We further demonstrated that these nucleases were well-tolerated and did not lead to off-target alterations of the exome in several corrected clonal cell populations. Collectively, these studies demonstrate the potential of engineered DNA-binding proteins to enable new approaches in medicine, science, and technology.The impact of reverse genetics, synthetic biology, and gene therapy has been restricted by the limitations of conventional genetic engineering technologies. To expand the capacity for genetic modification of mammalian cells, we are engineering artificial DNA-binding proteins, including zinc finger proteins, TAL effectors, and CRISPR/Cas9 to regulate and edit endogenous mammalian genes. For example, we have engineered both protein-based and RNA-guided transcriptional activators and repressors targeted to human genes relevant to medicine, science, and biotechnology. Delivery of combinations of transcription factors led to synergistic effects on gene activation and tunable expression levels. This approach recapitulates the previously intractable complexity of natural regulation of mammalian genes that is the product of cooperative actions of many transcription factors. We have also developed novel methods for controlling the activity of these proteins, such as optogenetic regulation of protein dimerization with blue light. Genome-wide analysis of the DNA-binding, gene regulation, and chromatin remodeling of these targeted epigenome modifiers has demonstrated their exceptional specificity. In other studies we have engineered synthetic nucleases to stimulate gene targeting to genomic safe harbor sites. This approach is particularly useful for generating isogenic cell lines. We showed that this method leads to a decrease in the variability of transgene expression within a clonal cell line and between multiple clones relative to conventional techniques. Finally, we have used similar methods to correct mutations causing genetic disease. We engineered synthetic nucleases targeted to the human dystrophin gene that is mutated in Duchenne muscular dystrophy patients. When we delivered these nucleases to cells from patients with this disease, the correct gene reading frame and expression of the functional dystrophin protein were restored Charles Gersbach is an inventor on patent applications and consultant in the area of genome editing.


The impact of signal-dependent transcription factors, such as glucocorticoid receptor and nuclear factor kappa-b, on the three-dimensional organization of chromatin remains a topic of discussion. The possible scenarios range from remodeling of higher order chromatin architecture by activated transcription factors to recruitment of activated transcription factors to pre-established long-range interactions.Using circular chromosome conformation capture coupled with next generation sequencing and high-resolution chromatin interaction analysis by paired-end tag sequencing of P300, we observed agonist-induced changes in long-range chromatin interactions, and uncovered interconnected enhancer–enhancer hubs spanning up to one megabase. The vast majority of activated glucocorticoid receptor and nuclear factor kappa-b appeared to join pre-existing P300 enhancer hubs without affecting the chromatin conformation. In contrast, binding of the activated transcription factors to loci with their consensus response elements led to the increased formation of an active epigenetic state of enhancers and a significant increase in long-range interactions within pre-existing enhancer networks. De novo enhancers or ligand-responsive enhancer hubs preferentially interacted with ligand-induced genes.We demonstrate that, at a subset of genomic loci, ligand-mediated induction leads to active enhancer formation and an increase in long-range interactions, facilitating efficient regulation of target genes. Therefore, our data suggest an active role of signal-dependent transcription factors in chromatin and long-range interaction remodeling.The online version of this article (doi:10.1186/s13059-015-0832-9) contains supplementary material, which is available to authorized users. HoxD locus serves as an example of pre-formed long-range interactions ) in control and stimulated cells. The constitutive P300 binding sites displayed a similar ChIP-seq and 4C signal pattern in vehicle-treated and ligand-treated cells Fig. . Importalls Fig. .The 4C assays support the presence of long-range interaction networks among P300 DBSs. Furthermore, they uncovered a significant increase in contact frequency at induced but not at pre-existing P300 DBSs Fig. . To furtIn order to accurately compare the pre-existing and induced subdomains in untreated and co-stimulated ChIA-PET libraries, we first estimated the local P300 concentrations  by summing up the RPKM values of P300 DBSs in ChIA-PET interaction subdomains harboring at least five P300 DBSs with a different degree of P300 induction Fig. . With feThus, preferential involvement of ligand-induced P300 DBSs in chromatin interactions implies that GR and/or NFκB binding to DNA via their cognate cis-acting elements opens up the closed chromatin by recruiting chromatin-remodeling complexes. Such open chromatin regions preferentially contact other P300 DBSs with a similar regulatory factor composition and chromatin state, resulting in an interaction network that is synchronized upon ligand-dependent GR and/or NFκB recruitment, resulting in increased contact frequency. We preferentially detected the networks that were highly affected by ligand stimulation. These networks are already established prior to stimulation (as detected by 4C-seq). However, the increase of the contact frequency at induced P300 DBSs upon ligand activation indicates the synchronization of such networks.We therefore consider that the (over) representation of induced interactions in our data sets may imply that a considerably larger proportion of cells in the population have the P300 protein network at these loci because the queried loci were synchronized by ligand treatment.Finally, we assessed the impact of agonist-induced regulatory elements and their special re-organization on transcriptional regulation using gene-body POLII density as a direct read-out . We quanIn this study, we have analyzed the impact of agonist-activated GR and p65 (NFκB) on chromatin state, 3D organization, and transcriptional regulation. Activated GR and p65 are mainly recruited to pre-existing regulatory elements that are pre-bound by P300, and display the epigenetic signature of active enhancers prior to TF activation, that is, they fall into the “permissive” model category. Our findings are in line with recent findings suggesting that signal-dependent TFs largely access the enhancer landscape that is set up by other lineage-specific TFs such as PU.1, C/EBPα, and AP1 . HoweverAn important question is how the agonist-induced enhancers and their target genes are spatially organized. We have compared the P300-mediated chromatin interactomes before and after GR and NFκB co-activation. Using ChIA-PET against P300, we observed the formation of P300-mediated long-range interactions at subdomains bearing induced P300 DBSs in response to ligand activation. With the current depth of ChIA-PET libraries, these interactions appeared to be either formed de novo (from latent enhancers) or stabilized upon ligand induction. In 4C analysis we reproducibly detected increased interactions at induced P300 DBSs; the induction in the 4C approach is, however, less pronounced as compared to ChIA-PET. This difference is likely due to intrinsic differences between the two assays: 4C detects long-range interactions irrespective of the presence or absence of P300 and hence is able to detect lower strength or lower frequency interactions and those that are P300 independent. In ChIA-PET, the immunoprecipitation step enriches for interactions mediated by P300 and does not pick up P300 independent interactions, providing an all or nothing picture. Collectively and in agreement with each other, our ChIA-PET and 4C results show that activation of GR and/or NFκB facilitates an induced interaction signal at a subset of DBSs. We interpret this increased signal as an increase in either the interaction frequency (stabilization of a network) or in the proportion of cells that engage in such interaction (synchronization of a network).Lcn2 gene locus is engaged in multiple long-range contacts with GR DBS. In agreement with our findings, it was shown that activated GR increases local chromatin interactions without dramatic change in 3D organization. In another report exploiting the 4C approach, activated GR was shown to bind a downstream enhancer of the Tsc22d3 gene, causing a 2-fold increase in long-range enhancer–promoter interaction and activation of transcription  were used in this study.Detection of putative P300 and POLII binding sites was performed using MACS (version 1.4.2)  with thehttp://www.uwencode.org/protocols). In brief, 5 × 106 nuclei were isolated using Buffer A  supplemented with 0.06 % IGEPAL CA-630 detergent. DNase I treatment (60 units) was performed for 3 min and the reaction stopped with stop buffer . The sample was further fractionated on 9 % sucrose gradient for 24 h at 25,000 rpm at 16 °C. Fractions containing DNA fragments smaller than 1 kb were purified and processed for sequencing according to the Illumina library preparation protocol. Normalized  DNase I data sets were used for the downstream analysis and visualization.DNase I libraries were prepared from DMSO-treated and TA + TNFα-treated Hela B2 cells as described  was taken for the further analysis after trimming the ends of each read. Subsequently, single-end sequenced reads were split at the linker ligation junction (linkerA/B-|-linkerA/B) and flipped to make the data compatible (similar to paired-end sequencing reads) for the ChIA-PET data analysis pipeline . The ave2 RPKM) in DMSO-treated and TA + TNFα-treated samples. All the subdomains are ranked by the average P300 concentration of DMSO and TA + TNFα treatments. We discarded the subdomains with less than five P300 peaks, and separated subdomains with at least one induced P300 peak (261 subdomains) and the ones with only constitutive P300 peaks (283 subdomains). We plotted the number of interaction clusters identified in DMSO and TA + TNFα data sets separately for individual subdomains. The subdomains were further filtered by the fold change of P300 concentration (>−0.3 and <0.3) and this resulted in 131 and 206 subdomains, respectively. The Mann–Whitney test was adopted to investigate the agonist-induced change in average chromatin interaction frequencies in comparable groups of subdomains that harbor only constitutive P300 DBSs against those having at least one agonist-induced P300 DBS.To minimize the bias induced by local P300 concentration on the chromatin interactions detected by ChIA-PET, we analyzed the changes of interaction frequencies per each P300 ChIA-PET defined subdomain as follows. For each subdomain, we counted the number of P300 peaks and calculated the P300 concentration  for 30 min. Nuclei were then digested by DpnII enzyme followed by inactivation of the restriction enzyme by incubating at 65 °C for 20 min. The digested chromatin was subsequently ligated (circularized) overnight at 16 °C with 50 U T4 ligase. Ligated chromatin was then reverse cross-linked by incubating with proteinase K at 65 °C and the RNA was removed by additional incubation at 37 °C with RNase A. The purified DNA was further digested with a second restriction enzyme of choice  followed by circularization of the DNA. The 4C product was subsequently amplified with bait-specific inverse primers  based on build version hg19 of the human genome. Then we estimated the mappability of the extracted sequences (each strand separately) and only uniquely mappable DpnII sites were considered for downstream analysis.All the reads from each library were parsed based on the bait-specific primer sequence and mapped to the reduced genome using bwa (version 0.6.2) with the default parameters. The mapping data of the individual libraries are summarized (Additional file GO analysis was performed using the DAVID web tool , 54. GenAll the ChIP-seq, ChIA-PET, and 4C raw data files have been submitted to GEO database [GEO: GSE61911]. Previously published GR, p65, and POLII ChIP-seq data can be accessed via [GEO: GSE24518].No approvals were required for the study, which complied with all relevant regulations.


