The results of this screen can then be compared against a reference uninsulated device to select a set of insulated promoters providing a precise level of expression. We verify this method by insulating the constitutive, inducible, and repressible promotors of a four transcriptional-unit inverter (NOT-gate) circuit, finding both that order dependence is largely eliminated by insulation and that circuit performance is also significantly improved, with a 5.8-fold mean improvement in on/off ratio.Variation in the DNA sequence upstream of bacterial promoters is known to affect the expression levels of the products they regulate, sometimes dramatically. While neutral synthetic insulator sequences have been found to buffer promoters from upstream DNA context, there are no established methods for designing effective insulator sequences with predictable effects on expression levels. We address this problem with Degenerate Insulation Screening (DIS), a novel method based on a randomized 36-nucleotide insulator library and a simple, high-throughput, flow-cytometry-based screen that randomly samples from a library of 4 This prhallenge , 9: at phallenge . Insteadhallenge , 11.The poor performance of an engineered genetic regulatory network can be due to many different factors, including interactions between the various devices making up the network, poorly understood interactions of devices with their operating environment, and physical or functional interactions between engineered components and the host genetic machinery , 12. In α-subunit (α-CTD) of the RNA polymerase (RNAP) to A/T-rich Upstream Promoter (UP) elements placed between -40 and -60 bp of the transcription start site (TSS) can increase transcription by 30-60x [α-CTD has been reported to bind to DNA sequences at least as far as 100 bp upstream of the TSS resulting in altered protein expression levels [Moreover, the effect of upstream DNA sequence can be quite strong, particularly for the minimal promoters often favored in synthetic biology, which are often only a few dozen base-pairs long. Binding of the y 30-60x , the RNAn levels , 15, andn levels .et al. showed that a synthetic insulator sequence inserted at the 5’ boundary of susceptible minimal promoters did successfully inhibit increased rate of transcription by an UP element [One approach to reducing the impact of upstream DNA sequence is to extend the 5’ end of the promoter with an “insulator” sequence, thereby increasing the separation of the active components of the promoter from the potentially variable portion of upstream sequence and thus hopefully reducing its impact. Davis  element . However36 potential insulated promoters in a high-throughput, flow-cytometry-based screen, comparing against a reference device for precise expression control. We test this method by applying it to insulate a minimal constitutive promoter , an inducible promoter  and a repressible promoter . Our experiments show that using DIS-insulated promoters largely eliminates the order dependence of component transcriptional units on the performance of a four transcriptional unit genetic inverter (NOT-gate). Regularizing promoter behavior also improves circuit performance, providing an incremental increase in on/off ratio with each additional insulated promoter to an ultimate 5.8-fold mean improvement when all promoters are insulated.To address these challenges, we have developed Degenerate Insulation Screening (DIS), a novel method for insulating promoters based on a randomized 36-nucleotide insulator library. DIS draws random samples from this library of 4Degenerate Insulation Screening (DIS) is a novel promoter insulation protocol based on amplification with degenerate primers and screening to compare against reference expression levels. This method is motivated by the fact that there are many possible mechanisms by which an upstream sequence might affect promoter expression , but any given such mechanism is likely to pertain to only a small fraction of sequences . We thus hypothesized that randomly generated sequences should have a reasonable probability of behaving as good insulators, but that it might also be necessary to screen many candidate sequences in order to find insulating sequences that closely match a reference expression level from an uninsulated device. Initial tests with a small number and hand-generated random DNA sequences produced results consistent with this hypothesis , leadingIn the remainder of this section, we first present the DIS protocol and results showing that DIS produces libraries with a wide distribution of device behaviors, supporting selection of multiple insulator options matching to a desired reference behavior. We then validate the insulation provided by DIS promoters by application to a four transcriptional-unit inverter circuit, finding that DIS-insulated promoters largely eliminate order dependence, as well as significantly improving circuit performance.Basic idea of DIS is to generate a custom set of insulators for each promoter by screening a large collection of constructs with random spacers inserted upstream of the promoter. The insulators are then those spacer constructs whose expression levels match a desired reference level of expression .E. coli and cultured to produce clonal populations of individual library elements, which may then be further split for testing multiple input conditions. All samples are then screened by flow cytometry to identify samples containing DNA spacers that empirically provide the precise desired expression level or levels ; these spacers are the desired library of insulators. Finally, the insulator library can be sequenced to identify effective spacer-promoter pairs and/or used directly in construction of larger systems . Additional details may be found in Materials and Methods and in Our implementation of this concept in the DIS protocol is shown in To test DIS, we applied it to insulate three commonly used promoters: a constitutive minimal promoter (J23100), an inducible promoter (pBAD), and a repressible promoter (pTet). For a baseline to compare against, we tested the impact of upstream sequence on these promoters against four MoClo assembly , 17 fusiThe distributions of geometric mean fluorescent expression from the DIS libraries for J23100, pBAD, and pTet are shown in To test the efficacy of DIS-generated insulators, we combined the three promoters to form an “inverter” circuit comprising four transcriptional units . In thisWe test the impact of local DNA context on circuit performance by using MoClo assembly , 17 to cEach permutation set was then cultured in triplicate overnight in both the uninduced  state and the induced  states, and the expression levels of GFP and RFP measured by flow cytometry. Finally, we compare expression levels by calculating the geometric mean expression of each sample and taking the ratio of the “on” state over the “off” state . Ideally, all permutations should show similar strong levels of GFP induction and RFP repression.As expected, the behavior of the uninsulated circuit is quite sensitive to local DNA context: Insulation with DIS-generated spacers markedly improves both the strength and regularity of performance: The in-context selection of DIS does indeed appear to be important for obtaining this effective insulation: randomly-generated spacers fail to effectively insulate from local DNA context, yielding performance nearly as bad as if there were no insulation at all . We alsoIf promoters are to be used as part of complex genetic designs, it is unavoidable that their upstream genetic context will change, since they may be placed after any number of other genetic constructs. Even small variations in the DNA sequence at the 5’ boundary of a promoter can greatly alter its behavior and cause design failures, and no method has yet been identified for reliably predicting the effect of upstream sequence on promoter behavior , 13, 16.Some potential challenges in generalization of this approach remain for future investigation. For example, we note that the degree of variation observed in a collection of random insulators differs significantly between different promoters : it is tAnother important question for future work is to determine how broad a class of DNA context effects can actually be insulated against by means of this approach. The promoters tested in this paper have mechanisms that are prototypical and representative of a large class of commonly used genetic components, so it appears likely that the method we have presented will effectively insulate against a wide range of transcriptional/translational components. At the same time, there are clearly limits to the efficacy of spacers: for example, spacers provide no mechanism to protect against side effects from nearby epigenetic modification or sequence editing.We also note that our screening method is defined in terms of a reference device to screen candidate insulators: in the absence of a defined reference that is desired as a target, our approach might instead be used for “tuning” to select a desired expression level from a given promoter or to adjust and optimize the performance of engineered biological systems. Likewise, although we have focused on bacterial promoters, there is nothing to prevent this technique from being applied to promoters in other organisms or to other components that prove to be sensitive to local genetic context.Finally, many widely used bacterial promoters are either known to or likely to suffer from the upstream genetic context issues that we have addressed in this paper. We thus expect that it will be valuable for the synthetic biology community to begin applying our method to develop and characterize insulated variants of a wide variety of bacterial promoters. Furthermore, in many cases we expect that it will be useful to prepare not just one but multiple variants of an insulated promoter, as is readily supported by our method, so that if a promoter is used multiple times in a design, different insulators can be used in each case in order to minimize undesired homology. Developing such insulated promoter libraries will enable faster, simpler, and more reliable use of those promoters in new biological engineering efforts in both academia and industry.A summary of key method information follows; additional details may be found in Supporting Information Basic parts  from the CIDAR-MoClo part collection  were assE. coli. Expression of samples from the insulated library was measured by flow cytometry and compared against a reference device.Randomized 36 nt insulated promoter libraries were generated by performing an inverse PCR reaction using divergent primers to the promoter to be insulated. A phosphate group and 18N extension was added to the 5’ end of both forward and reverse primer. Overnight digestion of PCR product with DpnI  was performed to remove traces of PCR template. PCR product was purified using a GenCatch PCR Purification Kit . PCR product was ligated in an overnight 16°C blunt end ligation using T4 DNA ligase . The purified PCR product was ligated and transformed into Spacers for constitutive promoter J23100 were selected by screening candidates from an insulated GFP expression cassette library of J23100_E (12.5X lower expression than J23100_A). Spacers for inducible and repressible promoters pBAD and pTet were obtained by screening insulated expression cassettes of pBAD_F : CGTTGTTCGCGACCCTGTTCTGAGGCAATGTTGGGCpBAD (second): TAGCACGCTTTTCTGCGTATGGTTGGTCAGCTTCTApTet: CGGAGAGAATACATAGTAAAGAAACCTCACTGTTGTThe 36 nt spacers ultimately selected to serve as insulators had the following sequences:Additional information related to these sequences may be found in Supporting