms by which an enhancer-associated lncRNA or transcription of its locus regulates the expression level of an adjacent protein-coding gene. However, long-range intra-chromosomal interactions between eRNA expressing loci and distantly located loci have also been documented trans at distal genomic locations. To address this and other issues, several techniques have been recently developed FoxC1 enhancer, has been shown to occupy 15 binding locations on multiple chromosomes, all well away from its endogenous locus; however, none of these were located within regulatory regions of E2-responsive genes and, thus, probably represent nonfunctional genomic interactions What has been studied less often is whether lncRNA transcripts can function in Hotair, a lncRNA transcribed from the homeobox (Hox) C locus, was shown initially to function in trans to repress the transcription of genes in the HoxD gene cluster on another chromosome Hotair depletion PCGEM1), which binds to the androgen receptor (AR), associates with 2142 binding locations on the genome, the majority (approximately 70%) of which correspond to AR-bound H3K4me1-modified enhancers. PCGEM1 association with AR-bound enhancers appears to increase AR-mediated gene activation without affecting AR levels Determination of the genomic binding profiles for several other types of lncRNA shows that a single lncRNA transcript can interact with multiple binding sites on different chromosomes away from its site of transcription. Paupar is an intergenic lncRNA that interacts with chromatin at over 2800 sites located on multiple chromosomes and controls large-scale gene expression programs in a transcript-dependent manner Pax6) gene and its depletion significantly alters the expression of Pax6 and 942 other genes distributed across the genome. Paupar binding sites, defined by CHART-Seq g transcription in trans, both when expressed from a transgene and also from its genomic locus, by interacting with WD repeat domain 5 (WDR5) and by altering Ifng histone H3K4 tri-methylation Xist might be a diffusible factor; when Xist is expressed from a transgene in female mouse embryonic fibroblasts, it diffused from the ectopic site of synthesis and acted on the endogenous Xist locus in transEvf-2, when expressed from a transiently transfected plasmid, cooperated with the DLX2 protein to activate the Dlx-5/6 enhancer in a luciferase reporter in transPaupar transcript modulated, in a dose-dependent manner, the transcriptional activity of a number of its genomic binding sites when inserted into transiently transfected reporters Several nuclear lncRNAs are able to regulate transcription when expressed in Therefore, coordination among sites of lncRNA synthesis, the spatial organisation of the genome in the nucleus, and specific lncRNA interactions with transcription and chromatin regulatory proteins are all likely to have roles in facilitating binding of lncRNA transcripts to their genomic targets .The genomic associations observed between lncRNAs and chromatin could be accomplished through direct base pairing between RNA and DNA sequences Xist to chromatin by binding DNA and Xist RNA through different sequence domains Gas5 and glucocorticoid receptor Panda and nuclear transcription factor Y (NFYA) Lethe and nuclear factor kB (NF-kB) Jpx and CCCTC-binding factor (CTCF) Paupar and PAX6 Rmst and SRY (sex determining region Y)-box (SOX2) Prncr1 and AR Gas5, Panda, Lethe, and Jpx lncRNAs appears to inhibit DNA binding of their associated transcription factors at several target sites, whereas knockdown of Paupar and Prncr1 levels had no effect on PAX6 or AR occupancy where tested. By contrast, Rmst appears to be required for the correct association of SOX2 with promoter regions of neurogenic target genes Six3OS and SRAlncRNAs that associate with sequence-specific DNA binding transcription factors could be targeted to the genome indirectly through RNA–protein–DNA interactions C. YY1, fXist appears to target active chromatin because domains that are initially occupied by Xist are unusually enriched in actively transcribed genes and open chromatin Pcgem1 and Prncr1 preferentially interact with enhancer-associated histone modifications in vitroChromatin modification and structure may also modulate how lncRNAs are recruited to the genome. in vitro in a size-dependent manner Several lncRNAs associate with chromatin-modifying complexes and transcriptional regulatory proteins in the nucleus. High throughput RNA-immunoprecipitation (RNA-IP) experiments have indicated that individual chromatin-modifying complexes, such as polycomb repressive complex 2 (PRC2) and mixed-lineage leukemia (MLL), interact with thousands of RNA transcripts, including lncRNAs Mistral, for example, which is transcribed from the intergenic region between the Hoxa6 and Hoxa7 genes, forms a RNA–DNA hybrid structure at its site of synthesis which recruits MLL1 complex proteins Fendrr can associate with PRC2 and regulate Pitx2 expression in trans. Fendrr is predicted to interact with a