Enhancers are closely positioned with actively transcribed target genes by chromatin looping. Non-coding RNAs are often transcribed on active enhancers, referred to as eRNAs (enhancer RNAs). To explore the kinetics of enhancer–promoter looping and eRNA transcription during transcriptional activation, we induced the β-globin locus by chemical treatment and analysed cross-linking frequency between the β-globin gene and locus control region (LCR) and the amount of eRNAs transcribed on the LCR in a time course manner. The cross-linking frequency was increased after chemical induction but before the transcriptional activation of gene in the β-globin locus. Transcription of eRNAs was increased in concomitant with the increase in cross-linking frequency. These results show that chromatin looping and eRNA transcription precedes the transcriptional activation of gene. Concomitant occurrence of the two events suggests functional relationship between them. Chromatin looping between enhancer and promoter was generated after chemical induction but before the transcriptional activation of gene in the β-globin locus. Transcription of enhancer RNAs was increased in concomitant with the increase of chromatin looping in this locus. Enhancers are regulatory elements activating the transcription of target genes. They are usually located at long distance from the genes in genome. The transcriptional activation of the genes depending on cell differentiation or experimental induction brings enhancers to promoters in close proximity, generating loop structure in nuclear environment ,2. This per se. eRNAs are transcribed in a transcription factor-dependent manner [3K4 at de novo enhancers by preceding these modifications [Earlier studies report that noncoding RNAs are transcribed on enhancers [referred to as enhancer RNAs (eRNAs)] and transcription of eRNAs correlates with transcription of target genes –9. Theset manner . The traications . A studyications . Transcrications .The β-globin locus has the locus control region (LCR) at upstream region of the globin genes. The LCR consists of several DNase I hypersensitive sites (HSs) that contain binding motifs for transcription activators and functions as an enhancer to regulate the spatio-temporal transcription of the globin genes. When the globin gene is actively transcribed, the LCR HSs are positioned in close proximity with the active gene, forming a chromatin loop –16. In aThe β major globin gene of the mouse β-globin locus can be transcriptionally-induced in murine erythroleukaemia (MEL) cells by chemical treatment. Chromatin structure in the β-globin locus in uninduced MEL cells, such as hypoacetylation of histones and weak association of RNA polymerase II and transcription activators, is strongly activated by the chemical induction ,25–27. I5/ml of density were treated with 5 mM of HMBA (10-[(3-Hydroxy-4-methoxybenzylidene)]-9(10H)-anthracenome) for 24, 48 and 72 h [MEL cells were grown in Dulbecco's Modified Eagle's medium (DMEM) containing 10% FBS. To transcriptionally activate the β-major-globin gene, MEL cells at 1.5×10and 72 h .6 MEL cells using the RNeasy Plus Mini Kit (Qiagen). A half microgram of RNA was reverse transcribed with random hexamers using the Superscript III first-strand synthesis system as suggested by the manufacturer (Invitrogen). A half microgram of RNA was reacted without reverse transcriptase. cDNA was amplified in a 10 μl of reaction volume by quantitative PCR using TaqMan chemistry. The relative intensity of specific cDNA sequences was compared with a genomic DNA standard using the comparative Ct method and then normalized with the relative intensity for the actin. Three independent preparations of RNA were analysed. The sequences of primers and TaqMan probes were provided in our previous study [RNA was prepared from 2×10us study .6 cells. Eight hundred units of HindIII restriction enzyme were used to digest DNA for overnight and the digested DNA was ligated with T4 ligase. The ligated DNA was purified after reverse cross-linking. The 3C products were quantitatively amplified by PCR using SYBR Green as a fluorescence dye. To correct the differences of ligation efficiency between fragments and the difference of PCR efficiency between primer sets, control templates were prepared by digesting and ligating equimolar amounts of bacterial artificial chromosome (BAC) vectors containing the β-globin locus and Ercc3 (excision repair cross-complementation group 3) gene and same amount of genomic DNA [Chromosome conformation capture (3C) assay was performed, as described, with the reduced number of cells ,30. MEL omic DNA . The ligomic DNA . Sequenc7) were cross-linked with 1% formaldehyde and then nuclei were isolated by cell lysis. Chromatin was fragmented by MNase digestion and sonication into mainly mononucleosomes and then was incubated with RNA polymerase II antibodies (Santa Cruz Biotechnology) or normal rabbit IgG (Santa Cruz Biotechnology) after pre-clearing. Protein–DNA complexes were recovered with protein A agarose beads and DNA was purified after reverse cross-linking.ChIP was carried out as described . BrieflyTo ask the kinetics of chromatin looping between enhancer and promoter during transcriptional induction, we induced the mouse β-globin locus in MEL cells by treating with HMBA for 3 days A. The β HindIII enzyme that separates the LCR HSs into different fragments , appears to promote the recruitment of RNA polymerase II to promoter and/or its elongation into the gene ,33. RNA The concomitant occurrence of the two events, chromatin looping and eRNA transcription, in the β-globin locus suggests functional relationship between them. Genome wide analysis shows that transcription of eRNAs is correlated with chromatin looping of enhancers with target gene promoters . Studies


BYpass of Ess1 (Bye1) protein is a putative S. cerevisiae transcription factor homologous to the human cancer-associated PHF3/DIDO family of proteins. Bye1 contains a Plant Homeodomain (PHD) and a TFIIS-like domain. The Bye1 PHD finger interacts with tri-methylated lysine 4 of histone H3 (H3K4me3) while the TFIIS-like domain binds to RNA polymerase (Pol) II. Here, we investigated the contribution of these structural features to Bye1 recruitment to chromatin as well as its function in transcriptional regulation. Genome-wide analysis of Bye1 distribution revealed at least two distinct modes of association with actively transcribed genes: within the core of Pol II- and Pol III-transcribed genes concomitant with the presence of the TFIIS transcription factor and, additionally, with promoters of a subset of Pol II-transcribed genes. Specific loss of H3K4me3 abolishes Bye1 association to gene promoters, but doesn’t affect its binding within gene bodies. Genetic interactions suggested an essential role of Bye1 in cell fitness under stress conditions compensating the absence of TFIIS. Furthermore, BYE1 deletion resulted in the attenuation of GAL genes expression upon galactose-mediated induction indicating its positive role in transcription regulation. Together, these findings point to a bimodal role of Bye1 in regulation of Pol II transcription. It is recruited via its PHD domain to H3K4 tri-methylated promoters at early steps of transcription. Once Pol II is engaged into elongation, Bye1 binds directly to the transcriptional machinery, modulating its progression along the gene.The  In vitro and in vivo studies indicate that Pol II movement is discontinuous and that nucleosomes provoke its pausing and backtracking during transcription at promoter-proximal regions and within the body of genes In eukaryotes, transcription is finely regulated through complex mechanisms, which promote RNA polymerases recruitment and progression through a chromatin matrix leading to efficient RNA synthesis. S. cerevisiae, the Pol II-interacting domain II of TFIIS shares a high homology with a central TFIIS-like domain (aa 232–365) of the putative transcription factor Bye1 that was discovered as a multicopy suppressor of the ESS1 gene deletion. ESS1 encodes a highly conserved peptidyl-prolyl isomerase implicated among other functions in the regulation of the whole transcriptional process, stimulating serine 5 dephosphorylation of the Pol II C-terminal domain repeats during transcription elongation in vitro and, globally, in vivo nearby the transcriptional start site (TSS) of actively transcribed loci In in vivo, we applied molecular and genetic approaches. Genome-wide analysis of Bye1 localization revealed that Bye1 is associated with bodies of Pol II and Pol III-transcribed genes  with distribution profiles resembling that of TFIIS. In addition, Bye1 is present at promoters of a subset of actively transcribed class II genes. This location depends on the presence of the H3K4me3 mark. We confirmed in vivo that Bye1 physically interacts with the jaw domain of Pol II and necessitates PHD, TFIIS-like and SPOC domains for strong binding. In general, BYE1 deletion alone has no consequence on cell viability. However, upon osmotic stress or carbon source changes, it becomes indispensable for normal growth and efficient transcriptional activation. We propose that Bye1 uses two distinct modes of recruitment to chromatin: through the binding to H3K4 tri-methylated nucleosomes and/or to the Pol II machinery. Hence, it is involved in regulation of transcription in at least two steps: (i) very early in the transcriptional cycle when it is recruited to the H3K4me3 mark around TSS and/or (ii) during elongation through interaction with Pol II in the body of genes. The two modes of Bye1 action might occur at the same genomic loci and have a positive outcome on the transcriptional process stimulating Pol II progression through the gene.To better understand Bye1 function in transcription and its relation to Pol II and chromatin modifications S. cerevisiae. All oligonucleotides significantly enriched in Bye1-bound chromatin  were further divided into three separate groups: (1) 4075 probes corresponded