Information μL of MoClo reaction mixture into 12-25 μL of Alpha Select Gold Efficiency chemically competent cells  following the manufacturer’s protocol. Cells were grown overnight at 37°C on MoClo assembly level-appropriate antibiotic selective LB agar supplemented with 80 μL of 20mg/μL 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-GAL) and 100 μL of 0.1M isopropyl-β-D-thiogalactopyranoside (IPTG)  for blue-white screening.Transformations were performed using 3Plasmids were purified using the QIAprep Spin Miniprep kit (Qiagen) following the manufacturers’ protocols. Inverters were checked for correct size by performing colony PCRs on MoClo transformation colonies using 2X Taq Mastermix  and VF (5’-tgccacctgacgtctaagaa) and VR (5’-attaccgcctttgagtgagc) primers. Sequences were analyzed using Benchling .E. coli cells (Bioline) containing the confirmed plasmid construct/clone from insulated promoter cassette library of interest. Colonies were grown in 200 μL LB broth (Sigma-Aldrich) with the appropriate antibiotic in sterile 96-well, deep well plates  in triplicate (for confirmed plasmid construct) or singly (for insulated promoter expression cassette library) for 14 hours at 37°C shaking at 300 rpm. Cells were diluted into phosphate buffered saline (BioExpress) in 96-well round bottom plates before measurement using a high-throughput sampler (HTS).All fluorescent expression devices were characterized using a BD LSRFortessa SORP flow cytometer. RFP fluorescence was measured using a solid-state Coherent Sapphire 561 nm laser at 100 mw strength with a PE-Texas Red 610/20 filter. GFP fluorescence was measured using a solid-state Coherent Sapphire 488 nm laser at 200 mw strength with a FITC 530/30 filter. LB agar  plates containing the appropriate antibiotic were inoculated with Alpha-Select Gold Efficiency E. coli cells , respectively. FITC channel measurements (for GFP) are calibrated to MEFL using SpheroTech RCP-30-5-A beads.Flow cytometry data was converted from arbitrary units to compensated MEFL  using the TASBE characterization method , 20. An S1 FileAdditional information about methods employed, including all DNA sequence information.(PDF)Click here for additional data file.S1 FigA preliminary test of the variable effect of random insulators on expression was performed with 36 nt spacer sequences designed by screening random DNA base composition resembling the intergenic bacterial DNA base composition for tandem promoter elements  and abse(PDF)Click here for additional data file.S2 FigConstitutive expression from (a) J23100 promoter, (b) pBAD inducible promoter, and (c) pTet repressible promoter, when downstream of MoClo A, E, F, and G sites. Error bars show +/- two std.dev. of mean fluorescence. The J23100 and pBAD promoters both show a significant difference in expression with different fusion sites, with more than a 10-fold difference in expression between the highest and lowest expression levels, while pTet expression did not vary significantly.(PDF)Click here for additional data file.S3 FigA preliminary test of the effect of insulator length on expression was performed with spacer sequences designed to several lengths  by screening random DNA base composition resembling the intergenic bacterial DNA base composition for tandem promoter elements  and abse(PDF)Click here for additional data file.S4 FigDistribution of expression levels for induced and uninduced expression levels from spacer screen for pBAD, corresponding with fold-change distributions reported in (PDF)Click here for additional data file.S5 FigDistribution of expression levels for induced and uninduced expression levels from spacer screen for pTet, corresponding with fold-change distributions reported in (PDF)Click here for additional data file.S6 FigComparison of GFP and RFP fold changes upon induction with L-arabinose of the 24-inverter permutation set in which each transcriptional unit is insulated with a different spacer from (PDF)Click here for additional data file.S7 FigComparison of GFP and RFP fold changes upon induction with L-arabinose of the 24-inverter permutation set in which only the J23100 promoter has been insulated with a DIS spacer. Insulation is denoted by a smaller gray box positioned between upstream MoClo fusion site and colored box denoting a transcriptional unit. Performance improves over uninsulated inverters, but is not as good as inverters with more insulators.(PDF)Click here for additional data file.S8 FigComparison of GFP and RFP fold changes upon induction with L-arabinose of the 24-inverter permutation set in which only the two pBAD promoters have been insulated, each with a different DIS spacer. Insulation is denoted by a smaller gray box positioned between upstream MoClo fusion site and colored box denoting a transcriptional unit. Performance improves over uninsulated inverters, but is not as good as inverters with more insulators.(PDF)Click here for additional data file.S9 FigComparison of GFP and RFP fold changes upon induction with L-arabinose of the 24-inverter permutation set in which both the J23100 and two pBAD promoters have been insulated with a DIS spacer. Insulation is denoted by a smaller gray box positioned between upstream MoClo fusion site and colored box denoting a transcriptional unit. Performance improves over a single insulator inverter, but is not as good as insulating all promoters, particularly for change in RFP expression levels.(PDF)Click here for additional data file.S1 DatasetAll data underlying the findings described herein.(ZIP)Click here for additional data file.


TERT, encoding the telomerase catalytic subunit. Genome-wide association studies (GWAS) have identified six single nucleotide polymorphisms (SNPs) in the TERT promoter associated with decreased breast cancer risk, although the precise causal variants and their mechanisms of action have remained elusive. Luciferase reporter assays indicated that the protective haplotype reduced TERT promoter activity in human mammary epithelial and cancer cells in an estrogen-independent manner. Using single variant constructs, we identified rs3215401 and rs2853669 as likely functional variants. Silencing of MYC decreased TERT promoter activity but neither MYC nor ETS2 silencing conferred allele-specificity. In chromatin immunoprecipitation experiments, the ETS protein GABPA, but not ETS2 or ELF1, bound rs2853669 in an allele-specific manner in mammary epithelial cells. Investigation of open chromatin in mammoplasty samples suggested involvement of three additional variants, though not rs3215401 or rs2853669. Chromosome conformation capture revealed no interaction of the TERT promoter with regulatory elements in the locus, indicating limited local impact of candidate variants on the TERT promoter. Collectively, our functional studies of the TERT-CLPTM1L breast cancer susceptibility locus describe rs2853669 as a functional variant of this association signal among three other potentially causal variants and demonstrate the versatile mechanisms by which TERT promoter variants may affect breast cancer risk.The multi-cancer susceptibility locus at 5p15.33 includes  Telomeres consist of DNA tandem repeats and telomere binding proteins, which together form secondary nucleoprotein structures at the end of chromosomes , 2. In hA major function of telomeres is to prevent the ends of chromosomes from being recognized as DNA strand breaks and therefore to protect against genomic instability. Also, due to the “end-replication problem”, the ends of linear DNA molecules are not fully replicated, and this limits the number of divisions a cell can undergo before entering the state of permanent cell cycle arrest referred to as senescence –10. TherSome cell types such as stem and germline cells maintain a constant telomere length –15. In tTERT upstream region includes many transcription factor consensus sequences and a CpG island. Presumably, these characteristics contribute towards its tightly regulated transcriptional activity [TERT promoter activity have been reported, such as transcriptional activators  and repressors , and sex hormones (estrogen and androgen) [Telomerase contains a protein catalytic subunit with reverse transcriptase activity, TERT, and an RNA subunit, TERC , 22, 23.activity . Many rendrogen) –40.TERT-CLPTM1L region on chromosome 5p15.33 as a breast cancer susceptibility locus with three independent genetic signals, which comprise variants in strong linkage disequilibrium [TERT promoter and harbors six closely correlated candidate variants: rs2736107, rs2736108, rs2736109, rs145544133 (synonym: rs10548207), rs3215401 and rs2853669, which for simplicity will be designated variants 1-6 in the figures and Table BRCA1 mutation carriers but most strongly with ER-negative breast cancer [TERT promoter activity in vitro, whereas rs2853669 had no effect [GWAS have identified the ilibrium . The stro effect .TERT transcription, and are predicted to alter numerous transcription factor (TF) binding motifs  underlying its association with breast cancer risk.The aim of the present study was to define the effect of the risk-associated haplotype in the TERT promoter activity [TERT promoter with either all the protective alleles (wildtype), or all risk-associated alleles of the six candidate variants (risk-associated haplotype). Luciferase assays showed that the risk-associated haplotype reduced TERT promoter activity significantly in Bre80 normal breast cells, ER-positive MCF-7 and ER-negative MDA-MB-231 breast cancer cell lines , though not with the first three . To clarify the roles of rs145544133, rs3215401 and rs2853669, we generated single variant constructs harboring individual risk-associated alleles. There was no detectable effect of rs145544133 but significant reduction of TERT promoter activity with either rs3215401 or rs2853669   assays were performed to detect interactions between the TERT promoter and NcoI fragments between hg19 co-ordinates chr5: 1,249,069-1,358,885 in Bre80 and MCF-7 cells.Since long-range DNA-DNA interactions play an important role in the regulation of gene expression, we also asked whether the effect of the protective haplotype is restricted to local TERT promoter as bait, we found one interaction peak that suggested chromatin looping between the TERT promoter and the CLPTM1L promoter in both cell lines  on 22 breast tissue samples, followed by genotyping using allele-specific SNP-type assays. In the heterozygous samples (8/22), FAIRE-enriched samples of rs2736108 and rs2736109 displayed a shift to their major alleles,  [All six candidate variants in risk association signal 1 are located within or in close proximity to transcription factor binding sites. The strongest candidate causal variant under our test conditions, rs2853669, is surrounded by three major transcription factor binding sites, i.e. E2F, ETS and E-box motif. ChIP data indicated an allele-specific effect of rs2853669 for MYC and the ETS family member, GABPA, but not for E2F1 and the ETS-family members, ETS2 and ELF1. MYC is a strong activator of  tissues , 28, 63. tissues , cooperaer cells  in an al in 2013 , 45  .TERT promoter activity in any of the three mammary epithelial cell lines. This does not rule out the possibility that ETS2 can induce TERT expression in other cell lines or under conditions that were not tested in our study [TERT promoter [ETS2 knock-down did not reduce ur study . Future promoter , which cpromoter , 45 and TP53 mutations [TERT promoter activity and was unaffected by ETS2 silencing in our analysis of these cell lines argues that it can act independently of ETS2. Besides, there are additional members of the ETS family of transcription factors which need further investigation