short stretch of complementary sequence, of fewer than 40 nt, in the Pitx2 promoter, which can form a RNA–DNA–DNA triplex in vitro. Given that Pitx2 promoter PRC2 occupancy and histone H3K27 tri-methylation are decreased in Fendrr knockout embryos, Fendrr may have a role in recruiting PRC2 to the Pitx2 promoter trans.lncRNAs that bind proteins specifically might act as guides to target chromatin-modifying complexes to the genome. The lncRNA Drosophila roX2 lncRNA appears to function as a critical complex subunit that is necessary for the correct assembly of a functional male-specific lethal (MSL) dosage compensation complex. Its stem loop-containing structured domains bind the MLE RNA helicase and MSL2 ubiquitin ligase components of the MSL dosage compensation complex in a sequential manner roX2 with the MLE RNA helicase results in an ATP-dependent conformational change in a roX2 stem-loop structure and a subsequent increase in its association with MSL2. Other lncRNAs may also promote the ordered recruitment of functional ribonucleoprotein complexes. For example, Pcgem1 and Prncr1 bind AR sequentially, thereby stimulating both ligand-dependent and ligand-independent AR controlled gene expression programs Prncr1 first associates with DOT1-like, histone H3 methyltransferase (DOT1L) and binds to acetylated enhancer-bound AR causing DOT1L to methylate the AR. AR methylation subsequently induces recruitment of Pcgem1, in complex with pygopus homolog 2 (PYGO2), which because of its H3K4me3-binding ability, may stimulate DNA looping interactions between AR-bound transcriptional enhancers and target promoters.Several recent studies suggest that lncRNAs modulate the structure and function of their associated protein complexes. The Hotair, for example, interacts with PRC2 through its 5′ end, whereas its 3′ region associates with (co)repressor for element-1-silencing transcription factor (CoREST) in vitroTug1) and metastasis associated lung adenocarcinoma transcript 1 with methylated and unmethylated Pc2 protein, respectively, and the subsequent relocation of growth control genes from Tug1-containing polycomb bodies, where they are repressed, to interchromatin granules for assembly of activator complexes, where they are activated These studies raise the possibility that a single lncRNA molecule contains multiple structural motifs, upon which multiple different proteins bind, which enhance the efficiency of genomic targeting and transcriptional regulation H19 and Xist lncRNAs in genomic imprinting and X chromosome inactivation, respectively. It has since become apparent that the genome encodes large numbers of nuclear localised intergenic transcripts. The number of these transcripts that derive from serendipitous transcription or that fail to have functions (as opposed to mere effects in vivo.Roles for lncRNAs as regulators of chromatin organisation and gene expression were initially described for


Among these means of regulation, transcription modulation is the most important, as evident in its tight regulation in cancer cell survival as well as pluripotent stem cell maintenance and differentiation. Here, we discuss how hTERT gene transcription is regulated, mainly focusing on the contribution of trans-acting factors such as transcription factors and epigenetic modifiers, as well as genetic alterations in hTERT proximal promoter.Embryonic stem cells and induced pluripotent stem cells have the ability to maintain their telomere length via expression of an enzymatic complex called telomerase. Similarly, more than 85%–90% of cancer cells are found to upregulate the expression of telomerase, conferring them with the potential to proliferate indefinitely. Telomerase Reverse Transcriptase (TERT), the catalytic subunit of telomerase holoenzyme, is the rate-limiting factor in reconstituting telomerase activity in vivo. To date, the expression and function of the human Telomerase Reverse Transcriptase ( The ends of human chromosomes are capped by telomeres which protect the chromosome termini from degradation, end-to-end fusion and recombination ,2. TelomAdult human somatic cells have limited telomere lengths ranging from 7 to 12 kb . TherefohTERT and restore telomere length homeostasis could potentially delay the clinical onset of these diseases as well as normal aging in humans.Defects in genes that regulate telomere length homeostasis may lead to various diseases, collectively termed telomeropathies ,21. PatiOn the other hand, telomerase activity is highly elevated in 85%–90% of human cancers and over 70% of immortalized human cell lines . This ishTERT gene are known to be regulated at various molecular levels. However, the transcription of hTERT has been suggested to be the dominant step in the regulation of telomerase activity -3-chlorocinnamic acid (3-C1-AHPC), an adamantyl-related molecule, has been shown to inhibit the expression of miR-202, which targets Mad1 gene, and hence lead to de-repression of hTERT gene and increased telomerase activity [hTERT expression was supported