to coding regions of Pol II-transcribed genes with occupancy by all three proteins: Pol II, Bye1 and TFIIS; (2) 130 oligonucleotides enriched in TFIIS and Bye1 bound chromatin but not in Pol II; and, finally, (3) 387 oligonucleotides hybridized uniquely to Bye1 bound chromatin  assessing in parallel genome-wide distributions of the HA-tagged TFIIS, the Myc-tagged Bye1 and Pol II in hromatin –S2.The first group represents actively transcribed class II genes, since all these loci showed high presence of Pol II . The PolThe results obtained by ChIP-chip experiments were confirmed by conventional ChIP on a number of Pol II- and Pol III-transcribed genes . All selrpb1-1 Pol II thermosensitive allele transformed either with an empty vector or a vector expressing the wild-type Rpb1 protein. We found that shifting the strain expressing rpb1-1 to 37°C for 30 min caused a dissociation of Pol II from coding regions of genes pointing to abolishment of transcription and, as a consequence, it resulted in a loss of Bye1 recruitment to both promoters and coding regions of class II genes  but is very quickly induced upon heat shock (37°C). Using ChIP, we showed that Bye1 is present at the HSP104 promoter and within the gene at 28°C and 37°C but its occupancy was increased at the high temperature suggesting that the enrichment of Bye1 as that of Pol II correlates with the transcription level (r) and Spearman (ρ) correlation coefficients between Bye1 and Pol II were improved with the increase of gene expression: (1) r = 0.545, ρ = 0.547 for lowly expressed, (2) r = 0.605, ρ = 0.635 for mildly expressed, and (3) r = 0.861, ρ = 0.846 for highly expressed genes .To investigate a dependence of Bye1 recruitment to chromatin on active Pol II transcription, we followed Bye1 occupancy at promoters and coding regions of class II genes by ChIP in a strain expressing the II genes . Importanditions 3. This eon level . We sepaon level . Indeed,ed genes . For lowHence, Bye1 recruitment to chromatin is dependent on active Pol II transcription and strongly correlates with Pol II presence and gene transcription within highly expressed genes.in vivo. We investigated this hypothesis by co-immunoprecipitation (IP) of Pol II and Bye1 from formaldehyde cross-linked chromatin . Y190 strains transformed by empty vectors or vectors encoding fusions of the Gal4 activation domain (GAD) with the jaw domain of Rpb1 and fusions of the Gal4 DNA-binding domain (GBD) with Bye1 or TFIIS were overlaid with X-Gal agarose to assess β-galactosidase activity. As expected, both TFIIS and Bye1 showed interaction with the jaw domain of Rpb1 in vivo  presents high homology with the domain II of TFIIS (aa 146–247). The observed co-localization of Pol II, TFIIS and Bye1 within a large number of transcribed regions suggested that, similarly to TFIIS, Bye1 may bind to the transcriptional machinery hromatin . Immunophromatin . On the  in vivo .via its PHD finger in vitro and, globally, to co-localize with the H3K4me3 modified +1 nucleosome downstream the TSS in vivo, we performed ChIP of Bye1-Myc in strains lacking either Set1 or Spp1 subunits of the Set1C complex. Set1 is the catalytic subunit of the complex responsible for all types of H3K4 methylation  while Spp1 is required only for tri-methylation of H3K4 Bye1 has been shown to interact with H3K4me3 SET2 deleted strain. This mutant had no detectable transcriptional defects in normal growth conditions and didn’t affect Bye1 chromatin occupancy  (data not shown). These observations suggested that Bye1 is dispensable for viability and/or redundant with other transcription factors. We investigated the latter possibility, looking for a synthetic growth defect in a strain deleted for BYE1 and DST1, the gene encoding TFIIS. A synthetic growth defect was revealed in a bye1 and dst1 double mutant strain in conditions of high NaCl (1M) or caffeine (5 mM) – both treatments that challenge cell wall integrity. The sickness was further enhanced at 34°C  even when they are transcribed at very low basal levels in the absence of the drug. We didn’t find any difference in transcriptional response to the caffeine treatment in strains deleted for BYE1 and/or DST1 when measuring steady-state mRNA levels for these two caffeine-induced genes (data not shown).Caffeine is known to induce pleiotropic responses in yeast, including activation of the TOR, PKC and Ras/cAMP signalling pathways with marked changes in transcriptome composition BYE1 did not change Pol II or TBP occupancy and transcription of all genes tested in this study upon normal growth conditions . The expression of GAL genes is controlled by carbon sources in the growth media, being repressed by glucose and transcriptionally activated by galactose. We demonstrated that BYE1 deletion resulted in slower and less efficient induction of transcription as monitored by measurements of steady-state mRNA levels by RT-qPCR and the Pol II occupancy of GAL10 by ChIP (GAL10 promoter and gene body (ORF) in repressed and activated conditions . This feature led us to propose that the loading of Bye1 to chromatin at the initiation or early elongation steps is important for efficient transcription activation. Indeed, in the absence of Bye1, GAL genes are induced more slowly and less efficiently. Previous studies have shown that the GAL locus is transcribed under repressed conditions into long non-coding RNAs GAL10 gene body that further interferes with GAL activation. We assume that Bye1 enters the H3K4me-dependent regulatory circuit later on after the glucose to galactose switch during the first rounds of GAL gene transcription, allowing H3K4 tri-methylation of the TSS-proximal nucleosome. Recently, analysis of the native Pol II associated transcripts revealed that in vivo Pol II tends to stall when it encounters nucleosomes or other barriers In addition to its role in transcription elongation, Bye1 has a function when recruited to promoters of a subset of class II genes. We noticed that, globally, these genes are transcribed at a quite low level in normal growth conditions, but can be activated upon stress  supplemented as indicated and containing 2% glucose or 2% galactose, NaCl , Caffeine .RNA extraction and RT-qPCR – Total RNA was extracted using the hot phenol extraction procedure. Reverse transcription was performed with 0.1–1 µg of total RNA and BioRad iScript kit. cDNA quantifications are the mean of at least 3 independent experiments. qPCR results were normalized using RPO21 as an internal control. Primers within the coding sequence of genes are listed in Chromatin immunoprecipitation (ChIP) – was performed as described previously RPO21-ORF or Intergenic Region). Primer sequences are listed in Co-immunoprecipitation – was performed as for ChIP. After IP and extensive wash beads were boiled in 2xSDS-PAGE loading buffer and loaded to 10% SDS-PAGE. Western blot was performed using the same antibodies as for IP. IP without antibodies was used as a Mock control.Microarray hybridization – was done with a use of Agilent Yeast Whole Genome ChIP-chip Microarray 4×44 K 014810 G4493A. Three biological replicates of Bye1-Myc ChIP-chip and four biological replicates of Pol II and TFIIS ChIP-chip have been used for all quantifications. The computational data analysis was performed in R using limma package from the Bioconductor project Yeast two-hybrid assay – was performed in the Y190 strain transformed with pVV212 and pVV213 empty or recombinant vectors listed in lacZ or HIS3 reporters was tested using X-Gal overlay plate assay or 3AT plate assay as described Figure S1Distribution of Pol II, TFIIS and Bye1 along class II genes: (A) ADH1 and PYK1, (B) NUP84 and HOS2 as assessed by ChIP-chip assay.(TIF)Click here for additional data file.Figure S2Distribution of Pol II, TFIIS and Bye1 along class III genes: tL(UAA)B1 and SCR1 as assessed by ChIP-chip assay.(TIF)Click here for additional data file.Figure S3Bye1-Myc protein level doesn’t change after abolishment of Pol II transcription following 30 min heat shock of the rpb1-1 mutant strain measured by Western blot.(TIF)Click here for additional data file.Figure S4Bye1 recruitment to class II genes depends on gene expression levels: (A) Genome wide dot-plot analysis of Bye1 fold enrichment and (B–D) scatter-plot analysis of Bye1-Myc and Pol II fold enrichment of coding regions of class II genes expressed at low (<1 copy/cell), middle (from 1 to 10 copies/cell) and high (more than 10 copies/cell) levels as assessed by ChIP-chip in the Bye1-Myc HA-TFIIS strain with Pearson (r) and Spearman (ρ) correlation coefficients.(TIF)Click here for additional data file.Figure S5Deletion of SET2 has no significant effect on Pol II and Bye1 recruitment to chromatin: Quantification of Pol II (A) and Bye1-Myc (B) enrichment of different genomic loci in WT, set1Δ and set2Δ strains relative to the intergenic region by ChIP. 3′ORF and mORF stand for the 3′end and middle coding regions.(TIF)Click here for additional data file.Figure S6Deletion of BYE1 doesn’t affect Pol II and TBP recruitment to class II genes in normal growth conditions: Quantification of (A) Pol II and (B) HA-TBP chromatin occupancy in WT and bye1Δ strains by ChIP. 3′ORF and mORF stand for the 3′end and middle coding regions.(TIF)Click here for additional data file.Table S1Bye1 occupancy of the yeast genome.(XLS)Click here for additional data file.Table S2Pol II occupancy of the yeast genome.(XLS)Click here for additional data file.Table S3TFIIS occupancy of the yeast genome.(XLS)Click here for additional data file.Table S4Genes showing a significant Bye1 occupancy of promoter and 5′-coding regions .(XLS)Click here for additional data file.Table S5GO term annotations for biological processes of genes showing Bye1 presence at promoters(XLS)Click here for additional data file.Table S6List of primers, plasmids and strains used in this study.(XLSX)Click here for additional data file.Table S7List of class III genes examined in this study.(XLSX)Click here for additional data file.