as they might influence TERT expression depending on rs2853669, GABPA being the best candidate at present.It may be possible that ETS2 binding requires additional events such as gain-of-function utations  which arTERT promoter regulation appears to be very sensitive to additional sequence variation. This is indicated by our findings, that 11 additional variants masked the effect of rs2853669 in previous work [TERT promoter activity in luciferase assays. The variant rs3215401 does not directly reside within a known TF binding site and so its role remains elusive at this stage.While rs2853669 had the strongest effect in our analyses, ous work , 42, andTERT expression in vivo. Due to their effect on chromatin configuration, an effect of open chromatin may not be detectable in transient transfection-based luciferase assays and additional experiments using a native chromatin state will be needed to define the functional interaction of rs2736108 and rs2736109 with the remaining variants of the risk-associated haplotype. This may also be true for estrogen induction of the TERT promoter. TERT expression is known to be activated by estrogen stimulation [TERT promoter activity using luciferase reporter assays. Reporter assays are artificial promoter systems and may not fully recapitulate the effects of variants on native chromatin structure. Further work in model systems will be required to elucidate how additional variants in the risk-associated signals may synergize (rs3215401) or antagonize (rs2736107) in concert with rs2853669 . Cell lines were authenticated using Geneprint 10 (Promega) conforming to ATCC standard ASN-0002-2011. STR DNA profiling was a provided service from the QIMR Berghofer core facility.The normal mammary epithelial cell line Bre80 , was grFor estrogen-induction assays, cells were treated with 10 nM fulvestrant  in phenol-red free DMEM , supplemented with 580 mg/L L-glutamine  48 hours prior to treatment with 20 nM estrogen and transfection.NcoI (New England Biolabs) in 1.2x restriction enzyme buffer . After 12-15 h the restriction enzyme was heat-inactivated (80°C for 20 min in 1.6% SDS). Free ends of cross-linked DNA were re-ligated under high-dilution conditions , 0.1 mg/ml BSA (NEB B9000S), 1 mM ATP (Sigma A7699) and 40000 U T4 ligase (HC) (NEB M0202T)) and the cross-linking was reversed by proteinase K  incubation. Ligated DNA products were isolated by phenol-chloroform extraction and precipitated . Further, the fragments were purified using the Amicon ultra- 0.5 30K filter unit (UFC503008) according to the manufacturer's instructions. The genomic control DNA was extracted separately by the salting-out method.Chromosome conformation capture (3C) analysis was conducted as previously described  in the BTERT promoter  as the bait and then repeated the analysis with another bait in the CLPTM1L promoter (hg19: 1342655-1358754). Interactions between the bait promoter fragment and all other fragments along the TERT-CLPTM1L locus  were analyzed by qRT-PCR  with following settings: initial denaturation at 95°C for 5 min, 55 cycles of 95°C for 45 s, 60°C for 45 s and 72°C for 60 s, final extension at 72°C for 5 min. The MyTaq HS DNA polymerase (Bioline BIO-21113) was used together with Syto9 (Life Technologies S34854) for qRT-PCR set-up. All qRT-PCR products were gel-separated in order to verify single amplicons.Initially, we used the fragment covering the Primers were designed using Primer Express (Applied Biosystems) with the following criteria: primer starts within 80-100 bp from restriction site, melting temperature between 68-70°C, 3’ GC clamp and 40-60% GC content. Primer sequences and genomic regions analyzed are shown in Allele-specificity was investigated by sequencing the qRT-PCR products. Deviations of less than one-third from the heterozygous condition were considered non-significant.TERT promoter sequences (3915bp) harboring all major or all risk-associated alleles of rs2736107, rs2736108, rs145544133, rs2736109, rs3215401 and rs2853669 were synthesized by GenScript and were cloned into a pGL3 reporter vector (Promega E1751). Combinations and single variants were subsequently introduced by cloning fragments containing the variant(s) of interest into the wildtype construct. The constructs were sequence verified after they were obtained from GenScript and following each completed cloning step.Wildtype and variant haplotype TERT promoter luciferase reporter constructs and 50 ng of pRLTK (Promega E2241) using Lipofectamine 2000 (Life Technologies 11668027), according to the manufacturer's instructions. After 24 h the cells were lysed using the Dual Glo Luciferase Assay System (Promega E2920), according to the manufacturer's instructions. Luminescence activity was measured with Glomax (Promega) or Synergy H4 hybrid reader (BioTek). All statistical tests were performed on the log-scale, however for ease of interpretation values were back transformed for plots presented. The log-scale was used as the underlying relationship is multiplicative. All further calculations were performed on the log scale using only addition and subtraction. This includes background correction  were obtained from GE Dharmacon and resuspended in 50 ul DNase- and RNase-free water for a 100 uM stock solution. Cells were harvested and reverse transfected with 20 nM siRNA using Lipofectamine 3000 (Life technologies L3000008), according to the manufacturer's instructions. After 24 h cells were harvested and reverse transfected with equimolar amounts of luciferase reporter constructs and 50 ng of pRLTK using Lipofectamine 3000. The next day, cells were lysed using the Dual Glo Luciferase Assay System, according to the manufacturer's instructions. Luminescence activity was measured with Glomax. All statistical tests were performed on the log-scale, however for ease of interpretation values were back transformed for the plots presented. Data from three or more independent experiments were analyzed using the three-way ANOVA with experiment as a blocking factor and an interaction term for silencing cell line. Multiple comparisons between groups of interest were adjusted using Bonferroni's multiple hypotheses testing correction.3VO4 (Sigma), 0.1 mM phenylmethanesulfonylfluoride , 2 mg/ml leupeptin (Serva Feinbiochemika), 2 mg/ml aprotinin (Serva Feinbiochemika), 0.2% Triton X-100, and 0.3% Nonidet P-40 (Sigma). Cells were incubated for 30 min on ice. Separation of protein extracts was achived by SDS-PAGE and proteins were transferred via immunoblotting. Primary antibodies were rabbit anti-ETS2 , rabbit anti-MYC  and mouse anti-β-actin . For detection anti-mouse IgG horseradish peroxidase-labeled secondary antibody  and ECL (Thermo Scientific/Pierce 32106) were used.For cell lysis a cell extraction buffer was used with following ingredients: 50 mM Tris pH 7.4 (Merck), 150 mM NaCl (Merck), 2 mM ethylene glycol tetraacetic acid , 2 mM ethylene diamine tetraacetic acid , 25 mM NaF (Sigma-Aldrich), 0.1 mM NaChIP experiments were conducted using the MAGnify™ Chromatin Immunoprecipitation System (Thermo Scientific 492024) following the instructions of the manufacturer. Exclusively, the normal mammary cell line Bre80 was used for ChIP as these cells represent the normal state in beast tissue. Cells were cross-linked with 1% formaldehyde followed by cell lysis. Approximately 250 000 cells were required per sample. The sonicator Bandelin Sonoplus HD 2070 generated chromatin fragments with a final size of 200-400 bp. For the immunoprecipitation with Protein A/G magnetic beads the following antibodies were used: 4 ul MYC , 2 ul E2F1 , 5 ul ETS2 (GeneTex GTX104527), 3 ul GABPA (Santa Cruz sc-22810-X) and 3 ul ELF1 (Santa Cruz sc-631-X). DNA was isolated, PCR-amplified and sequenced via Sanger sequencing with following primer pair: 5’-GGAGGCGGAGCTGGAAGGTGAAGG-3’ and 3’-CCAGTGGATTCGCGGGCACAGAC-5’.ChIP). Data were normalized to the negative rabbit IgG antibody  was subtracted from the input Ct value and the percent of input were calculated using the formula 100*2^(Adjusted input Ct - Ct y Figure .FAIRE was conducted as previously described . In brieAll statistical analysis were performed using GraphPad Prism 6 apart from three-way ANOVA, which was conducted via STATA. Log-transformations were done in Microsoft Excel.


In mammals, genomic DNA that is roughly 2 m long is folded to fit the size of the cell nucleus that has a diameter of about 10 μm. The folding of genomic DNA is mediated via assembly of DNA-protein complex, chromatin. In addition to the reduction of genomic DNA linear dimensions, the assembly of chromatin allows to discriminate and to mark active (transcribed) and repressed (non-transcribed) genes. Consequently, epigenetic regulation of gene expression occurs at the level of DNA packaging in chromatin. Taking into account the increasing attention of scientific community toward epigenetic systems of gene regulation, it is very important to understand how DNA folding in chromatin is related to gene activity. For many years the hierarchical model of DNA folding was the most popular. It was assumed that nucleosome fiber (10-nm fiber) is folded into 30-nm fiber and further on into chromatin loops attached to a nuclear/chromosome scaffold. Recent studies have demonstrated that there is much less regularity in chromatin folding within the cell nucleus. The very existence of 30-nm chromatin fibers in living cells was questioned. On the other hand, it was found that chromosomes are partitioned into self-interacting spatial domains that restrict the area of enhancers action. Thus, TADs can be considered as structural-functional domains of the chromosomes. Here we discuss the modern view of DNA packaging within the cell nucleus in relation to the regulation of gene expression. Special attention is paid to the possible mechanisms of the chromatin fiber self-assembly into TADs. We discuss the model postulating that partitioning of the chromosome into TADs is determined by the distribution of active and inactive chromatin segments along the chromosome.This article was specially invited by the editors and represents work by leading researchers. The concept of hierarchic chromatin organization in the eukaryotic cell nucleus has been developed rather long ago and is commonly accepted now –3. A matIt was always clear that higher-order compaction levels must follow the 30-nm chromatin fiber, but the mode of chromatin packing at these levels was long unknown. One of the most common model suggestes that 30-nm fibers are organized in loops, which are attached to the nuclear matrix , 17–20. Several studies published in the recent years questioned the existence of 30-nm chromatin fibrils in living cells –26. A prDrosophila, the number of TAD boundaries coinciding in embryonic and culture cells constitutes 40-50% [Drosophila [Drosophila TADs were observed in plants [Drosophila chromosomes.The above results do not contradict the mere existence of higher-order hierarchic levels in chromatin compaction, but indicate that these levels are not based on assembly of regular structures, such as the 30-nm fiber. An important contribution to understanding the principles of hierarchic chromatin folding was made in studies that employed the so-called C methods, which address the physical proximity of particular genome regions in the three-dimensional space of the cell nucleus. The methods are based on ligation of DNA fragments located close together. The procedure was proposed as early as the 1990s , 32, buts 40-50% . Substans 40-50% . It shous 40-50% –45. As ts 40-50%  . The prIn the case of transcription, the definition of a functional domain is also not a trivial question. Before the era of whole-genome