by the observation that the overexpression of pre-miR-202 and inhibition of miR-202 resulted in the decreased and increased repressive activity of Mad1 on hTERT expression, respectively. In addition, EGCG (a green tea polyphenol), sulforaphane (SFN), 15d-PGJ2 , selenium, and 12-O-tetradecanoylphorbol-13-acetate have been shown to enhance Mad1’s binding to and repression of hTERT promoter [Mad1 is an antagonist of Myc protein in that it competes with the latter for E-box motif binding in promoter region of target genes and mediates the repression (as opposed to Myc’s activation) of these genes. The binding of Mad1 to r, Sin3A . This bir, Sin3A . The Madcontrols . The autactivity . The reppromoter ,217,218.hTERT, resulting in the repression of the gene. This binding is important but not sufficient to govern hTERT expression; chromatin environment, such as histone modification patterns, is also essential [hTERT proximal promoter leads to the recruitment of a histone deacetylase (HDAC), HDAC1, to the promoter region and, in turn, modifies the epigenetic landscape of the region, resulting in the silencing of the hTERT gene [hTERT gene [hTERT promoter, and a decrease in RNA polymerase II recruitment to the promoter due to lower histone acetylation levels in the region. Ceramide has also been shown to reduce Sp1 binding to the hTERT promoter, thus abrogating its activating effect on hTERT transcription [hTERT mRNA levels or telomerase activity [Sp3 is a GC-box-binding protein with an antagonistic function to Sp1. Sp3, together with Sp1, has been shown to bind to a recognition site in the proximal promoter of ssential . In factERT gene . This reERT gene . This incription . Howeveractivity .hTERT gene was reported to suppress its expression in telomerase-negative cells but not in cancer cells [hTERT regulatory region. The treatment of telomerase-positive cells with a strong demethylating agent (5-azadC) led to the reactivation of CTCF binding to its response element and repression of hTERT expression. This observation was supported by another study whose results showed that the introduction of sulforaphane (SFN), a potent histone deacetylase inhibitor, in human breast cancer cells resulted in CpG demethylation and hyperacetylation of the region proximal to the first exon of hTERT and this facilitated the binding of hTERT repressors, such as MAD1 and CTCF, in this region [hTERT transcription. In addition to binding to the first exon of hTERT, CTCF was also found to negatively modulate hTERT expression by binding to an enhancer element approximately 4.5 kb upstream of the hTERT transcription start site and this was confirmed by chromosome conformation capture (3C) assay [CTCF is chromatin-binding factor which affects transcription of numerous genes. It mostly acts as a transcription repressor but is known to activate the transcription of several genes. The binding of CTCF to the first exon of the er cells . It prefs region . ConversC) assay .hTERT in human squamous cell carcinoma cells by binding to and modulating hTERT proximal promoter [hTERT promoter activity and telomerase activity. The effect of E2F1 on hTERT promoter has been proposed to counteract the activating effect of c-Myc on the regulatory region [hTERT inhibition [E2F1 is a transcription factor which binds to E2 recognition sites found in the promoter of numerous genes, especially those involved in cell cycle regulation and DNA damage response. It has also been shown to play a direct role in the transcription regulation of promoter . The ovey region  and act hibition .hTERT translation start site and repress the expression of the gene [hTERT transcription. However, another report has argued against the direct role of VDR on hTERT transcription repression. Instead, it was proposed that the inhibitory effect of VD3 stems from its suppression of hTERT mRNA by destabilizing it [hTERT expression through the induction of miR-498, which in turn targets the 3’ untranslated region (3’ UTR) of hTERT transcripts and thus decreases its expression [Vitamin D(3) receptor (VDR) is the nuclear receptor for 1,25-dihydroxyvitamin D(3) (VD3) and it can form a heterodimer with another nuclear receptor, retinoid X receptor (RXR), whose ligand is 9-cis-retinoic acid (9-cis-RA). In prostate cancer cells, this heterodimer was found to bind directly to a region approximately 2.5 kb upstream of the gene . In vivoizing it . This wapression ,223.hTERT transcription is androgen receptor (AR). Treatment of prostate cancer cells with AR agonist resulted in the inhibition of hTERT promoter activity and, conversely, treatment of AR antagonist failed to recapitulate this inhibition [hTERT promoter.Another nuclear receptor that has a repressive effect on hibition . On top hTERT gene expression in telomerase-positive cells but suppresses it in telomerase-negative ones. It does this by binding to five GC-box motifs found in the proximal promoter of hTERT [hTERT activation or repression in various cell lines. However, the expression and hTERT