TFF1 gene, the recruitment of TLE3 to the chromatin is FoxA1-dependent and prevents ERα and RNA polymerase II recruitment to TFF1 gene regulatory elements. Moreover, the interaction of TLE3 with HDAC2 results in the maintenance of acetylation at a basal level. We also provide evidence that TLE3 is recruited at several other regulatory elements of ERα target genes and is probably an important co-regulator of the E2 signaling pathway. In sum, our results describe a mechanism by which TLE3 affects ligand dependency in ERα-regulated gene expression via its binding restricting function and its role in gene regulation by histone acetylation.Chromatin constitutes a repressive barrier to the process of ligand-dependent transcriptional activity of nuclear receptors. Nucleosomes prevent the binding of estrogen receptor α (ERα) in absence of ligand and thus represent an important level of transcriptional regulation. Here, we show that in breast cancer MCF-7 cells, TLE3, a co-repressor of the Groucho/Grg/TLE family, interacts with FoxA1 and is detected at regulatory elements of ERα target genes in absence of estrogen. As a result, the chromatin is maintained in a basal state of acetylation, thus preventing ligand-independent activation of transcription. In absence of TLE3, the basal expression of ERα target genes induced by E2 is increased. At the  Estrogen is the ligand for the nuclear receptor estrogen receptor alpha (ERα) and is implicated in various pathologies such as osteoporosis and breast, ovarian and endometrial cancers . Upon itChromatin has a general repressive effect on basal transcription, but it can also play more specific roles in transcriptional regulation . For exaGenome-wide chromatin immunoprecipitation (ChIP)-seq experiments revealed that more than 50% of ERα-binding sites are localized in the vicinity of a Forkhead site (FKH) recruiting the transcription factor FoxA1 (Forkhead box protein A1) ,8. FoxA1Thus, the control of ERα-mediated transcription is established at several levels. Chromatin modifications and co-factor binding have to be regulated to prevent inappropriate gene activation. It is possible that this regulation can be achieved prior to ligand stimulation, by co-repressor complexes containing histone modification enzymes that prevent premature gene expression.Groucho/Grg/TLE is a family of such transcriptional co-repressors known to be interacting partners of histone deacetylases (HDACs) ,12,13 anHere, we show that TLE3 is indeed an important regulator of ERα target genes transcription. It is an essential factor that maintains transcription in a basal state in the absence of hormone. TLE3 is recruited to the chromatin by FoxA1 at several ERα binding sites throughout the genome. It mediates repression via interaction with HDACs to regulate histone acetylation, which in turn affects the recruitment of transcription factors, RNA polymerase II (RNAPII) and co-activators to regulatory regions.−7, 10−5 and 10−5 M, respectively, unless otherwise stated.MCF-7 cell lines were maintained in DMEM (Wisent) containing 10% fetal bovine serum (FBS) and antibiotics (penicillin and streptavidin). Prior to their use in E2 treatment experiments, MCF-7 cells were grown in DMEM without phenol red (Wisent) and supplemented with 5% charcoal-dextran-treated FBS for at least 3 days. These cells were mycoplasma free and were kept in a humidified chamber at 37°C in 5% CO2. 17β-estradiol, TAM and ICI 182780 (Sigma) were used at concentrations of 10The shRNA sequences (listed in Supplementary Table S1 for shTLE3 and  for shFo−7 M) for 30 min. After 10 min of crosslink with formaldehyde 1.1%, the cells were harvested in 200 μl of SDS lysis buffer  and put on ice for 30 min. Cell lysis was followed by two rounds of sonication , to decrease DNA viscosity. The volume of each sample was completed to 1 ml with IP dilution buffer  and 50 μl of each sample were spared for the input. The antibodies were added as shown in Supplementary Table S2, and incubated overnight at 4°C. 40μl of magnetic beads  were added in each IP reaction for 4 h. Beads were washed two times with TSE 150 , then two times with TSE 500  and finally two times with TE1x. IPs and inputs were eluted in 200 μl of elution buffer  for 30 min at room temperature and the crosslink was reverted by incubating overnight at 65°C. SDS loading dye was added to the samples and incubated 5 min at 95°C before loading the gels used for western blot.MCF-7 cells were hormone-deprived for at least 3 days and then treated with vehicle  or E2 (10−7M) for 30 min. ChIP assays were performed essentially as described previously (MCF-7 cells were hormone-deprived for at least 3 days and then treated with vehicle (EtOH 0.1%) or E2 (10eviously  with a p−7M). Total RNA was isolated using GenEluteTM Mammalian Total RNA Miniprep Kit (Sigma Aldrich) and the RNeasy kit (Qiagen). RNA was reverse-transcribed into first-strand cDNA using M-MLV reverse transcriptase (Enzymatics). Samples were subjected to q-PCR using a Bio-Rad C1000TM Thermal cycler. The relative abundance of all the tested genes was calculated after normalization to GapDH  mRNA, where relative expression levels were calculated as 2-ΔCT where ΔCT = CT test gene – CT GapDH. Primers used are listed in the Supplementary Table S3.Cells grown for 3 days in estrogen-free medium were treated for 3 h with vehicle (ethanol 0.1%) or estrogen  and GOBO . This co-expression was also observed in breast cancer primary tumors . These observations suggest that TLE3 and ERα expression are associated despite the fact that the TLE3 gene is not regulated by estrogen (E2) .To explore the link between ERα status and TLE3 expression in breast tumors, we performed an TLE3 and ERα expression levels are correlated and these factors are also interaction partners  and Supp and procIGFBP4, NR5A2 and SGK3 compared to the control, whereas TFF1 mRNA levels increased 7-fold under the same conditions. Upon induction with E2, the expression of these genes increased in both control and TLE3-depleted cells  that are located in the nearest ERα-binding sites around IGFBP4, NR5A2 and SGK3, three genes whose expression is induced by the depletion of TLE3 in absence of hormone, .To determine the effects of TLE3 knockdown on the conformation of chromatin at the enhancer in a basal state, we first assessed the recruitment of ERα, FoxA1 and RNAPII to s Figure . This aut Figure . HoweverTFF1 locus (primer c), but not at the control region when compared to control cells  of histone H3 lysine 27 (H3K27), and the general level of histone H4 acetylation (H4panAc). As a control of histone loading, we performed a ChIP assay of H3 . ChIP in TLE3-depleted cells cultured in E2-stripped medium revealed that acetylation of H3K27 and H4pan was enhanced at the distal estrogen response element (ERE) of the s Figure . These rTFF1 regulatory elements . We then validated these results using ChIP experiments for HDAC2 at TFF1 regulatory elements, in TLE3-depleted MCF-7 cells grown in hormone-deprived medium. In absence of TLE3, HDAC2 recruitment to chromatin was decreased, suggesting that TLE3 regulates acetylation by its interaction with HDAC  activity, or both. The interaction of TLE3 with HDAC has already been shown  and our C Figure . To evalnhancers  and are nhancers ,40,41. Inhancers  and 6C.Together, these results indicate that TLE3 is a co-regulator of ERα activity in absence of E2. The restriction of ERα and RNAPII binding to the chromatin and also histone acetylation prevention by favoring HDAC recruitment appear to be two mechanisms involved in this function.Drosophila (Groucho), mouse (Grg3) and human (TLE3). In human, TLE3 function has been explored in various cell lines, namely, adipocytes .Several studies have examined the role of TLE3 and homolog factors in many organisms including yeast (Tup1), ipocytes , prostatipocytes , ovarianipocytes  and breaipocytes . In breaipocytes . HoweverTFF1 basal expression. TFF1 mRNA level was lower in TLE3-depleted cells after treatment with ICI , demonstrating the importance of ERα in the basal induction of its target genes in TLE3-depleted cells. In breast cancer cells, the phosphorylation of ERα is an essential mechanism for ligand-independent transcriptional activation of ERα  in absence of TLE3 and examined n of ERα . Indeed,n of ERα . Furthern of ERα  and thisn of ERα . Thus, wn of ERα  and induIn several hormone therapy-resistant cancer cells, ERα is recruited to the DNA and activates its target genes in absence of E2 . The resTFF1, despite the fact that the expression of this gene is induced by the hormone. As we focused on ERα target genes repressed by TLE3, we expected a decrease of TLE3 recruitment to the enhancer and the promoter. Taken together, these results suggest that TLE3 may still be expressed in cancer cells because of its dual role in transcription.TLE3 and its homologs have commonly been described as co-repressors because they down-regulate their target genes expression. However, recent studies in adipocytes and breast cancer cells showed that theses co-factors can be co-activators as well as co-repressors. In breast cancer cells, TLE1 binds the chromatin at ERα-binding sites and is required for optimal ERα recruitment. The absence of TLE1 leads to a decline in ERα binding, thereby preventing ERα target genes expression. Thus, TLE1 appears to function as a positive regulator of ERα-mediated transcription (19). Likewise, TLE3 can also be a co-activator of transcription. In the pre-adipocyte, TLE3 is recruited at the promoter of peroxysome proliferator-activated receptor gamma (PPAR-γ) target genes and promotes their transcription. At the same time, it competes with β-catenin for transcription factor 4 (TCF4) interaction to relieve the repression by the Wnt pathway, and induces the genes responsible for adipocytes differentiation . Thus, iDrosophila have shown that the repressive function of TLE3 is weakened when it is phosphorylated (Previous studies in orylated . Severalorylated . MoreoveTFF1. However, TLE3 might not interact with only one specific HDAC. Indeed, in Drosophila, Groucho proteins interact with Rpd3 (TLE3 prevents the inappropriate acetylation of histones via its interaction with HDACs and our ChIP assays demonstrated the presence of HDAC2 on ith Rpd3 , a homolTFF1 regulatory elements since the acetylation of their target residue, H3K27, was strongly enhanced in absence of TLE3. However, the depletion of TLE3 could also increase the acetylation of other H3 residues, or the recruitment of other HATs that could also acetylate H4 lysines. Although we focused our study on the acetylation level of H3K27, p300 is also responsible for the acetylation of H3K14, H3K18, H4K5 and H4K8. Further analysis on the acetylation of those residues should give a better picture on the role of p300. Likewise, H3K9, K14 and K18 are acetylated by GCN5 and PCAF, whereas Tip60 is responsible for H3K14 and H4K5, K8, K12 and K16 acetylation (We also tested the recruitment of p300 and CBP to tylation , suggestIn conclusion, we report that TLE3 is an essential factor for the regulation of ERα target gene transcription via its function on histone acetylation. A previous study showed that HMTs and histone demethylases are responsible for the ligand-dependent transcription of ERα target genes . Here weSupplementary Data are available at NAR Online.