research, a limited number of genomic models were used in the majority of experimental studies, the mammalian and avian globin gene loci being the most common ones , 62–64. Additional line of evidence supporting the idea that TADs represent structural and functional units of the genome arises from the studies of cell differentiation and reprogramming. In the model system of ESC differentiation into several distinct lineages, TADs were found to be largely stable along the genome, but demonstrated a high flexibility in both inter- and intra-TAD interactions . TADs coIt should be noted that the establishment of enhancer–promoter communication should depend on how fast the enhancer and its target promoter are brought close together in the nuclear space. A restriction of the search area to a TAD will certainly reduce the time it takes to establish enhancer–promoter communication. Lack of rigidity in the TAD structure is of importance in this context. Alternative configurations of the chromatin fiber continuously interchange within a TAD . This isMany models were proposed in the literature to describe the mechanisms of TAD formation. Computational simulations showed that entropic forces primarily drive the formation of compact contact domains in a polymer model confined to a limited space. The profile of polymer partitioning into contact domains may further be modulated by additional factors, such as bridges between distant polymer regions . The finIt is crucial to understand what factors determine the relatively specific profiles of chromosome partitioning into TADs. Two alternatives are possible here Fig. . One is Drosophila [It is assumed in the boundary element hypothesis that insulators play a main role in TAD separation –83. Insuosophila , 92. In osophila , 94. CTCosophila –98.Drosophila is less clear. In particular, loop domains limiting spatial contacts between distant genomic elements were not observed in Drosophila cells. We studied the distributions of several insulator proteins relative to TAD boundaries in four Drosophila cell lines of different origins and did not detect an appreciable enrichment in binding sites for dCTCF and Su(Hw) for TAD boundaries [Drosophila does not have loop domains [The CTCF ability to organize DNA in loops certainly contributes to the TAD formation. High-resolution Hi-C maps constructed for various human and mouse cells  were colundaries . On the undaries , 44, 48.undaries . The potundaries , 12. Theundaries , 102. Asundaries , 103. Anundaries , 105. Ouundaries . With voundaries . In the  domains  that are domains .Drosophila, which is known to have several architectural proteins in addition to CTCF.Other models of TAD formation emphasize the role of architectural proteins, which are thought to pull parts of a linear segment of a chromatin fiber together to produce a compact TAD by interacting with each other. To explain the existence of isolated TADs, the models assume a multiplicity of architectural protein groups, each ensuring the formation of a particular TAD –111. TheSaccharomyces cerevisiae [Arabidopsis thaliana [Drosophila genome, while annotated genes are almost twice as many as in Drosophila.If TADs indeed are predominantly inactive chromatin domains separated by active regions, then the TAD size must depend in a certain way on the gene sizes, the gene distribution through the genome, and the relative sizes of the active and inactive genome fractions. Indirect evidence for this assumption can be found in the literature. For instance, the average size of contact domains is 2–10 Kb in revisiae , in whicthaliana , 112, whDrosophila, promoter-specific H3K4 monomethylation, ubiquitously transcribed genes and P-element integration events are highly enriched within TAD boundaries [As mentioned above, genome organization in contact domains is hierarchic. The question of the level at which contact domains should be considered to be TADs or sub-TADs is solved to a great extent intuitively, based on the common views of average TAD sizes in various organisms . In mammundaries , denotinundaries . A modelundaries , 116. Thundaries . The macundaries . The situndaries , clusterundaries .Drosophila [We think that genetic information that is not in demand is stored in TADs in the simplest variant. This TAD function is prevalent in osophila . Howeverosophila . A genomosophila , and thiDrosophila, inactive chromatin domains became assembled in compact masses (TADs) due to electrostatic interaction of nucleosomes located on neighboring fibers [Although the importance of the 3D genome organization for regulation of gene expression has long been envisaged , the expg fibers . These dg fibers , 113. Whg fibers , mammalig fibers . Althougg fibers , 116. Hog fibers . The rel


Notably, biochemical and chromatin immunoprecipitation analyses reveal constitutive binding of CREB to target gene promoters in the absence of neuronal activity, whereas recruitment of CRTC1 to proximal CRE/TATA promoters depends on neuronal activity. Neuronal activity induces rapid CRTC1 dephosphorylation, nuclear translocation and binding to endogenous CREB. These results indicate that neuronal activity induces a preferential binding of CRTC1 to the transcriptional complex in CRE/TATA-containing promoters to engage activity-dependent transcription in neurons.Gene expression mediated by the transcription factor cAMP-responsive element-binding protein (CREB) is essential for a wide range of brain processes. The transcriptional coactivartor CREB-regulated transcription coactivator-1 (CRTC1) is required for efficient induction of CREB target genes during neuronal activity. However, the mechanisms regulating induction of specific CREB/CRTC1-dependent genes during neuronal activity remain largely unclear. Here, we investigated the molecular mechanisms regulating activity-dependent gene transcription upon activation of the CREB/CRTC1 signaling pathway in neurons. Depolarization and cAMP signals induce preferential transcription of activity-dependent genes containing promoters with proximal CRE/TATA sequences, such as  By regulating hundreds of neuronal genes in response to a variety of extracellular stimuli, CREB mediates neuronal excitability, synaptic plasticity and long-term memory2. Synaptic transmission activates Ca2+ and cAMP signaling pathways leading to phosphorylation of CREB at Ser133, a required step for transcription of multiple genes3. CREB phosphorylation mediates binding of CREB to the histone acetyl-transferase (HAT) paralogues CREB binding protein (CBP) and p300, which leads to induction of transcriptional activity4. However, CREB phosphorylation is essential but not sufficient for gene transcription5, which suggests that multiple cellular mechanisms, including recruitment of coactivators act in concert with CREB phosphorylation to engage the transcriptional machinery.One of the most relevant features of the nervous system is its ability to sense, adapt and respond to environmental changes. These cellular responses occur in most cases through regulation of genetic programs that ensure appropriate changes in gene expression, a process mediated by transcription factors such as the cAMP-responsive element (CRE) binding protein (CREB) that are ubiquitously expressed, although CRTC1 is the main isoform expressed in neurons8. In resting conditions, CRTC1 is phosphorylated by the salt-induced kinase 1/2 (SIK1/2) promoting its interaction with 14-3-3 proteins and sequestration in the cytoplasm10. By contrast, CRTCs are activated by dephosphorylation and nuclear translocation in response to Ca2+ and cAMP signals. Synaptic activation increases cAMP levels and activates PKA, which phosphorylates and inhibits the SIK1/2 kinase, thereby preventing CRTC1 phosphorylation11. Simultaneously, Ca2+ influx through L-type voltage gated calcium channels activates the Ca2+-dependent phosphatase PP2B/calcineurin, which dephosphorylates CRTC1 inducing its translocation to the nucleus14. Once in the nucleus, CRTC1 binds to the bZIP domain of CREB, enhancing the interaction with the TATA box-binding protein (TBP)-associated factor TAFII130, a subunit of the transcription factor TFIID9. In neurons, CRTC1 dephosphorylation and nuclear translocation mediates activation of CREB target genes16, although how synaptic activity mediates CRTC1 recruitment and activation to specific gene promoters is still unclear.CREB-regulated transcription coactivators (CRTCs) regulate multiple biological processes, including metabolism, cell transformation, memory and lifespan, and when disrupted cause age-related brain diseases19. Notably, the specific contribution of each of these factors to activity-induced gene expression in neurons is unknown. Here, we report that neuronal activity triggers rapid and robust expression of CREB target genes containing both consensus CRE sites and TATA box sequences (CRE/TATA) in their proximal promoter region. On the contrary, genes with promoters containing consensus CRE sites lacking close TATA box sequences (CRE/TATA-less) are not efficiently induced by neuronal activity. Activity-dependent gene transcription in neurons involves CRTC1 dephosphorylation, binding to CREB complexes and recruitment to proximal CRE/TATA promoter regions. These results suggest that binding of CRTC1 to CREB/CBP into proximal CRE/TATA-containing promoter regions is a required step for robust activity-dependent gene transcription in neurons.The molecular mechanisms by which CRTC1 selectively binds and activates specific gene promoters in neurons are still largely unclear. The number of CRE sites, their distance to the transcription start site (TSS) and the presence of proximal TATA boxes within the promoter region, and the presence of coactivators seem to be the key determinants of transcriptional responsiveness to CREB in non-neuronal cellshttp://natural.salk.edu/CREB; ref.18], and regardless whether they are located proximal (−500 bp to +300 bp) or distal (>−500 bp) from the transcription start site (TSS). The number of CRE sites for the analized genes are: c-fos (6), Dusp1 (2), Nr4a1 (6), Nr4a2 (6), Psen1 (3), Psen2 (2) and Ptgs2 (2) (Table 2+ and/or cAMP with depolarizing concentrations of KCl and the adenylate cyclase activator forskolin (FSK), respectively. Treatment of cortical neurons with FSK or KCl resulted in a differential time-dependent increase of mRNA levels of c-fos  = 298.9, P < 0.0001; time, F = 45.48, P < 0.0001; interaction, F = 20.49, P < 0.0001), Dusp1  = 324.2, P < 0.0001; time, F = 19.11, P < 0.0001; interaction, F = 14.32, P < 0.0001), Nr4a1  = 154.3, P < 0.0001; time, F = 12.91, P = 0.0003; interaction, F = 8.10, P = 0.0002), Nr4a2  = 99.40, P < 0.0001; time, F = 7.48, P = 0.004; interaction, F = 4.32, P = 0.007) and Ptgs2  = 68.02, P < 0.0001; time, F = 2.44, P = 0.1152; interaction, F = 3.55, P < 0.017), but not Psen1  = 2.90, P = 0.064; time, F = 5.73, P = 0.0119; interaction, F = 1.27, P = 0.318) or Psen2  = 20.49, P < 0.0001; time, F = 29.61, P < 0.0001; interaction, F = 4.92, P < 0.004)  = 0.40, P = 0.756; time, F = 3.11, P = 0.071; interaction, F = 0.4189, P = 0.856). Post hoc analyses revealed that FSK plus KCl induced in combination a significant additive or synergistic effects on expression of c-fos (~30–45 fold), Dusp1 (~15–20 fold), Nr4a1 (~20–30 fold), Nr4a2 (~20–40 fold) and Ptgs2 (~20–40)  or absence (CRE/TATA-less) of a TATA box within 300 bp downstream of the CRE site, according to the CREB Target Gene database [Psen1 and Psen2, which contain promoters with distal consensus CRE/TATA sites  and proximal CRE/TATA-less sites (Psen1: 244 bp) from the TSS in both mouse and human genes  reaching a peak at 15 min  = 2.4, P < 0.05; Fig. 1/2 ≈ 1 min) and persistent