promoter-binding of Sp1 alone is insufficient to drive the desired effect. Epigenetic environment, especially the presence of suitable histone marks, is crucial in actualising these effects.Sp1 is a transcription factor that binds to GC-box motifs in the promoter of its target gene and regulates its expression, either activating or repressing the transcription process depending on cellular context. Sp1 is known to activate of hTERT ,165. MuthTERT expression on its own or in conjunction with specific co-activators. Sp1 may work cooperatively with c-Myc and bind their respective response elements in hTERT proximal promoter to upregulate the transcription of the gene [hTERT transcription activity. Kaposi’s sarcoma-associated herpesvirus (KSHV) latent protein, latency-associated nuclear antigen (LANA), was also found to bind directly to Sp1 in KSHV-infected body-cavity-based lymphoma (BCBL) cells and enhance the binding of Sp1 to its cognate binding sites in the hTERT proximal promoter [hTERT promoter region and causes the activation of the target gene [hTERT expression [hTERT proximal promoter at a site flanked by two GC-boxes and this allows for the synergistic interaction between NFAT1 and Sp1, resulting in the activation of hTERT transcription [hTERT and this leads to an increase in the expression of the gene [hTERT gene expression in various cell lines , a HDAC inhibitor [hTERT was shown to be enhanced by the overexpression of Sp1 [hTERT promoter and suppress hTERT activation in human lung adenocarcinoma cells [As alluded to earlier, epigenetic environment is important in Sp1-mediated regulation of s region ,226. Thenhibitor . TSA-indn of Sp1  or HDAC1n of Sp1 . In contma cells .hTERT transcription has been contentious. It was initially reported to repress hTERT expression in HeLa cells, where the transient overexpression of its components (c-Fos and c-Jun or c-Fos and JunD) strongly represses hTERT promoter activity [hTERT translation start site. Mutation of these binding sites led to the abrogation of the repressive effect of AP-1 on hTERT. On the other hand, ectopic expression of the viral protein HBZ and JunD in HeLa cells was shown to activate hTERT promoter [hTERT promoter.AP-1 is a transcription factor complex which consists of components belonging to the c-Jun, c-Fos, Activating Transcription Factor (ATF) and J Domain-Containing Protein (JDP) families. Its effect on activity . This supromoter . This inhTERT transcription is two-fold—it can act as an activator and also a repressor depending on the tissue of origin. Ectopic expression of EGR-1 in villous cancer cells led to an increase in hTERT expression, both in the mRNA and protein levels [hTERT. Furthermore, expression of EGR-1 was found to correlate with that of hTERT during trophoblastic development and in patients with preeclampsia. On the contrary, the examination of mRNA levels in primary cervical cancer tissues revealed a negative correlation between the expression of EGR-1 and that of hTERT. Further studies involving epidermoid carcinoma and squamous cell carcinoma cell lines supported this observation—overexpression of EGR-1 resulted in the suppression of hTERT expression [hTERT gene transcription.EGR-1 is a transcription factor whose activity is crucial for mitogenesis and cellular differentiation. The effect of this protein on n levels . This efpression . These rhTERT gene is solely positive, HIF-2α plays dual role in modulating hTERT expression. In several renal carcinoma cell lines tested, HIF-2α overexpression led to an increase in hTERT promoter activity and its depletion resulted in lower hTERT mRNA levels [hTERT proximal promoter and the recruitment of p300 and histone acetyltransferase to the regulatory site. In contrast, ectopic expression of HIF-2α in three glioma cell lines led to repression of hTERT gene expression [hTERT gene regulation in different cellular context.HIF-2α is a transcription factor which is involved in cellular oxygen regulation. Unlike another hypoxia-inducible factor, HIF-1, whose regulatory effect on A levels . This popression . This suhTERT expression. KLF2 was shown to bind to a putative site in the first exon of the hTERT gene in resting T cells, resulting in the repression of the latter [hTERT expression in telomerase-negative, alternative lengthening of telomere (ALT) cells and human fibroblast cells via direct binding to a response element in the proximal promoter region of hTERT [hTERT expression significantly. Furthermore, telomerase expression was found to be sufficient in replacing KLF4 function in the maintenance of self-renewal in human embryonic stem cells, showcasing that hTERT is one of the primary targets of KLF4 in stem cell maintenance. Interestingly, a recent study revealed that the knockdown of hTERT via shRNA in HeLa cells led to a decrease in KLF4 expression [hTERT is a complex and important one.KLFs are a group of proteins which share a common structure, namely a C-terminal domain consisting of three zinc finger configurations. The