HMGCS1, FDFT1 and IDI1) were negatively correlated with time, while injury genes  were positively correlated with time after SCI. PCNA, MCM2, JUN and SNAP25 were the hub proteins of the PPI network. The transcription factors LEF1 and SP1 were observed to be associated with the regulation of two DEGs that were involved in the cholesterol-associated metabolism as well as injury responses. A number of microRNAs  were observed to target cholesterol metabolism-associated DGEs. The hub genes PCNA, MCM2, JUN and SNAP25 presumably have critical roles in rats with SCI, and the transcription factors LEF1 and SP1 may be important for the regulation of cholesterol metabolism and injury responses following SCI.The present study aimed to investigate the molecular mechanisms of spinal cord injury (SCI) in rats. First, the differentially expressed genes (DGEs) were screened based on GSE45006 microarray data downloaded from Gene Expression Omnibus using the significant analysis of microarray (SAM) method. Screening was performed for DEGs which were negatively or possibly correlated with time and subsequently subjected to gene ontology (GO) functional annotation. Furthermore, pathway enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes was also performed. In addition, a protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins database. Finally, GeneCodis was used to seek transcription factors and microRNAs that are involved in the regulation of DEGs. A total of 806 DEGs were upregulated and 549 DEGs were downregulated in the rats with SCI. Cholesterol metabolism-associated genes (e.g.  Spinal cord injury (SCI) refers to any injury to the spinal cord, and the symptoms may vary widely, from pain to paralysis to incontinence. The more serious and profound consequences of SCI are microscopic events following initial tissue injury, including inflammation, necrosis, apoptosis and glial scar formation . MicroarIn addition, microRNAs (miRNAs) that can post-tran-scriptionally regulate the entire set of genes exhibited altered expression following traumatic SCI . PreviouIn the present study, microarray data (GSE45006) were used to screen differentially expressed genes (DEGs). Based on the screened DEGs, protein-protein interaction (PPI) network was then constructed and the roles of transcription factors and miRNAs in the regulation of DEGs were further investigated with the objective to expand the current knowlege on the molecular mechanisms of SCI.http://www.ncbi.nlm.nih.gov/geo/). The platform was GPL1355 [Rat230_2] Affymetrix Rat Genome 230 2.0 Array. Data from a total of 24 tissue samples from the epicenter area of normal (n=4) and injured (n=20) rat thoracic spinal cords (T7) were used, and the latter contained four samples from rats with spinal cord injury after one day, three days as well as 1, 2 and 8 weeks, respectively.The raw microarray data (GSE45006) were downloaded from the Gene Expression Omnibus database  method . Using tet al , DEGs thet al .Changes in gene expression levels reflected by the microarray may be caused by either biological factors or the background noise . To exclThe Pearson correlation coefficient between the expression levels of screened gense and the time after spinal cord injury was calculated using R/Bioconductor software, with P=0.01 defined as the significant correlation level. As the sample size was 24 in the present study, the correlation coefficient was approximated to be >0.5 or <−0.5 at this significance level. Positively and negatively DEGs meeting this criterion were submitted to DAVID to analyze the enriched gene ontology (GO) biological processes.To elucidate the interaction of the DEGs, the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database was utilized to build an interaction network of encoding products of DEGs . A STRINThe DEGs were submitted to GeneCodis  to evaluIn total, 806 upregulated DEGs and 549 downregulated DEGs were screened. According to the KEGG biological pathway enrichment analysis, it was found that the upregulated DEGs were significantly enriched in 13 pathways (P<0.05), including lysosome, complement and coagulation cascades and extracellular matrix-receptor interaction . HoweverCorrelation analysis revealed that the levels of 314 DEGs were enhanced with increasing time after SCI (correlation coefficient >0.5), while the expression levels of 253 DEGs were decreased over time (correlation coefficient <−0.5).CYP51, EBP, HMGCR, DHCR7, HMGCS1, MVK, IDI1 and FDFT1, whereas those with expression levels positively correlating with time were mainly involved in injury response , MAZ  and LEF1 , respectively. The top three transcription factors with affinity for binding sites in the downregulated DEGs were LEF1 , E12 , and MAZ , respectively. LEF1 and SP1 were observed to have target DEGs that were involved in cholesterol-associated metabolism (e.g. FDFT1 and HMGCS1) and in injury responses (e.g. C1S and RAB27A). Further transcription factors, NFAT, AP4, SREBP1 and STAT5B, were also observed to target upregulated DEGs that were involved in injury responses, and NFY, TATA and MEIS1 were observed to target downregulated DEGs that were involved in cholesterol metabolism.Transcription factors were observed to participate in the up- and downregulation of DEGs. A total of 185 and 1,215 transcription factors were screened for the up- and downregulated DEGs, respectively. Among them, the top three transcription factors with affinity for binding sites in the upregulated DEGs were SP1 , and miR-200a and miR-141 regulated 23 downregulated DEGs each, with P-values of 8.7×10−8 and 1.4×10−8, respectively. In addition, a number of miRNAs, including miR-16, miR-210, miR-15b, miR300-3p, miR-540, miR-325-5p and miR-487b, were observed to have target DEGs involved in cholesterol-associated metabolism, e.g. IDI1 and FDFT1.In addition, 151 miRNAs were predicted for the down-regulated DEGs. miR-429 was indicated to regulate 26 downregulated DEGs  protein is one of the highly conserved MCMs, which form the hexameric protein complex that is involved in the initiation and the elongation of eukaryotic genome replication, particularly the formation and elongation of the replication fork  with lower total cholesterol levels in the serum. Therefore, these miRNAs are also indicated to have important roles in the regulation of cholesterol and sterol biosynthesis after SCI, which requires further experimental verification. miR-429, miR-141 and miR-200a belong to the same miR-200 family. Benoit et al (Several studies have reported the disturbed cholesterol metabolism in spinal cord-injured patients ,32. In tit et al  have repRAB27A, which is involved in the injury response, suggesting its role in the regulation of injury-associated DEGs after SCI. This agrees with the finding that SP1 or SP1-associated proteins are involved in regulating the expression of peripherin intermediate filament gene, which is activated after nerve injury via binding to the intron 1 site (C1S, which is involved in complement systems, was observed to be upregulated, confirming its role in injury responses after SCI.The transcription factors LEF1 and SP1 were observed to be associated with the regulation of the DEGs that were involved in cholesterol-associated metabolism and in injury responses; thus, it may be presumed that these two transcription factors have critical regulatory roles in gene expression after SCI. SP1 is a ubiquitous transcription factor. It has been reported to activate the LCAT promoter, which modulates the transportation rate of cholesteryl ester to the liver . Furthern 1 site . Thus, wn 1 site . Function 1 site . In the n 1 site . In addin 1 site . In the PCNA, MCM2, JUN and SNAP25 presumably have critical roles in rats with SCI, and the transcription factors LEF1 and SP1 may be important for the regulation of cholesterol metabolism and injury responses after SCI.In conclusion, the present study revealed that expression of cholesterol metabolism-associated DEGs was downregulated over time, while injury-associated DEGs were upregulated over time after SCI. Furthermore, the hub genes


Methods that use high-throughput sequencing have begun to reveal features of the three-dimensional structure of genomes at a resolution that goes far beyond that of traditional microscopy. Integration of these methods with other molecular tools has advanced our knowledge of both global and local chromatin packing in plants, and has revealed how patterns of chromatin packing correlate with the genomic and epigenomic landscapes. This update reports recent progress made in this area in plants, and suggests new research directions. Arabidopsis thaliana, which has a small genome with chromosome arms of around 10 megabases, the positioning of genes within the nucleus can affect their expression levels [A. thaliana [Some time ago, cytological studies not only showed that chromosomes are arranged in species-specific ways during interphase but also suggested that chromosome length is an important determinant of overall chromosome conformation in the nucleus . Some pln levels . The phythaliana  also sugIn non-plant species, the application of a 3C (chromatin conformation capture)-based method named Hi-C has greatly advanced our understanding of genome packing. In particular, Hi-C has revealed that TADs  are a predominant structural feature in most organisms –12. Eachb1 gene, which controls pigmentation. Several additional loops have recently been found at four A. thaliana genes that have roles in flowering and hormone signaling [Within the DNA sequence of a chromosome, transcriptional enhancers can often be located far away from transcription units, but chromatin loops can bring distal regulatory elements into direct contact with the promoters that they control. The first plant chromatin loop to be found using the 3C method was at the maize ignaling –20.b1 gene in maize encodes a transcription factor that regulates anthocyanin pigment production. Two epialleles of the b1 gene, B-I and B′, are distinguished by their level of RNA expression, which is much higher in B-I than in B′. In husk tissues specifically, both epialleles form chromatin loops between the gene body and an enhancer located 100 kb upstream [B-I allele, this enhancer has open chromatin that is thought to induce the formation of a multiloop structure between the b1 transcription start site (TSS) and additional upstream regions that mediates high b1 expression. By contrast, the B′ enhancer has compact chromatin, which prevents the formation of a multiloop structure and ultimately results in low b1 expression [The upstream . In the pression .FLOWERING LOCUS C (FLC) of A. thaliana has emerged as an ideal platform for the discovery of many epigenetic regulatory mechanisms, involving histone modifications, small RNAs and long non-coding RNAs [FLC transcribed region was found to interact with sequences immediately downstream of the polyadenylation signal [FLC gene is comparable to examples outside plants, its formation does not directly correlate with RNA expression: it can be detected in a wide range of genetic backgrounds that have very different levels of FLC expression. Its disruption does appear, however, to be an early response to prolonged cold exposure, or vernalization, which ultimately