dephosphorylation of CRTC1 in cultured neurons  = 2.9, P < 0.03), which is associated with translocation of CRTC1 from the cytosol to the nucleus  = 8.5, P < 0.0003; acute, F = 14.0, P < 0.0001; Fig. Crtc1 shRNA that decreases Crtc1 mRNA and protein levels (~75%) was able to reduce significantly activity-induced CREB transcriptional activity  = 30.1, P < 0.0001; Fig. )15. In agreement with the above results, neuronal activity rapidly (t1/2 < 1 h) and significantly increased transcript levels of the canonical CREB target genes c-fos (fold change at 4 h: 40), Dusp1 (10–12 fold), Nr4a1 (40 fold), Nr4a2 (50 fold) and Ptgs2 (20 fold), whereas changes in Nr4a3 (4 fold) and Psen1/Psen2 (0.6 fold) were modest , Nr4a1, Nr4a2 and Ptgs2 (Treatment effect: P < 0.001), but not those of Nr4a3, Psen1 or Psen2 , Dusp1 , Nr4a1 , Nr4a2 , Nr4a3 , Psen1 , Psen2  and Ptgs2 . We then explored the possibility that CREB and CRTC1 could bind differentially to the proximal promoter gene regions depending on the presence of CRE sites in close proximity to TATA boxes (CRE/TATA sites) by performing quantitative chromatin immunoprecipitation (ChiP-qPCR) analyses using CREB and CRTC1 antibodies and different set of primers amplifying distinct regions of CRE-containing promoters  containing TATA boxes (CRE/TATA) in the promoter regions differ substantially among the analyzed genes both in human and mouse genomes Table . For theers Fig. . In agre4a2 Fig. . Notably4a2 Fig. . Conversion Fig. . To examTTX Fig. . These rPsen1 and Psen2. CRTC1 does not bind to the promoter regions of Psen1 and Psen2 in basal or stimulated conditions  in basal but not stimulated conditions . Similarly, CREB occupancy was significantly enriched close to the most distal CRE/TATA-less site of the Psen2 promoter (−4412 bp) in basal but not stimulated conditions (P < 0.05 Veh vs FSK/KCl). Since CREB binds to the proximal promoter region of Psen1 in basal conditions, it is possible that CREB could regulate Psen1 gene expression independently of CRTC1. To test this idea, we developed four different ShRNAs (ShRNA 1–4) targeting the mouse CREB gene (Creb1). Creb1 ShRNA1 and 2 significantly reduced Creb1 protein  = 76.86, P < 0.0001) and mRNA  = 14.59, P < 0.001) in primary neurons  protein levels but did not affect PS1 protein levels, as assayed with an antibody that recognize the PS1 C-terminal fragment (CTF), in human HEK-293T cells and cortical neurons  half CRE site of a SARE enhancer element of Arc24. In contrast to binding to proximal CRE/TATA-containing promoters of target genes , CRTC1 does not bind to CRE/TATA-less promoter regions of Nr4a3, Psen1 and Psen2. This result is consistent with the requirement of proximal CRE/TATA sequences for efficient CREB-mediated transcription in non-neuronal cells17. The absence of CRTC1 recruitment to Psen1 and Psen2 promoters contrasts with basal binding of CREB to the proximal promoter regions of Psen1, a result suggesting the possibility that CREB could regulate Psen1 transcription depending on the physiological conditions. For instance, neuronal activity reduces binding of CREB to the Psen1 promoter while is recruited to other target gene promoters, a result resembling that observed when overexpressing CRTC1 in vivo24. However, whereas neuronal activity does not affect Psen1 mRNA levels in cortical neurons,  glutamate and BDNF activate Psen1 expression in a CREB-dependent manner in neuroblastoma cells25. Notably, multiple consensus transcription binding sites are present in the promoter proximal region (−811 bp to +140 bp) of the human PSEN1 gene including those for CREB, Lyf-1, CdxA, AML-1a, Nkx-2, Pbx-1, E47, MZF1, Sp1 and Elk-125. Our biochemical studies showing that CREB inactivation does not affect Psen1 levels in neurons reinforce the view that CREB is unlikely to be the main transcription factor regulating Psen1 expression. Likewise, we also detected recruitment of CREB to a full CRE site located distally (−4412 bp) from the transcription start site of the Psen2 promoter. It is relevant, however, that presenilins regulate CREB-mediated transcription in the adult brain likely through a mechanism involving the transcriptional coactivators CBP and CRTC128. Future experiments will be necessary to elucidate the presenilin-regulated mechanisms of CREB-dependent transcription, and the role of proximal and distal CRE sites as regulatory elements  of the presenilin genes.The first interesting finding is that neuronal activity induces robust activation of CREB target genes containing proximal CRE/TATA consensus promoter sequences. Notably, whereas CREB binds constitutively to target gene promoters, robust activity-induced transcription is associated with increased recruitment of CRTC1 to the promoter proximal regions containing conserved CRE/TATA sequences. Basal recruitment of CREB to gene promoters is largely independent of neuronal activity since blocking spontaneous activity with TTX does not abolish its binding to the 30. However, although CREB phosphorylation is important it does not always correlate with activation of gene expression suggesting that phosphorylation kinetics and/or other transcriptional effectors regulate gene transcription32. Our results indicate that Ca2+ and cAMP signals have differential effects on expression of CREB target genes in neurons, whereas simultaneous Ca2+/cAMP signals result in additive or synergistic effects on gene expression. This differential effect on gene expression is unlikely due to differences on CREB phosphorylation. For instance, we found that Ca2+/cAMP signals induce gradual increase of CREB phosphorylation but rapid CRTC1 dephosphorylation in cortical neurons . It is known that besides inducing similar levels of CREB phosphorylation, cAMP is more effective than stress signals in promoting CREB/CBP complex formation and gene transcription34. CBP recruitment to CREB is sufficient for CREB-mediated gene activation, whereas efficient transcriptional induction does not only requires CREB/CBP complex formation but also optimal cAMP signaling and histone acetylation36. Indeed, CRTCs potentiate CREB transcriptional activity through binding to CREB independently of CREB Ser133 phosphorylation21. Accordingly, our biochemical analysis showed that the Ca2+/cAMP-induced potentiation of gene transcription involves recruitment of CRTC1 to CREB/CBP complexes in target promoter regions. This result agrees with previous reports showing that Ca2+ and cAMP signals act synergistically on gene expression by activating CRTC1 or CRTC2 in neurons and non-neuronal cells, respectively37. Indeed, neuronal activity regulates CRTC1 nuclear translocation while cAMP modulates its persistence into the nucleus11. Conversely, Ca2+ and cAMP signals do not always cooperate to activate gene expression in response to neuronal activity38. The fact that genetic inactivation of CRTC1 blocks Ca2+/cAMP-mediated transcription strongly suggests that CRTC1 recruitment to CREB is essential for activity-induced CREB-dependent transcription in neurons. Importantly, neuronal activity and memory training activate preferentially CREB-dependent transcriptional programs40. In agreement, CRTC1 selectively regulates expression of CREB target genes involved in synaptic plasticity and memory, including among others Bdnf, c-fos, Dusp1, Fgf1, Nr4a1 and Nr4a241.CREB-dependent transcription depends, at the molecular level, on CREB phosphorylation at Ser13317. CRTC1 dephosphorylation and nuclear translocation are key molecular mechanisms triggering activity-dependent CREB target gene transcription in neurons42. Conversely, deficient dephosphorylation and nuclear translocation of CRTC1 cause transcriptional and memory deficits in experimental models of Alzheimer’s disease43. Future investigations on the mechanisms and effectors involved in CREB-dependent gene transcription may shed light on novel pathways and therapeutics in age-related cognitive disorders.In summary, this study shows that binding of CRTC1 to neuronal gene promoters depends on neuronal activity resulting in induction and/or maintenance of CREB-dependent transcription in neurons. Despite basal binding of CREB to gene promoters, efficient CREB-dependent transcription depends on activity-induced recruitment of CRTC1 to promoters containing proximal CRE/TATA elements. These results are consistent with the idea that the presence of consensus CRE/TATA sequences within the core promoter is required for efficient activation of CREB-dependent transcription mediated by interaction of CREB and the transcriptional complex in non-neuronal cellsCrtc1 ShRNA lentivirus was generated as described15). Creb1 shRNA sequences were designed using the siDESIGN Center tool  (http://dharmacon.gelifesciences.com/design-center/). The complete sequences of the oligos used to generate the Creb1 shRNAs are as follows (target sequence is underlined).TCGTAGAAAGAAGAAAGAATTTCAAGAGAATTCTTTCTTCT TTCTACGTTTTTTGGAAC-3′Creb1_shRNA_1-Forward: 5′-Creb1_shRNA_1-Reverse: 5′-TCGAGTTCCAAAAAACGTAGAAAGAAGAAAGAATTCTCTTG AAATTCTTTCTTCTTTCTACGA-3′GAGCAATACAGCTGGCTAATTCAAGAGATTAGCCAGCTG TATTGCTCTTTTTTGGAAC-3′Creb1_shRNA_2-Forward: 5′-TCreb1_shRNA _2-Reverse: 5′-TCGAGTTCCAAAAAAGAGCAATACAGCTGGCTAATCTCTTG AATTAGCCAGCTGTATTGCTCA-3′ACTGATGGACAGCAGATTCTATTCAAGAGATAGAATCTG CTGTCCATCAGTTTTTTTGGAAC-3′Creb1_shRNA_3-Forward: 5′-TCreb1_shRNA_3-Reverse: 5′-TCGAGTTCCAAAAAAACTGATGGACAGCAGATTCTATCTCTT GAATAGAATCTGCTGTCCATCAGTACAGCAGCTCATGCAACATCATTTCAAGAGAATGATGTTG CATGAGCTGCTGTTTTTTGGAAC-3′Creb1_shRNA_4-Forward: 5′-TCreb1_shRNA_4-Reverse: 5′-TCGAGTTCCAAAAAACAGCAGCTCATGCAACATCATTCTCTT GAAATGATGTTGCATGAGCTGCTGA-3′.Creb1 or scramble or pLVTHM-Crtc1 or scramble shRNAs with psPAX2 (packaging), and pM2G (envelope) vectors using X-tremeGENE transfection reagent (Roche) as described15. Lentivirus-containing medium was collected 48 h later and lentivirus were purified and stored at −80 °C.Oligos were annealed and cloned into the HpaI-XhoI sites of the lentiviral vector pLLX, downstream of the U6 promoter. Lentiviral particles were generated by co-transfecting HEK293T cells with pLLX-4. Neurons were plated in poly-D-lysine (PDL) coated dishes at a density of 375 cells/mm2 using DMEM containing 5% FBS penicillin-streptomycin  and glutamine . Two hours after seeding, DMEM medium was replaced by Neurobasal medium containing B27 supplement (Thermo Fisher Scientific), penicillin-streptomycin  and glutamine . For gene inactivation, 4 DIV neurons were infected with Crtc1 or Creb1 ShRNA lentiviral vectors (1–2 transducing units/cell). Neuronal activity was induced by treating 7–10 days in vitro (DIV) neurons with 10 μM forskolin (FSK), 55 mM potassium chloride (KCl) or both for 0, 2, 4 and 8 hr as indicated15. In some experiments, neuronal activity was blocked by adding 1 μM tetrodotoxin  at 11 DIV.Mouse embryonic brains  were dissected and cortices were digested in cold Hank’s Balanced Salt Solution (HBSS) containing 0.025% trypsin-EDTA (Thermo Fisher Scientific) and DNAse (Sigma). Trypsin activity was inhibited by adding 20% FBS and the tissue was dissociated in HBSS containing DNAse and MgSOCrtc1 and Creb1 ShRNAs lentiviral vectors (1–2 transducing units/cell) and treated with vehicle or KCl (30 mM) plus forskolin  for 0–8 h at 12 DIV. For luciferase assays, 7–15 DIV neurons in 24-well dishes were transfected using LipofectAMINE 2000 (Thermo Fisher Scientific) for 24 h with pCRE-luc reporter vector , containing 4xCRE sequences and a TATA-box element, and pRL-TK Renilla luciferase plasmid , which encodes Renilla luciferase gene driven by HSV-TK promoter in a pGL4.74 vector . Neurons were treated with vehicle or FSK/KCl chronically (0–12 h) or acutely (1 h) and then washed and incubated for 0–12 h. Samples