human genome contains 17 KLF family genes and they may act as activators or repressors of their target genes. Two of the members of the KLF family proteins are known to play opposite roles in the regulation of e latter . Howeverof hTERT . Converspression . This shhTERT gene expression. NFX1-123, with c-Myc as a co-activator, was shown to bind to an X-box motif located adjacent to a known E-box motif, the canonical response element of c-Myc and other E-box proteins, and activate hTERT promoter activity [hTERT expression by binding to the same X-box motif and recruiting Sin3A and histone deacetylases (HDACs) to the proximal promoter of hTERT [hTERT gene in human fetal kidney cells.NFX1 is traditionally known as a transcription repressor. It plays a key role in determining the duration of inflammatory response via its interaction with and transcription regulation of the promoter of MHC class II genes. Three isoforms of NFX1 have been identified and two of these have opposite roles in the regulation of activity . In contof hTERT . This rehTERT gene expression. USF1/2 heterodimers were found to bind directly to E-box motifs found on the proximal promoter of hTERT in both telomerase-positive and -negative cells. However, they specifically activate hTERT expression only in telomerase-positive cells [hTERT expression in telomerase-positive oral squamous cancer cells and E6-expressing immortalized cells [hTERT expression upon the forced expression and siRNA-induced knockdown of the USF genes, respectively. Moreover, binding of USF1 and USF2 to the proximal E-box motif in the hTERT promoter diminishes in cells expressing E6 and this is accompanied by an increase in c-Myc binding to the same site [hTERT expression is dependent on the relative abundance of the splice isoforms in the cells. They showed that both full-length and truncated forms of USF2 can bind to the hTERT proximal E-box. However, the latter has a dominant-negative effect on the former, therefore depleting the full-length isoform and abrogating its activating effect on hTERT expression [USFs are a group of proteins belonging to the basic helix-loop-helix leucine zipper protein family. They function as transcription regulators and are known to bind to E-box motifs found in regulatory elements of their target genes. USF1 and USF2 have been shown to play both activating and repressive roles in the regulation of ve cells . This poed cells ,202. Thiame site . Interespression . This copression .hTERT expression. hTERT promoter is eventually silenced when the cells are terminally differentiated. On the other hand, reprogramming of somatic cells to induced pluripotent stem cells results in the activation of hTERT expression. The epigenetic regulation of hTERT promoter plays an important role in these two opposite hTERT transcriptional states. The following section will focus on the known modes of epigenetic regulation of hTERT expression including methylation of hTERT promoter, histone modifications, and modulation by sirtuins and non-coding RNAs. However, it remains unclear how hTERT expression is differentially regulated during cancer development, cellular differentiation and reprogramming.Differentiation of pluripotent stem cells is accompanied by the downregulation of hTERT and decreases acetylated lysine-12 of histone H4 (H4K12Ac) levels at this site. Treatment of telomerase negative cells with histone deacetylase inhibitor (HDACi), trichostatin A (TSA), could activate transcription of hTERT gene [hTERT expression in umbilical cord mesenchymal stem cells as well [hTERT expression [hTERT. These findings have been recapitulated by other groups with additional mechanism. Deletion of SIRT1 leads to significant reduction in telomerase expression, thus causing telomere dysfunction-induced foci and nuclear abnormality [hTERT promoter [hTERT promoter via reduction in H3K4 trimethylation and histone H3 acetylation.In telomerase-positive cells, telomerase expression is associated with hyperacetylation of histone H3 and H4 and methylation of lysine-4 of histone H3, whereas in telomerase-negative cells (ALT cells), the silencing of the expression of telomerase is associated with hypoacetylation of H3 and H4 and methylation of lysine-9 of histone H3 . InductiERT gene .TSA was  as well . Sirtuin as well , stress  as well . SIRT1,  as well . Deacety as well , and thipression . Knockinormality ,235. ThehTERT promoter region contains clusters of CpG islands with dense GC-rich regions. This suggests that DNA methylation may play a role in the regulation of hTERT expression [hTERT transcription start site (TSS) in most cancer cell lines and embryonic stem cells [hTERT expression in cancer cells [hTERT promoter, data interpretation, and categorization, and the DNA methylation detection methods is summarized in DNA methylation is involved in gene silencing ,237. Thepression . Howeverem cells ,240. Coner cells ,241,242.hTERT alleles, specifically in its promoter region (see below for a detailed discussion of hTERT promoter mutations