causes stable, Polycomb-mediated repression of FLC [FLC loop relies on SWI/SNF chromatin-remodeling activity, as inferred from knockdown of the SWI-SNF subunit BAF60, which stabilizes the FLC chromatin loop [The flowering repressor ing RNAs . In recen signal . In non-n signal , or by pn signal . Althougn of FLC . Disrupttin loop .FLC involves the long noncoding RNA (lncRNA) COOLAIR [FLC chromatin loop allows access of RNA polymerase II or of transcription factors to COOLAIR regulatory elements, which (through an intricate series of further events) ultimately leads to Polycomb-mediated silencing of FLC [PINOID (PID) auxin-response gene, which has a chromatin loop in its promoter region [PID expression is low. Formation of the loop depends on LHP1, an H3K27me3-binding plant homolog of HETEROCHROMATIN PROTEIN 1 [APOLO (AUXIN-REGULATED PROMOTER LOOP), which is divergently transcribed relative to PID. Auxin stimulation induces DNA demethylation at the APOLO locus, which reduces the frequency of H3K27me3 marks and consequently LHP1 binding, and thereby leading to the opening of the loop. Simultaneously, APOLO expression is activated, which ultimately triggers a new round of RNA-dependent DNA methylation (RdDM) and deposition of associated H3K27me3 chromatin marks that repress the expression of PID. The full model thus suggests an elegant mechanism for transient induction of PID: auxin induces DNA demethylation, leading to ejection of LHP1 and disruption of the chromatin loop and thus activation of PID expression. Because APOLO lncRNA expression is activated at the same time, a new round of RdDM is initiated, leading to PID downregulation.Regulation of  COOLAIR . It has g of FLC . This scr region . This loROTEIN 1 , 27. TheFLOWERING LOCUS T (FT) integrates many different environmental cues, including vernalization (by virtue of being a direct FLC target), photoperiod, age and ambient temperature. Much of this integration occurs at the FT locus itself, and its regulatory sequences are accordingly complex, as is the pattern of FT expression during the life cycle of the plant  is regulated very differently than FT, but also has complex regulatory sequences that include an enhancer located downstream of the transcription unit [TFL1 [The flowering gene iewed in ). Sequen the TSS , 19. Thi the TSS , a B-boxpression . These rpression . The FT ion unit . Bindingit [TFL1 .A. thaliana seedlings have revealed patterns that correspond well with cytological observations  and crwn4 mutants, which both have much smaller nuclei than wild-type plants, suggesting independence between nuclear morphology and chromosome packing [A. thaliana differs from most other organisms in that obvious TADs are not a predominant feature of A. thaliana genome organization. The lack of animal-like TADs in A. thaliana correlates with the absence of homologs of canonical insulator proteins such as CTCF [A. thaliana [A. thaliana Hi-C map led to the identification of over 1000 TAD-boundary-like and insulator-like regions [A. thaliana Hi-C maps was increased from the 20 kb to the 2 kb range [Saccharomyces cerevisiae, in which TADs were only noticed when utilizing a high-resolution variant of Hi-C that uses DNA digestion by micrococcal nuclease instead of restriction enzymes [At the megabase scale, the or loose . The coror loose . Formati packing . At a mo as CTCF , 34. Morthaliana . Neverth regions , 36. The enzymes .Apart from more local interactions, there are prominent intra- and inter-chromosomal interactions among heterochromatic regions dispersed throughout the otherwise euchromatic chromosome arms –34, 39 . These rA. thaliana Hi-C map [The relationships between various chromatin modifications and chromatin packing have also been explored. H3K27me3, which is associated with Polycomb Repressive Complexes (PRCs), correlates with compact chromatin, and mutants lacking this histone mark have dramatically reduced chromatin contacts within such regions , 33. H3KHi-C map . ChromatA. thaliana has short chromosomes that adopt a rosette conformation. By contrast, species with long chromosomes feature what is known as the ‘Rabl’ conformation, and such differences are expected to be visible in Hi-C maps. Similarly, chromosomes can be quite differently organized, even in species that have similar chromosome number or genome size. The A. thaliana relatives Arabidopsis lyrata and Capsella rubella both have genomes that are about 50 % larger than that of A. thaliana [A. lyrata, the increase in genome size in C. rubella is confined to the centromeres. It will be interesting to see how these differences are reflected in Hi-C maps of these species. Such closely related species that have rampant structural variation also afford a great opportunity to determine at a more fine-grained scale how deletions or insertions affect local chromatin–chromatin interactions.Plant genomes are very diverse, and so are their three-dimensional (3D) structures . A. thalthaliana , 42. HowA. thaliana, which has a very high gene density of about one gene per 5 kb. If local chromatin loops are as widespread in A. thaliana as they are in humans, many chromatin loops that have roles in the regulation of transcription would have a comparatively small size. The identification of such small loops is a technically and computationally challenging task. First, conventional 3C-based methods need to be coupled with additional steps to increase the sequencing depth of query regions, as this is a prerequisite to achieving a more accurate estimate of background signals or random chromatin interactions that are associated with loci of interest. Approaches that can help to provide this resolution include selective amplification-based methods, such as 4C and 5C [The most impressive recent Hi-C study was the one by Rao and colleagues , who proC and 5C –46, the C and 5C , and theC and 5C . Micro-CC and 5C . On the C and 5C , might bC and 5C . Other bC and 5C .FLC-LacO transgene loci was observed in connection with Polycomb-mediated silencing [LacO arrays that can be recognized specifically by bacterial LacI protein labeled with fluorescent proteins. Today, more sophisticated genome-editing techniques such as CRISPR/Cas9 would allow the non-random insertion of LacO arrays into the genome. A CRISPR/Cas-based chromatin-imaging method has already been used in mammalian cell lines for visualization of non-repetitive genomic loci [To complement sequencing-based methods, there are cytological tools that can visualize and monitor the behavior of chromatin loci in the nucleus. For example, padlock fluorescent in situ hybridization (FISH)  in combiilencing . Both stmic loci . The recmic loci .Many environmental and developmental factors, such as light intensity, temperature, microbe infection, and cell differentiation, can trigger global rearrangement of chromatin in plants –58, and


Evidence suggests that some human endogenous retroviruses and endogenous retrovirus-like repeats (here collectively ERVs) regulate the expression of neighboring genes in normal and disease states; e.g. the human globin locus is regulated by an ERV9 that coordinates long-range gene switching during hematopoiesis and activates also intergenic transcripts. While complex transcription regulation is associated with integration of certain exogenous retroviruses, comparable regulation sustained by ERVs is less understood.We analyzed ERV transcription using ERV9 consensus sequences and publically available RNA-sequencing, chromatin immunoprecipitation with sequencing (ChIP-seq) and cap analysis gene expression (CAGE) data from ENCODE. We discovered previously undescribed and advanced transcription regulation mechanisms in several human reference cell lines. We show that regulation by ERVs involves long-ranging activations including complex RNA splicing patterns, and transcription of large unannotated regions ranging in size from several hundred kb to around 1 Mb. Moreover, regulation was found to be cooperatively sustained in some loci by multiple ERVs and also non-LTR repeats.Our analyses show that endogenous retroviruses sustain advanced transcription regulation in human cell lines, which shows similarities to complex insertional mutagenesis effects exerted by exogenous retroviruses. By exposing previously undescribed regulation effects, this study should prove useful for understanding fundamental transcription mechanisms resulting from evolutionary acquisition of retroviral sequence in the human genome.The online version of this article (doi:10.1186/s12977-015-0161-9) contains supplementary material, which is available to authorized users. In mammalian genomes retroviral elements have been adapted to fulfil essential biological functions as epitomized by the domestication of envelope fusion proteins, the syncytins, which mediate fusion of trophoblasts during placenta formation. Such diverse elements which are generally divided into human endogenous retroviruses and endogenous retrovirus-like elements (here collectively ERVs) comprise ~8% of the human genome and originate from cumulative germ line infections and retrotranspositions in our ancestors . Whilcis-linked genes that coordinate gene switching during normal hematopoiesis. The ERV9 also activates intergenic RNAs at low levels as a result of transient DNA looping with multiple intergenic sites at the globin locus [TP63 (p63), a member of the tumor suppressor TP53 (p53) family. In testis of Hominidae an ERV9 LTR functions as a strong promoter affecting novel isoform expression of TP63 [FAB7) gene that is normally active in brain, is sustained by an LTR (LTR2-FABP) in diffuse large B-cell lymphoma [While most ERVs are disrupted by fragmentation and mutations in the retroviral genes, the long terminal repeats (LTRs) preserve their function as either promoters or enhancers that may regulate adjacent human genes. In the human globin locus, an ERV9 modulates long-range transcription factor occupancies at several in locus . In Hodgin locus . LTR-medlymphoma .chimeric is here used to denote transcription covering ERV and adjacent sequence.ERV9 family members belong to the human ERV-I class and were repeatedly amplified during primate evolution . The ERVWe identified chimeric transcription in available non polyadenylation-selected directional long paired-end RNA-sequencing (RNA-seq) data from ENCODE/Cold Spring Harbor Lab (CSHL)  Table 11, using R = 0.88 and p-value = 3.29e–4, Pearson’s correlation, N = 11)  and cap analysis gene expression (CAGE) data from ENCODE projects, as follows: ENCODE/CSHL Table  and ENCOWe observed transcriptional regulation of genomic loci spanning from several hundred kilobases to around one megabase that do not contain gene annotations in common databases including UCSC. Unannotated transcription was found to proceed in both directions, including transcription of opposite genome strands, and initiating either bidirectionally, from a single position, and/or from distinct positions containing ERVs and other repeats  locus in H1 hESC and K562 cells Figure B shows tWe have exposed regulatory patterns that attribute a pivotal role of ERVs in sustaining complex and pervasive transcription of the human genome, in some cases involving cooperative effects by several ERVs and non-LTR repeats. This was demonstrated by RNA-seq and CAGE that showed complete shifts of transcription polarity, and/or initiation of transcription at LTRs and non-LTR repeats (e.g. Figures Interestingly, in some loci recurrent association with ChIP-seq regulatory motifs implied that transcription is sustained by a limited number of major regulators, whereas the presence of multiple potential regulators in other loci showed that coregulation by LTRs and non-LTR repeats may be more pervasive (e.g. compare Figures While bidirectional transcription separates activation into distinct patches or sub-regions of transcription in the forward and reverse directions, respectively (e.g. Figures Transcription of unannotated regions encompassing several hundred kilobases to almost one megabase is of fundamental interest as the sheer amount of genome sequence and associated RNA splicing patterns suggest transcription of multiple unknown RNAs. We detected concurrent unannotated and annotated transcription at the CT49 locus of negative and positive polarity in both K562 and H1 hESC cells Figure B. In K56Coregulation of transcription by multiple endogenous LTRs and associated non-LTR repeats in large unannotated and gene-containing loci reveals a higher order complexity of human genome regulation that to our knowledge was not shown before. We have previously exposed transcription patterns of similar complexity sustained by exogenous retroviruses in mouse tumors using RNA-seq and ChIP-seq . It shou