were analyzed by triplicate in at least three independent transfections with the dual-luciferase activity assay (Promega) in a Synergy HT luminometer (Bio-Tek) as described15.Primary neurons were infected at 4 DIV with scramble or TM II reverse transcriptase (Thermo Fisher Scientific) at 25 °C for 10 min, 42 °C for 60 min and 72 °C for 10 min. Quantitative real time RT-PCR (qRT-PCR) was performed in duplicate in at least 3–5 samples using an Applied Biosystems 7500 Fast Real-Time PCR system (Thermo Fisher Scientific). Data analysis was performed by the comparative ΔCt method using the Ct values and the average value of PCR efficiencies obtained from LinRegPCR software44. Gene expression was normalized to Gapdh for cultured neurons or the geometric mean of Gapdh, hypoxanthine guanine phosphoribosyl transferase (Hprt) and peptidylprolyl isomerase A (Ppia) for brain samples using the NormFinder, BestKeeper and geNorm algorithms45.For quantification of mRNA, RNA from cultured neurons or hippocampal tissue was purified using the PureLink RNA Mini Kit (Thermo Fisher Scientific). RNA integrity number (RIN) was measured using the Agilent 2100 bioanalyser (Agilent Technologies). RNA  was reverse-transcribed in 50 μl of a reaction mix containing 1 μM of Oligo (dT) primers, 1 μM random hexamers, 0.5 mM dNTP, 0.45 mM DTT, RNAseOut (10 units) and SuperScript3VO4, 50 mM NaF, 1 mM PMSF) containing protease and phosphatase inhibitors . Proteins were quantified with the BCA protein assay kit (Thermo Fisher Scientific), resolved on 8–14% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to PVDF membranes before blotting with the following antibodies: rabbit anti-CRTC1 , CREB (1:250) and phosphorylated CREB (Ser133) , NURR1 , β-actin  and anti-GAPDH  and PS1 Loop  or β-tubulin . We used distinct percentages of acrylamide in the gels for separation of phosphorylated/dephosphorylated CRTC1 . Protein (2 mg) was immunoprecipitated overnight at 4 °C with or without antibodies against CREB or CRTC1  followed by incubation with Dynabeads protein G (Invitrogen). The beads were spun to obtain a supernatant (unbound fraction) and a pellet, which was washed in ice-cold buffer (x3) and eluted with 2x SDS sample buffer (immunoprecipitated fraction). Quantitative analysis of immunoblots, normalized to control loading protein, was performed by densitometry using ImageJ . Original blots scans are shown in Supplementary Information.Neurons were lysed in cold lysis buffer  and KCl (30 mM) for 15 min, or pretreated with tetrodotoxin  for 12 h. Cells were crosslinked with 1% formaldehyde, lysed in ChIP buffer  and chromatin was sheared between 200 and 500 bp by sonication using a BioruptorPlus . Fragmented chromatin was analyzed using the High Sensitivity DNA Kit (Agilent Technologies). Immunoprecipitation (2.5 μg DNA) was performed overnight in diluted ChIP buffer  with or without anti-CRTC1 or CREB antibodies . Input and immunoprecipitated DNA were decrosslinked and amplified by real-time qPCR using specific primers, and the fold enrichment of the amplified target regions was calculated over an irrelevant region in the chromosome 4.Chromatin immunoprecipitation (ChIP) was performed essentially as describedP < 0.05 were considered significant.Statistical analysis was performed using t-test, one-way or two-way analysis of variance (ANOVA) for multiple comparisons followed by Bonferroni or Dunnett’s tests using GraphPad Prism software. Differences with This study was performed in accordance with the experimental European Union guidelines and regulations (2010/63/EU). Experimental protocols and experiments involving vertebrate animals were conducted in accordance with the ethical protocol approved by the Animal and Human Ethical Committee (CEEAH) of the Universitat Autònoma de Barcelona (protocol number: CEEAH 2896) and the local Governmental Ethical Committee of the Generalitat de Catalunya (protocol number: DMAH 8787).All data generated during this study are included in this published article or are available from the corresponding author upon reasonable request.Supplementary Information


Spag16) uses alternative promoters to produce two major transcript isoforms (Spag16L and Spag16S) and encode proteins that are involved in the cilia/flagella formation and motility. In silico analysis of both mouse and human SPAG16L promoters reveals the existence of multiple putative SOX5 binding sites. Given that the SOX5 gene encodes a 48-kDa transcription factor (S-SOX5) and the presence of putative SOX5 binding sites at the SPAG16L promoter, regulation of SPAG16L expression by S-SOX5 was studied in the present work.The mammalian sperm-associated antigen 16 gene , encoding another axonemal protein, is activated by S-SOX5. Therefore, S-SOX5 may regulate formation of motile cilia/flagella through globally mediating expression of genes encoding axonemal proteins.S-SOX5 regulates transcription of human  Sox genes are classified into 10 groups from A to J [The family of Sox transcription factors is defined by the presence of a conserved high mobility-group (HMG) domain that mediates DNA-binding and is highly similar to that of the sex-determining region (SRY) protein , 2. Basem A to J . They dim A to J . Availabm A to J . Selectim A to J .SOX5 is primarily expressed in the short (S-SOX5) and long form (L-SOX5) of transcripts [L-SOX5 cDNA is predicted to encode a 763-amino-acid protein that exceeds S-SOX5 by 416 residues [S-Sox5 was originally cloned from testis [Spag6), whose translated product is enriched in the tissues with motile cilia, particularly in the testis [The SOXD group is composed of SOX5, SOX6 and SOX13 [nscripts , 8. L-SOresidues . Mouse Sm testis . The resm testis –13. Latem testis , and it e testis .Spag16) is the orthologue of Chlamydomonas reinhardtii pf20 that encodes an axonemal protein essential for flagellar motility. Chlamydomonas mutants carrying pf20 mutation display paralyzed flagella with defects in axonemal central apparatus [SPAG16 genes are expressed as two major transcripts of 1.4 and 2.5 kb with different expression patterns. The human 1.4 kb transcript was detected in multiple tissues whereas the human 2.5 transcript was highly expressed in testis [Mammalian sperm-associated antigen 16  was co-transfected with S-SOX5 expression plasmids (S-SOX5/pcDNA3) into human bronchial epithelial cells BEAS-2B and the relative luciferase activity was measured. Endogenous expression of S-SOX5 has been reported in BEAS-2B cells, suggesting this cell line can be used to study S-SOX5-mediated gene regulation [SPAG16L expression was detected, which is about 10× higher than that of the control empty pGL3-basic plasmids. When co-transfected with S-SOX5, the relative luciferase activity was elevated about 100-folds  or infected with S-SOX5 adenovirus (Ad/S-SOX5) were generated, and expression of S-SOX5 protein in these cells was confirmed by Western blot analysis. Even though, S-SOX5 was undetected in the controls, the S-SOX5 protein (48 kDa) was produced in both cell lines and clearly increased when infected with S-SOX5 adenovirus  and ChIP assay was conducted with an antibody specifically against S-SOX5 or rabbit IgG. Two primer sets flanking putative SOX5 binding sites were designed; as a control, another primer set located about 3 kb upstream of the transcriptional start site without flanking any SOX5 binding site was also designed . As expected, co-transfection of S-SOX5 largely stimulated the SPAG16L promoter activity when both putative SOX5 binding sites (P-I & P-II) were present  led to higher baseline promoter activities; however, the promoter activities were not increased in the presence of S-SOX5  in the absence of S-SOX5 and is also essential for activation of SPAG16L by S-SOX5.To study if S-SOX5 activates Cilia are evolutionarily conserved, filamentous cellular structures that are present on the cell surface and have been implicated in the sensing of environmental signals and cellular motility . Based oFine-tuned regulatory mechanisms mediate expression of stage-specific genes for the formation of distinct types of cilia during ciliogenesis. The sophisticated genetic program of ciliogenesis is modulated probably through the precise presence and maintenance of essential proteins in a time- and tissue-dependent manner , 23. EmpFoxj1 demonstrate the requirement of this gene for biosynthesis of motile cilia in mouse tissues [FOXJ1 belongs to the forkhead/winged-helix family of transcriptional factors . Loss-of tissues , 34. A n tissues , 36.Spag6 [SPAG16L, because the SPAG16L proximal promoter region also contains multiple putative SOX5 binding sites. Our findings demonstrated that S-SOX5 does regulate SPAG16L transcription through binding to the SOX5 binding sites. However, it should be aware that not all the putative SOX5 binding sites predicted by bioinformatic analysis are functional. One of the two putative SOX5 binding sites analysed in this study is not bound by S-SOX5. Thus, experiments must be conducted to verify if these putative binding sites are functional. Overall, this study presents another example that S-SOX5 regulates another gene essential for motile cilia function, and supports the notion that S-SOX5 is a general transcription factor to control formation and function of motile cilia.Our earlier studies demonstrated that S-SOX5, together with FOXJ1, regulates expression of an axonemal gene, Spag6 . To explκBβ, ZNF230 and Catsper1 [SOX5 locus is associated with male infertility [Sperm flagella are special motile cilia. During spermiogenesis, germ cells undergo dramatic morphological changes as they develop into functional sperm. These changes include formation of flagella. Sperm flagella contain a “9 + 2” axoneme. Besides this core axoneme structure, other affiliated structures, including the fibrous sheath and outer dense fibers, are also assembled into the sperm flagella . S-SOX5 Catsper1 , 13, 38 Catsper1 , we hypoertility , and theL-SOX5 allows us to make mutant mice with disruption of S-SOX5 only. Using this model, we will be able to study the function of S-SOX5 in vivo, and probably identify the target genes regulated by S-SOX5 globally.The in vivo function of S-SOX5 is still not known. However, the unique exon not present in SPAG16L by S-SOX5. S-SOX5 activates transcription of SPAG16L through specifically interacting with SOX5 binding sites at the SPAG16L promoter. The data suggest that S-SOX5 plays a regulatory role in the formation of cilia/flagella.This study demonstrates the molecular mechanism underlying the regulation of human SPAG16L promoter luciferase reporter fusion was constructed by cloning a 2 kb human SPAG16L proximal promoter region including the transcriptional start site and multiple putative SOX5 binding sites into the pGL3-basic vector . Similarly, another luciferase reporter fusion was made by cloning a shorter region of SPAG16L proximal promoter into the pGL3-basic vector. This transcriptional luc fusion construct contained the transcriptional start site of SPAG16L and adjacent two putative SOX5 binding sites. The primers for construction of the transcriptional luc fusions were listed in Table The S-SOX5 expression plasmids, the adenovirus expressing S-SOX5, and the RNAi constructs targeting SOX5 transcripts were generated previously in the laboratory [boratory . OligonuSPAG16L promoter construct were mutated using a QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies) following the manufacturer’s instructions. Mutations at the SPAG16L promoter were verified by DNA sequencing. The mutagenic primers for construction of mutated SOX5 binding sites are shown in Table Two SOX5 binding sites in the Equal amount of proteins (50 µg/lane) were