and their effect on hTERT expression). This is attributed to the reversal of hTERT gene silencing on the one active allele while the other one remains silenced. The promoter of the active gene was found to be decorated by the permissive histone H3K4me2/3 mark, while the promoter of the silenced gene showed higher deposition of H3K27me3, a histone mark indicating transcriptionally repressed heterochromatin regions [hTERT promoter region, the point mutation allows for the binding of the transcription factor GA Binding Protein Transcription Factor Alpha and -Beta 1(GABPA/B1) heterodimer to the newly created site, which in turn facilitates the recruitment of RNA Polymerase II to the promoter, resulting in the transcription of hTERT gene.Several cancer cell lines contain point mutations in only one of its  regions . ConcomihTERT transcription regulation is complex. It involves the interplay between the various components of molecular and cellular biology. This is not surprising given the crucial role telomerase plays in the maintenance of stem cell and cellular transformation. Despite the wealth of knowledge in the roles transcription factors and epigenetic modifiers play in the regulation of hTERT expression, a lot more has to be done to elucidate the mechanism underlying the switching off and on of hTERT gene during cellular differentiation and cellular reprogramming, and during cellular transformation, respectively.It is clear from the multitude of evidence presented above that hTERT promoter have further confirmed the importance of telomerase activation in human cancers [Recent advances in DNA sequencing technologies have enabled large-scale genome sequencing studies across various tumor types. Many alterations in protein-coding genes have been identified ,249. On  cancers ,252.hTERT promoter mutations first came into attention with the discovery of highly recurrent mutations in this region in melanomas. In particular, two hotspot C > T point mutations were observed at the nucleotide position 124 bp and 146 bp upstream of the translation start codon (ATG) and these specific mutations were termed C228T and C250T, respectively. Other less common mutations were also detected in the hTERT promoter region such as the CC > TT mutations at −124/−125 and −138/−139 positions. The observation that the frequency of mutations detected in hTERT promoter is higher than those in the B-Raf Proto-Oncogene Serine/Threonine-Protein Kinase (BRAF) gene, and that the presence of highly recurrent point mutations in just two nucleotide positions strongly suggests that these are driver mutations which play key roles at various stages of tumorigenesis in melanoma [hTERT activation in human cancers. In addition, the discovery of driver alterations in the non-coding portion of the human cancer genome was a novel advancement in understanding the role of mutated non-coding sequences in transcriptional deregulation and tumorigenesis.Transcriptional regulation at the level of melanoma ,252.TheshTERT promoter mutations in melanomas and other types of cancers, particularly at the two hotspot locations, C228T and C250T  across the different tumor types  transcription factors whose recruitment to these sites may result in increased promoter   in the hTERT promoter region, specifically at the two hotspots, making it the most frequently mutated gene identified so far in this region. Hence, these hTERT promoter mutations have the potential to become a good biomarker for clinicians to conduct early detection of bladder cancer and subsequent follow-up tests for cancer progression or recurrence in patients. In hepatocellular carcinomas, about 42% mean mutation frequency (n = 1463) in the hTERT promoter region was observed. Similarly, their high prevalence suggests that these hotspot mutations may be utilized as candidate biomarkers for early detection and monitoring. Lastly, the high prevalence of hTERT promoter mutations in various subtypes of gliomas also suggest that the mutations can serve as useful biomarkers to aid in classification and prognostication via extraction of samples from cerebrospinal fluid. Further studies to confirm such a causal relation in various tumor types will be necessary before genome-based clinical classifications based on hTERT promoter mutations can find their way into the clinics.As shown in hTERT promoter is activated or repressed, novel strategies to therapeutically target telomerase can be developed. For instance, new specific small molecular inhibitors may be developed to interfere with the binding of ETS/TCF transcription factors to the CCGGAA/T binding motif that is only present in cancer cells that carry the specific C228T or C250T mutation. In addition, as mentioned above, the presence of hTERT promoter mutation in a tumor may be used as a biomarker to predict subsequent clinical response to a telomerase inhibitor drug.Activation of telomerase enzyme is classified as one of the classic hallmarks of cancer as it confers cells with replicative immortality . Hence,