Adult neurogenesis is the process by which new neurons are generated in the brains of adults. Since its discovery 50 years ago, adult neurogenesis has been widely studied in the mammalian brain and has provided a new perspective on the pathophysiology of many psychiatric and neurodegenerative disorders, some of which affect memory. In this regard, adult hippocampal neurogenesis (AHN), which occurs in the subgranular zone (SGZ) of the dentate gyrus (DG), has been suggested to play a role in the formation and consolidation of new memories. This process involves many transcription factors, of which cyclic AMP (cAMP)-responsive element-binding protein (CREB) is a well-documented one. In the developing brain, CREB regulates crucial cell stages , and in the adult brain, it participates in neuronal plasticity, learning, and memory. In addition, new evidence supports the hypothesis that CREB may also participate in learning and memory through its involvement in AHN. This review examines the CREB family of transcription factors, including the different members and known signaling pathways. It highlights the role of CREB as a modulator of AHN, which could underlie its function in memory consolidation mechanisms. Cyclic AMP (cAMP)-responsive element-binding protein (CREB) belongs to the family of leucine zipper transcription factors, which are expressed in a variety of tissues , and activating transcription factor-1 (ATF-1). This family belongs to the superfamily of basic region/leucine zipper (bZIP) transcriptional regulators, and all its members mediate cAMP-responsive transcription. CREB and ATF-1 are expressed ubiquitously, whereas CREM is mainly expressed in the neuroendocrine system. Each transcription factor is encoded by a single gene and contains the highly conserved leucine zipper sequence with an adjacent basic region that binds to a CRE DNA sequence. Members of the CREB family also have a regulatory central kinase-inducible domain (KID), although the Q1 and Q2 glutamine-rich regions adjacent to KID are less conserved among different family members  the well-conserved bZIP domain at the C-terminus, which facilitates dimerization between different family members and participates in the recognition of and binding to CRE sites; and (b) KID (mentioned above), which encloses sites for phosphorylation by protein kinase A (PKA) and other kinases. KID is delineated by two glutamine-rich domains (Q1 and Q2), which are responsible for basal transactivation activity. Q2 connects with TAFII 130 (TBP [TATA-binding protein]-associated factorII 130) and recruits the transcription machinery to the promoter region. Constitutive (Q2) and inducible (KID) domains work together in response to different stimuli that trigger CREB-dependent gene expression  levels through voltage- and ligand-dependent channels results in increased cAMP levels via activation of G-protein-coupled receptors. Growth factors can also activate receptor tyrosine kinases to augment cAMP levels. All these pathways affect CREB phosphorylation levels. Downstream of neuronal activity, the PKA, mitogen-activated protein kinase , and CaMKIV  kinase pathways result in CREB phosphorylation at Ser133. PP-1 (protein phosphatase-1) and PP2-A are the major CREB phosphatases, and Ca2+-dependent signals can induce intracellular events that lead to phosphorylation or dephosphorylation. The effect of stimulation depends on the nature of the stimulus and its cellular context. For example, activation of N-methyl-D-aspartate (NMDA) receptors leads to CREB dephosphorylation in extrasynaptic neurons, while in synaptic sites, it leads to CREB phosphorylation and CREB-dependent gene expression  in KID regulates CREB activity. This phosphorylation establishes an effector role for CREB, initiating the translation of extracellular stimuli into gene expression  has recently been described as crucial, and the CBP paralog p300 enhances CREB functionality. CBP and p300 are both chromatin remodelers (histone acetyltransferases) involved in unwinding promoter chromatin and providing the RNA polymerase II (Pol II) transcription machinery access to promoter DNA  are relatively new concepts. Moreover, ideas about neurogenesis have evolved since its discovery 50 years ago Figure .This review focuses on the most relevant and recent studies on CREB’s role in memory regulation and its participation in neurogenic processes. CREB influences cognitive processes directly by affecting memory and indirectly by affecting adult hippocampal neurogenic capacity. CREB’s affects memory consolidation through its regulation of adult hippocampal neurogenesis (AHN), which mainly occurs in the hippocampal subgranular zone (SGZ) of the dentate gyrus (DG). This mechanism provides a novel perspective on memory consolidation within the adult hippocampus.The existence of “adult neurogenesis” in mammals was first confirmed 50 years ago  are found in the SGZ, a specific DG subregion . miRNAs are ncRNAs (non-coding RNAs) that generally contain 19–23 nucleotides and regulate translation through one of two mechanisms: degradation of target mRNAs or inhibition of the translation machinery -GPR40-CREB signaling  have been reported, and there is evidence that subjects with these diseases also have impairments in AHN , and BDNF . Appropriate gene expression is crucial in determining phenotype, and the transcription of different genes affects diverse mechanisms including cell quiescence, proliferation, and death. Finally, can CREB modulate adult neurogenesis through the control of other adult neurogenesis regulators such as neurotransmitters, steroid hormones, or cytokines (Ortega-Martinez, SO-M is the sole author of this paper and is responsible for its content.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Listeria monocytogenes is a Gram-positive human-pathogen bacterium that served as an experimental model for investigating fundamental processes of adaptive immunity and virulence. Recent novel technologies allowed the identification of several hundred non-coding RNAs (ncRNAs) in the Listeria genome and provided insight into an unexpected complex transcriptional machinery. In this review, we discuss ncRNAs that are encoded on the opposite strand of the target gene and are therefore termed antisense RNAs (asRNAs). We highlight mechanistic and functional concepts of asRNAs in L. monocytogenes and put these in context of asRNAs in other bacteria. Understanding asRNAs will further broaden our knowledge of RNA-mediated gene regulation and may provide targets for diagnostic and antimicrobial development. Listeria monocytogenes is a Gram-positive, facultative foodborne pathogen that causes a severe life-threatening disease (listeriosis) in susceptible humans and animals. Complex regulatory mechanisms allow L. monocytogenes to adapt and survive in a wide range of environmental conditions  and infect a variety of hosts including mammalia and insects  in which the major virulence determinants are organized   elements in various bacterial genomes added a further layer of complexity in our understanding of bacterial gene regulation. In the last decade a myriad of non-coding RNAs (ncRNAs) of different genomic origin, length, function, and mechanisms of gene regulation were identified  the thermosensor forms a complex secondary structure that prevents translation of PrfA by interfering with the Shine-Dalgarno (SD) region . An important example in Trans-encoded small RNA (sRNA) could be considered as the second category. Those transcripts regulate genes located elsewhere on the genome and share only limited complementarity with the target. They often interact with multiple different target transcripts, and therefore function analogous to human microRNA , is located on the opposite DNA strand of their target and therefore share a high degree of complementarity with it. There is growing evidence that asRNAs are present in several Gram-positive and Gram-negative bacterial species and families with a high variability in prevalence and genomic density , distinct media, and organs  and under stress conditions (hypoxia and low temperature). This study demonstrated the influence of regulatory RNAs in response to different microenvironments.The reliable detection of antisense RNA is challenging because of technical difficulties. A major problem using microarrays, for instance, is artificially generated products during cDNA synthesis from RNA -detection they identified 86 additional ncRNAs, including 50 novel asRNAs , differences in origin and mechanisms  short, (ii) long, (iii) 3′UTR, (iv) 5′UTR, and (v) excludon Figure .L. monocytogenes are for example rliE, rli23, rli25, rli29, rli30, and rli35  was 2149 nucleotides in length and spans across neighboring genes partially including lmo2098 . It might represents an effective way to regulate neighboring genes. For example, transcription of 7 Figure .lmo0733 and lmo0743 both encoding putative transcription regulators, interact through 3′UTR . For example, R Figure . The traL. monocytogenes—the excludon  transcription, (ii) transcript stability, or (iii) translation.Although next generation sequencing was instrumental in the identification of several novel asRNAs in Staphylococcus aureus, Streptococcus pyogenes, and Vibrio anguillarum   encodes a transcriptional regulator, which activates transcription of the neighboring pdu and cob genes in the presence of propanediol. Pdu and Cob are essential for the catabolism of 1,2-propanediol catabolism and vitamin B12 biosynthesis. Propanediol is a byproduct of the metabolism of commensal intestinal bacteria. The ability to metabolize propanediol is important for pathogenicity and provides a survival advantage for bacterial during infection. In the process of propanediol catabolism vitamin B12 is required as a cofactor for involved