heated to 95 °C in sample buffer for 10 min, resolved in 10% SDS–polyacrylamide gels and then electro-transferred to polyvinylidene difluoride membranes (Millipore). After blocking in TBS-T buffer  for 1 h, the membranes were incubated with antibodies against SOX5  or rabbit β-actin  overnight at 4 °C. After being washed in TBS-T, the membranes were incubated with an anti-rabbit immunoglobulin conjugated with horseradish peroxidase (1:2000 dilution) at room temperature for 1 h. SOX5 or β-actin proteins were detected with SuperSignal West Pico Chemiluminescent Substrate (Pierce).ChIP assays were conducted using a ChIP assay kit (Millipore) according to the manufacturer’s instructions. Briefly, BEAS-2B cells were (CRL-9609) purchased from the American Type Culture Collection and infected with AdS-SOX5 for 48 h. After infection, the protein-DNA complexes from the cells were cross-linked by addition of 1% formaldehyde. The cells were suspended in SDS lysis buffer and were sonicated to shear DNA to 200–1000 bp fragments. Samples were precleared with protein A agarose/salmon sperm DNA (50% slurry) and were immunoprecipitated with antibodies against SOX5 or IgG. After washing the immunocomplexes with appropriate buffers, DNA was recovered by reverse cross-linking and purified by phenol/chloroform extraction followed by ethanol precipitation. The DNA fragments were used as a template for PCR reaction with primer sets (Table SPAG16 promoter using FuGENE6 transfection reagent (Roche). Co-transfection was performed with either empty vectors or S-SOX5 expression vectors (S-SOX5/pcDNA3). The cells were cultured for 48 h and the promoter activity was measured by the Dual-Luciferase Reporter Assay System (Promega). Luciferase activity was normalized to Renilla luciferase activity (control vector).Human bronchial epithelial BEAS-2B cells were cultured in BEBM and were plated 24 h before transfection. The cells were transfected with plasmids containing the wild-type or mutated SPAG16L, SOX5 and GAPDH with primers listed in Table Total RNA was extracted from BEAS-2B cells infected with indicated plasmids using TRIzol Reagent (Invitrogen) and was reversed transcribed to cDNA. The cDNA was used for PCR amplification of


Kip1 is a member of the Cip/Kip family of cyclin-dependent kinase (Cdk) inhibitors. It interacts with both the catalytic and the regulatory subunit (cyclin) and introduces a region into the catalytic cleave of the Cdk inducing its inactivation. Its inhibitory capacity can be modulated by specific tyrosine phosphorylations. p27Kip1 also behaves as a transcriptional regulator. It associates with specific chromatin domains through different transcription factors. ChIP on chip, ChIP-seq and expression microarray analysis allowed the identification of the transcriptional programs regulated by p27Kip1. Thus, important cellular functions as cell division cycle, respiration, RNA processing, translation and cell adhesion, are under p27Kip1 regulation. Moreover, genes involved in pathologies as cancer and neurodegeneration are also regulated by p27Kip1, suggesting its implication in these pathologies.The protein p27Kip1 can associate with different proteins, including transcriptional regulators. In contrast, its NH2-terminal region specifically interacts with cyclin-Cdk complexes. The general mechanistic model of how p27Kip1 regulates transcription is that it associates by its COOH region to the transcriptional regulators on the chromatin and by the NH2-domain to cyclin-Cdk complexes. After Cdk activation it would phosphorylate the specific targets on the chromatin leading to gene expression. This model has been demonstrated to apply in the transcriptional regulation of p130/E2F4 repressed genes involved in cell cycle progression.The carboxyl moiety of p27Kip1 in the regulation of transcription, on the transcriptional programs under its regulation and on its relevance in pathologies as cancer and neurodegeneration.We summarize in this review our current knowledge on the role of p27 Kip1 (p27) is a member of the Cip/Kip family of cyclin dependent kinase (Cdk) inhibitors that also includes p21Cip1 (p21) and p57Kip2 (p57) [The protein p27p2 p57) . These tp2 (p57) , 5. Desp7 . Thesep2 (p57) . These rp2 (p57) . In p27 p2 (p57) . Whereasp2 (p57) . The C-tThe cellular levels of p27 are regulated by both transcriptional and post-transcriptional processes. A number of transcription factors (TFs) including FKHR-L1, AFX, FOXO, Sp1, E2F1, and Menin have been identified as transcriptional activators of p27, whereas, in contrast, c-myc, Id3 and Ap1 act as transcriptional repressors –17. HowePhosphorylation of p27 at S10 induces its translocation from nucleus to the cytoplasm Figure . This phRecent evidence indicate that p27 is involved in the regulation of cytoskeleton reorganization and gene expression . It has All the members of the Cip/Kip family have the ability to associate with a number of different cyclin-Cdk complexes and act by binding to both cyclins and Cdks . The claCyclin-dependent kinases (Cdks) are serine/threonine kinases whose activity depends on their association with specific regulatory subunits named cyclins . Cdks inInk4a, p15Ink4b, p18Ink4c and p19Ink4d [Cdk activity is regulated by different mechanisms that include: 1) association with cyclins that activate Cdks, 2) an activating phosphorylation, 3) inhibitory phosphorylations, 4) inactivation by acetylation, and 5) association with inhibitory proteins. As mentioned above, the association of a Cdk with its cyclin partner is needed for a minimal activity. This activity is enhanced by the specific phosphorylation of a threonine residue (T160 in Cdk2) by the cyclin H-Cdk7 complex (Cdk-activating kinase (CAK)) that stabilizes the activated form of the Cdk . Moreovep19Ink4d . These mThe extracellular signals regulating cell cycle trigger the activation of different pathways that finally act by regulating cyclin-Cdk activity through any of these different mechanisms. Also the intracellular cell cycle checkpoints act controlling the activity of Cdks through these mechanisms.In the current model of cell cycle regulation, the main targets of the G1 cyclin-Cdk complexes (Cyclin D-Cdk4/6 and cyclin-Cdk2) are the members of the pocket proteins family: the retinoblastoma protein (pRB), p130 and p107 . During In addition to these main targets, cyclin-Cdk complexes are able to phosphorylate a number of TFs thereby linking cell cycle and transcription . Among tThere are several Cdk subfamilies that are directly involved in the regulation of transcription . The main role of these Cdks is to regulate the activity of RNA polymerase II (RNAPII) and this is performed by phosphorylating specific sites of the C-terminal domain (CTD) of the largest subunit (Rpb1) of RNAPII. Also cell cycle-related kinases Cdk1 and Cdk2 phosphorylate the CTD , 62. DesQuiescent cells have high levels of p27 in the nucleus. However, when cells are induced to proliferate, most of this nuclear p27 translocate into the cytoplasm where it is subsequently degraded. These facts suggested that p27 was playing a role in the nucleus of quiescent cells that had to be related to the inhibition of cell division cycle since proliferating cells needed to reduce or eliminate this nuclear p27. The identification of several chromatin associated proteins, as for instance the transcriptional co-activator PCAF, as p27-binding proteins strongly supported the putative role of p27 in the regulation of transcription . PreviouΔ51 mutant that has a deletion of the first 51 aa and the p27CK− mutant including mutations in the regions of interaction with cyclins and Cdks [CK− mutant additionally showed a significant number of down-regulated p27-TGs that were not decreased in p27KO or p27Δ51 cells [The nuclear localization of p27 in quiescent cells and its interaction with chromatin-bound proteins suggested that p27 could be associated with chromatin and there to play a role as a transcriptional regulator. ChIP on chip analysis performed in quiescent NIH3T3 cells and directed to identify p27-binding sites (p27-BSs) on gene promoters firstly demonstrated that p27 specifically associates with 427 gene promoters. This association was further validated in a number of genes by ChIP followed by qPCR . Gene Onand Cdks , 75) alsand Cdks . Interesand Cdks . In all 51 cells . These r51 cells .Two different articles have reported ChIP-seq analyses that have been performed to identify the p27-BSs in the chromatin of two different cellular types. One has been performed in quiescent HCT116 colorectal cancer cells  and the These ChIP-seq analyses also revealed that 7SK RNA can be putatively regulated by p27. This RNA regulates the activity of P-TEFb that stimulates transcriptional elongation . FinallyRemarkably, GO classification of the protein encoding genes putatively regulated by p27 revealed that in both cellular types, the four more significant biological processes were cell adhesion, neuron differentiation, cell signaling and ion transport Figure . The traTaken together all these results indicate that the role of p27 in transcription is mostly performed by its association to chromatin regions distal from gene promoters and intronic regions. The genes putatively regulated by p27 through the interaction with these regions are preferably involved in the four biological processes mentioned above. However, despite the global association of p27 with gene promoters seems to be limited, the ChIP on chip analysis performed in NIH3T3 cells indicates that several relevant cellular functions as cell cycle progression, mRNA processing and splicing, translation and respiration are regulated by the p27 association with specific gene promoter sequences. A possible interpretation of these data may be that different sets of genes can be regulated by the association of p27 to different chromatin domains.There is no evidence for p27 directly associating with DNA, thus its interaction with the chromatin has to be through chromatin-binding proteins. The identification of the p27-binding proteins that mediate its association with chromatin was firstly achieved by sequence analysis of the p27-BSs on the promoters detected by ChIP on chip . This anΔ51 and p27CK− - MEFs the genes repressed by p130/E2F4 were up-regulated [CK− - MEFs a significant number of p27-TGs was down-regulated. Remarkably, these down-regulated genes were ETS-dependent [Δ51 did not show the down-regulation of these p27-TGs [Δ51 or when p27 is absent in the p27KO). In contrast, when this sequence is present (p27WT) and able to associate the cyclin and Cdk complexes, transcription is temporally regulated [CK−, it may be postulated that despite the first 51 aa are present, the mutated form of this fragment, that prevents its association with cyclin-Cdks, might adopt a conformation that totally unable the accessibility of cyclin-Cdk complexes to phosphorylate the transcriptional regulators on the promoters. In this case it additionally plays a role as a strong repressor.Interestingly, the analysis of the p27-TGs, identified in the ChIP on chip studies, whose expression is altered in cells lacking p27 or in cells with the different p27 mutants gave interesting information . It was egulated . These regulated . Anotherependent . Surpris p27-TGs . These regulated . HoweverDetailed investigations have been performed to analyze the mechanisms by which p27 regulates transcription through E2F4. In quiescent cells E2F4 mainly associates with p130 to form transcriptional repressor complexes on promoters of genes involved in cell cycle progression , 84, 85.IP experiments demonstrated that p27 interacts with members of these complexes as p130, E2F4, several HDACs and