enzymes. The reported asRNA anti1150 (aspocR) overlaps with the pocR gene. Interestingly, aspocR is controlled by a vitamin B12 dependent riboswitch that prematurely terminates transcription of aspocR in presence of vitamin B12 and thereby generates only a small transcript previously known as rli39. Subsequently, PCR-based experiments confirmed that pocR transcription was negatively regulated by aspocR. Additional experiments using ectopically transcribed aspocR showed inhibitory action in trans on pocR expression. These findings emphasize that the utilized mechanism is rather transcription attenuation or inhibition of translation than transcription interference or modulation of transcript stability in this case. Given that pocR is important for vitamin B12 biosynthesis, here antisense regulation seems to be rather a fine-tuning mechanism than an on- off-switch  is most likely involved in regulating central metabolic pathways in L. monocytogenes hundreds of ncRNAs, including even more than hundred asRNAs possibly implicated in the regulation of 102 Listeria genes, were discovered to date.Technological and methodological advances transformed the field of RNA-mediated gene regulation in bacteria and provided insight into an unexpected complexity. In L. monocytogenes show the dependency of some antisense transcripts on transcription factors or even the absence of a metabolite, the importance of experimental conditions is highlighted.This number seems rather low compared to the scope reported from other bacteria and will presumably rise with further studies. Yet, as recent findings in L. monocytogenes has proven to be a valuable model organism for studying asRNA regulation and given rise to novel discoveries like the excludon concept that could then be transferred to other bacteria.Also despite the rather low extent of asRNAs reported to date, L. monocytogenes likely act through different mechanisms and could either function as an on-off switch or fine regulators of a particular network. Thereby, asRNAs might be involved in regulating metabolic processes, virulence and determinants of host inflammatory response. In addition, the impact of asRNA regulation is spread as many targets of antisense regulation then again affect the expression of other genes .It might be speculated that asRNAs in However, our understanding about mechanisms and function remains limited to few individual transcripts. Mechanistic and functional validation of ncRNAs, including asRNAs, will shed further light into the extent of RNA-mediated regulation in bacteria. This understanding may then allow to develop new approaches for therapeutics.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


TCF7 and TCF7L2 genes have both been implicated in the progression of colorectal cancer in animal models and humans. TCFs recognize specific DNA sequences through their high mobility group (HMG) domains, but invertebrate TCFs and some isoforms of vertebrate TCF7 and TCF7L2 contain a second DNA binding domain known as the C-clamp. This review will cover the basic properties of C-clamps and their importance in Wnt signaling, using data from Drosophila, C. elegans, and mammalian cell culture. The connection between C-clamp containing TCFs and colorectal cancer will also be discussed.T-cell Factor/Lymphoid Enhancer Factor (TCF/LEF) transcription factors are major regulators of Wnt targets, and the products of the  Drosophila, depletion of the sole TCF gene (TCF/Pangolin or TCF/Pan) results in severe patterning defects in the embryo and larval imaginal discs, indicative of loss of Wnt signaling [C. elegans, the single TCF gene POP-1 has similar phenotypes to Wnt or β-catenin mutants in gonadal stem cell specification and left Q (QL) neuroblast migration [TCF loss-of-function mutant has been found that resembles the gastrulation defect phenotype seen in mutants of other positive acting factors in the Wnt pathway—e.g., Wnt3, β-catenin, and LRP5, LRP6 double mutants [The T-cell Factor/Lymphoid Enhancer Factor  family of transcription factors are major nuclear mediators of the Wnt/β-catenin signaling pathway ,2,3. In ignaling ,5,6. Simigration ,8, and rigration ,10,11,12 mutants ,14. WhilTCF ortholog, mammals have four TCF genes: TCF7 , LEF1, TCF7L1 , and TCF7L2  [TCF genes undergo extensive alternative splicing and have multiple promoters [Unlike invertebrates, which typically have a single romoters ,16, whilromoters ,17. TCF7romoters ,18,19. Tromoters ,21,22, wromoters ,24,25,26All TCF family members share two highly-conserved domains: the β-catenin binding domain and the High Mobility Group (HMG) DNA-binding domain . OriginaThe HMG domains found in TCFs belong to the HMGB subgroup of the larger HMG superfamily ,34. ThesMany TCFs also contain a second DNA binding domain, termed the cysteine clamp or C-clamp, which is C-terminal to the HMG domain . Origina12-Cys-X2-Cys-X5-Cys spacing of C-clamps is distinct from all other classes  [LEF1 and CDX1 genes, which require an intact C-clamp for activation by Wnt signaling [SP5, CDX2, and MYC) that are upregulated in colorectal cancer (CRC) [Like the HMG domain, the C-clamp can recognize DNA in a sequence-specific manner. The presence of a C-clamp in mammalian TCF1E and TCF4E allows these proteins to recognize a motif of RCCG in addition to the classic HMG binding site ,46,53. Iper site ,48. In fW = A/T) . Helper W = A/T) ,55. Helpignaling ,56. Helpignaling ,53, incler (CRC) ,53. TakeIn addition to enhancing DNA specificity, the architecture of HMG–Helper site pairs also contributes to more nuanced transcriptional regulation of Wnt-regulated enhancers. The spacing and orientation of these motifs were systemically examined for TCF/Pan. Some constraints were identified , but there was also a remarkable level of flexibility in DNA recognition, with the presence of a Helper site in any orientation near a HMG site improving TCF binding and Wnt target activation . This isDrosophila and C. elegans, where a single TCF is responsible for both basal repression and β-catenin-dependent activation, activation of Wnt targets requires both HMG and Helper sites [TCFs are thought to regulate many Wnt targets through a transcriptional switch mechanism, and there is data indicating that this regulation involves differential use of Helper sites. In this model, targets are repressed in the absence of signaling by TCF-dependent recruitment of co-repressors to Wnt target gene chromatin ,57. β-caer sites . While Her sites . It is ier sites ,59,60,61Drosophila hematopoietic cells for genes that are repressed by Wnt/β-catenin signaling. In these cases, TCF/Pan activates the gene’s expression in the absence of signaling, while β-catenin promotes transcriptional repression [In addition to the classic TCF transcriptional switch, another type of switch has been described in pression ,63. The It is important to note that the above-mentioned DNA binding properties of TCF have been studied primarily in invertebrates. However, the primary sequences of the HMG and C-clamps of TCF1E and TCF4E are very similar to TCF/Pan , suggestDrosophila has only one other C-clamp-containing protein, known as fly Glut4EF, which regulates wing position in adult flies [In addition to TCF1E and TCF4E, the C-clamp is present in three other proteins in mammalian genomes ,66,67,68lt flies . HDBP2—mlt flies . Howeverlt flies ,76. The lt flies .TCF7L2 whole animal or conditional knockouts result in a loss of proliferative capacity of the intestinal epithelia [TCF7L2 inhibits growth and results in apoptosis [TCF7L2 fusion that presumably allows it to activate transcription in a β-catenin-independent manner [Elevated Wnt signaling is thought to be oncogenic in many human cancers, but perhaps the most compelling case is in CRC ,79. TCF7pithelia ,82. In spoptosis ,83. Const manner . While the short summary above reflects the prevailing view , it shouTCF7 resulted in an increase in adenoma formation in the adult gut and markedly increased tumor incidence in multiple intestinal neoplasia (min) mice, which are heterozygous for a stop codon in the tumor suppressor gene APC (minApc) [TCF7 has also been implicated in CRC and intestinal biology, acting in an antagonistic manner to TCF7L2. In non-pathological conditions, intestinal cells largely express a truncated TCF7 from an intronic promoter, producing a protein lacking the N-terminal β-catenin binding domain (dnTCF7) ,29,45. D(Apcmin) ,29. At odds with the mouse knockout data, TCF7 has also been reported to have an oncogenic role in one CRC cell line . One expLef1 and Cdx1 promoters [SP5, TGIF, YAP1), suggesting a model where C-clamp containing TCF activates their expression to bypass a p21 block in the cell cycle of CRC cells [SP5, was found to be dependent on C-clamp–Helper site interactions, suggesting a direct role for the C-clamp in the modulation of the cell cycle in intestinal cancer cells [While most studies on the role of TCF7 and TCF7L2 in CRC do not indicate whether the isoform used contains a C-clamp, Waterman and colleagues have examined this issue in some detail. Originally, they noted that TCF1E and TCF4E could specifically activate reporters containing regulatory DNA from the romoters ,86. Subsromoters ,49. Overromoters . This efromoters . Transcrer cells . Consister cells .TMEPAI (transmembrane prostate androgen-induced RNA) gene [TMEPAI activation is dependent on both the TGF-β and Wnt pathways [Min/+Apc mice [TMEPAI reporter construct in the presence of TGF-β stimulation, but not isoforms lacking this domain [TCF7L2 mutants with either a C-clamp deletion or C-clamp point mutation (C463A) were no longer able to potentiate reporter activity, indicating that the C-clamp is necessary for this regulation [Another research group explored a broader role for the C-clamp across multiple cancers through transcriptional regulation of the NA) gene . TMEPAI pathways , and TMEn/+ mice ,90, as wn/+ mice ,92,93,94s domain . Moreovegulation .While the importance of the C-clamp in Wnt/TCF regulation of CRC remains to be established, the existing data indicates that more attention needs to be paid to this DNA binding domain. For example, one recent report on the reciprocal regulation of TCF7L2 and the transcription factor ZEB1 in CRC progression utilized a TCF7L2 isoform containing the C-clamp, but failed to consider whether this domain played a role in the regulation of several Wnt target genes . Moving TCF7 locus [TCF1E, TCF4E knockout. TCF7 and TCF7L2 compound null mouse embryos exhibit severe hindgut defects, with anterior transformation of the gastrointestinal tract [TCF1E, TCF4E mutants would display more nuanced phenotypes. If these mice had normal intestines, they could be placed in CRC mouse models such as minApc [CDX2P-NLS Cre recombinase transgene and a loxP-targeted Apc allele [TCF1E, TCF4E double mutant into either background could provide important insights into the tumor-promoting versus tumor-suppressing roles of C-clamp-containing TCF isoforms.A rigorous test of the C-clamp’s importance in Wnt transcriptional regulation would be to engineer a mouse mutant where the TCF1E or TCF4E isoforms are not expressed. Deletion of the C-clamp-containing exon 9 in the F7 locus , and exoF7 locus  would geal tract . It is ts Apcmin , or micec allele . Crossin