mSin3A . MoreoveInterestingly, the association of p27 with the promoters depends on p130. In fact, in p130 KO cells, p27 is not able to interact with these regulatory regions . This asChIP experiments performed at different times after proliferative activation allowed to generate a model describing the basic mechanisms through which p27 regulates the transcription of genes repressed by p130/E2F4 complexes during the G1 phase of the cell cycle . Since tIn quiescent cells, p27 is associated with the promoters of target genes bound directly to E2F4 and p130 by its carboxyl terminus. Simultaneously, cyclin D2/D3-Cdk4 are bound to the KID domain of p27, sited on its amino moiety. At that time, Cdk4 activity is inhibited Figure . When ceInk4a and inducing Cdk4 inhibition [Removal of cyclin D2/D3-Cdk4 complexes from the promoters can be achieved by degrading p27, since these complexes are associated to the promoters through p27. Several mechanisms can participate in this elimination of p27. It has been shown that phosphorylation in Y74 and Y88 of p27 by tyrosine kinases allows the activation of the associated cyclin D-Cdk4 complexes that furtherly can phosphorylate T187 of p27 inducing its subsequent degradation . Additiohibition . Thus, aThe analysis of the mechanism of how cyclin-Cdk2 complexes are incorporated to the promoters revealed that p21 plays an important role in this process . In factAt mid-late G1 of the cell cycle p27-cyclin D2/D3-Cdk4 complexes are substituted by p21-Cyclin D1-Cdk2 Figure . At thatInterestingly, the expression kinetics of p27 and p21 during cell cycle progression is quite complementary. Whereas in G0 the amount of p27 is high, it subsequently decreases at early-mid G1 due to its degradation. However, the p21 levels that are very low in quiescent cells started to increase just when p27 was decreasing at early-mid G1. These different kinetics are due to the fact that p27 regulates the expression of p21 through the TF Pitx2. It has been shown that p27 represses Pitx2 expression, thus, when p27 is degraded, Pitx2 expression increases and since it is a transcriptional activator of p21, it started to rise at that time . This exIt has to be taken into account that in cells lacking p27, at early G1, cyclin-Cdk complexes could be active, although not recruited on the promoters, and able (perhaps with low efficiency) to phosphorylate p130. In such scenario, transcription of target genes would be facilitated and as a consequence also cell cycle progression. This p130 phosphorylation can occur unless p21 was upregulated due to the absence of p27. In this case p21 could inhibit cyclin-Cdk complexes and block transcription or p130/E2F4 repressed genes or at least to delay it. This phenomenon, can explain why in p27KO cells only the up-regulation of Ets-dependent genes was observed in the expression microarrays. Up-regulation of p21 would block transcription of p130/E2F4 depending genes. Thus, the simultaneous deletion of p27 and p21 strongly induce transcription of targets genes. In this case cyclin-Cdk complexes would be not inhibited and would be more efficient to phosphorylate p130 .Ink4a and inducing Cdk4 inhibition [It has also been extensively studied the collaboration between p27 and the co-activator PCAF. The acetyltransferase PCAF  has beenhibition . Thus, Phibition .Recently, it has been found that PCAF is also able to acetylate p27 at lysine 100. This acetylation induces its ubiquitylation and its degradation via proteasome. During cell cycle PCAF acetylates p27 mostly at early G1 and this acetylation participates in p27 degradation observed at that time . This deA first approach to the identification of the transcriptional programs regulated by p27 was the analysis of the genes, whose promoters included specific p27-BSs, identified by ChIP on chip and that were functionally classified by GO analysis . ResultsA recent expression microarray analysis performed in p27KO-MEFs versus controls deeply amplified the functional aspects that could be regulated by p27 . SpecifiIn addition to that, the KEGG pathway analysis of the expression data revealed a number of very interesting pathways putatively regulated by p27. For instance, in addition to those mentioned above, metabolic pathways as glutathione metabolism, purine and pyrimidine metabolism, fatty acid metabolism, pyruvate metabolism and amino acid metabolism are also regulated by p27. Other functions as axon guidance, mismatch repair and nucleotide excision repair are as well under the control of p27. Another very interesting aspect is that cells lacking p27 deregulates a significant number of genes involved several pathologies as cancer and different neurodegenerative diseases as Huntington's disease, Alzheimer's disease and Parkinson's disease .Finally, it is important the fact that in the absence of p27 a significant number of TFs are deregulated. Specifically, the up-regulation (> 3 fold change) of 42 TFs and the down-regulation (> 3 fold change) of 24 TFs was observed in p27KO cells. The alteration of TF expression in p27KO cells could be responsible for most of the expression changes observed in these cells . The anaAlteration of TF expression in p27KO brings up the possibility that the around of 4000 genes whose expression is altered in these cells could be a consequence of different transcription waves. First, a limited number of genes directly regulated by p27 would be initially expressed in p27KO cells, and among them, some TFs, as for instance Mef2c, Sox6, Zfpm2 and Shox2 that have p27-BSs in their vicinity . As a coRemarkably, p27 may be also involved in splicing. A number of specific splicing factors are deregulated in p27KO cells (unpublished results). Interestingly, these results together with those mentioned above regarding the putative regulation of the five major spliceosomal RNAs by p27 suggest an unexpected new role of p27 in this relevant cellular process. However, this putative new role of p27 still remains unexplored.It is very well described that in many different types of human cancer the decrease of p27 or its cytoplasmic localization is associated with increased malignancy and with a worse outcome –114. IniThe expression microarray data reveal that decreased levels of p27 might deregulate the expression of genes involved in at least four of these cancer hallmarks Figure . One of The decrease of p27 levels can also facilitate the acquisition of a second hallmark: deregulating cellular energetics. Specifically, it has been shown that at least 18 genes involved in oxidative phosphorylation and belonging to different complexes of the respiratory chain are down-regulated in p27KO cells. Interestingly, seven of these genes belongs to complex I. Thus, these results suggest that in p27-deficient cells there is a decrease of oxidative phosphorylation. The decrease of respiration is a key mechanism leading to aerobic glycolysis, a characteristics of tumor cells –124. In A number of cytokines and chemokines are upregulated in p27KO cells. Interestingly, some of these chemokines as CXCL1 and CXCL5 act through the binding to the CXCR2 receptor that has been involved in angiogenesis, invasion and metastasis –127. Thuad hoc niche by producing specific components as for instance tenascin C, that amplifies Notch and Wnt signaling, TGF-β, which stimulates fibroblasts to secrete periostin, and Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase 2 (Plod2) [ad hoc niche are under transcriptional repression by p27 [Tumors are thought to arise from mutant stem cells in their native niches. These niches are rich in developmental and self-renewal signals, among them Wnt and TGF-β , 129. Al (Plod2) –136. Alln by p27 . Thus, ln by p27 , 137 plan by p27 . ConsideAs mentioned above, one of the relevant transcriptional programs regulated by p27, identified in the expression microarray analysis, is neuron differentiation . This isAs oxidative stress and mitochondrial dysfunction are also involved in the induction of AD and HD –143, theAltogether these data strongly suggest a participation of p27 in the etiology of these neurodegenerative diseases.Data accumulated during the last years clearly indicate that in addition to its classical role as a regulator of cyclin-Cdk activity, p27 also plays a role as a transcriptional regulator. This role is mediated by its interaction with specific TFs that facilitates its association with defined chromatin regions. p27 mainly associate with intergenic distal and intronic regions, although it also binds to a significant number of gene promoters. Up to now, a number of TFs that binds to p27 have been reported. It interacts with p130/E2F4 complexes, Ets1, Pax5, Ngn2, AHR, Pax4 and MyoD. It also interacts with the acetyltransferase and transcriptional co-activator PCAF. A mechanistic model of the participation of p27 in the regulation of p130/E2F4 repressed genes has been developed showing that p21 also collaborates in this mechanism. In this model the sequential recruitment of different cyclin-Cdk complexes on the promoters of target genes is accomplished first by p27 and later on by p21. In this model p27 and p21 associate with E2F4/p130 by its carboxyl moiety while they recruit the cyclin-Cdk complexes by the KID domain located at their NH2 half. It can be hypothesized that this model could be extended to other TFs by postulating that p27 can associate with the TF partners by its carboxyl domain and thus in such a way binds to the chromatin. By interacting with cyclin-Cdks by its amino region it facilitates the proximity of the Cdk with substrates associated to the chromatin. However, the extension of this model to other TFs, still remains to be validated. ChIP on chip, ChIP-seq and expression microarray analysis have allowed the identification of the transcriptional programs regulated by p27. In addition to cell cycle progression and functions as mRNA processing and splicing, translation and oxidative phosphorylation, firstly identified by Chip on chip data, a number of other cellular processes as: cell adhesion, cell-signaling, neuron differentiation and ion transport have been identified by ChIP-seq and expression microarrays. Among genes deregulated in p27KO cells there is a number of TFs that probably generates subsequent waves of transcription that, in this case, would not be directly regulated by p27. Interestingly, a reduced number of TFs as Mef2c, Sox6, Zfpm2 and Shox2 that have p27-BSs at their vicinity (as detected by ChIP), are also deregulated in expression microarrays suggesting that they are direct targets of p27. They can be subsequently involved in the triggering of a second transcriptional wave. Many other TFs that do not have p27-BSs in the proximity and that were found deregulated in p27KO cells might be involved in subsequent transcriptional waves.Finally, altered levels of p27 can participle in different pathologies as cancer and neurodegenerative diseases as PD, AD and HD. In the case of cancer, it is well known that reduced levels of p27 in human tumors are associated with a poor outcome of the patients. The increased malignancy in these tumors can be induced by transcriptional alteration of p27-TGs involved in different cancer hallmarks. Potentially interesting are data indicating that p27 might regulate the expression of genes involved in different neurodegenerative diseases as for instance SNCA, ApoE and genes encoding different subunits of the different oxidative-phosphorylation complexes that could induce oxidative stress. The putative participation of p27, through its transcriptional regulatory role, in the onset and progression of these different neurodegenerative diseases needs to be extensively studied in the next future.