Isl1, Lhx3, and Lmo4 are direct target genes of the Isl1-Lhx3 complex. Our results show that specific genomic loci associated with these genes recruit the Isl1-Lhx3 complex to activate the transcription of Isl1, Lhx3, and Lmo4 in embryonic MNs of chick and mouse. These findings support a model in which the Isl1-Lhx3 complex amplifies its own expression through a potent autoregulatory feedback loop and simultaneously enhances the transcription of Lmo4. LMO4 blocks the formation of the V2 interneuron-specifying Lhx3 complex. In developing MNs, this action inhibits the expression of V2 interneuron genes and increases the pool of unbound Lhx3 available to incorporate into the Isl1-Lhx3 complex. Identifying the pathways that regulate the expression of these key factors provides important insights into the genetic strategies utilized to promote MN differentiation and maturation.Motor neuron (MN) progenitor cells rapidly induce high expression of the transcription factors Islet-1 (Isl1), LIM-homeobox 3 (Lhx3), and the transcriptional regulator LMO4, as they differentiate. While these factors are critical for MN specification, the mechanisms regulating their precise temporal and spatial expression patterns are not well characterized. Isl1 and Lhx3 form the Isl1-Lhx3 complex, which induces the transcription of genes critical for MN specification and maturation. Here, we report that  The precise temporal and spatial regulation of transcription factor expression is critical for embryos to generate the appropriate number and variety of motor neurons (MNs). This process dictates the formation of motor circuits, which regulate coordinated movement and homeostasis. When MN specification is impaired, it leads to serious medical conditions such as spinal muscular atrophy. Understanding MN development is crucial for effectively treating pediatric MN disorders and neurodegenerative disorders, such as amyotrophic lateral sclerosis. Here, we show that three essential factors for MN development, Islet-1 (Isl1), LIM-homeobox 3 (Lhx3), and LMO4, are induced directly by the Isl1-Lhx3 complex. Characterizing the pathways that direct the expression of these factors provides key insights into the genetic mechanisms that regulate MN development.Combinatorial expression of specific transcription factors establishes discrete progenitor domains, in the embryonic spinal cord, which each generate distinct types of neurons . The p0-Hb9, and genes that are required for cholinergic neurotransmission, such as VaCHT  and LIM-homeobox 3 Lhx3; . Both pras VaCHT .To efficiently transition from a progenitor state to a terminally differentiated state, pMN cells must rapidly upregulate and maintain the expression of Isl1 and Lhx3. Deletion of Isl1 or Lhx3, or disruption of Isl1-Lhx3 complex assembly, severely impairs MN specification . FollowiLhx3, as well as a known Isl1 enhancer . LMO4 is expressed in embryonic MNs, and is important for inhibiting the formation of the Lhx3 complex, indirectly increasing the probability that Lhx3 will incorporate into the Isl1-Lhx3 complex  and de-crosslinked by incubating at 65°C overnight. Eluate was incubated at 50°C for more than 2 h with proteinase K. Next, DNA was purified with phenol/chloroform and DNA pellet was precipitated by ethanol and resolved in water. The purified final DNA samples were used for qPCRs using the SYBR green kit  and CFX Connect (Bio-Rad). The total input was used for normalization. All ChIP experiments were repeated independently at least three times. Data are represented as the mean of duplicate or triplicate values obtained from representative experiments; error bars represent SEM. Following immunoprecipitation, qPCR was performed to detect peaks using the following primers. Lhx3-Peak-A Fwd: GGTCTGCCTCCCGTAAAACT and Rev: CACCATCAATGCTTTGTTCAG, Lhx3-Peak-B Fwd: CAATGCAGGGTGACCTGG and Rev: GTGGGATTGACTGGGGTC, Isl1-Peak Fwd: CTGCCACTCCACTTAATAACCTAA and Rev: ATGGACACACCAGCTGGATAAATC, and LMO4-Peak Fwd: ATCACTCGAGGACGTGGGTCCCTTTAAGATCC and Rev: CTGAGTCGACGGATTCTGCCTCCTCTCCTC.We isolated spinal cords from E12.5 mouse (ms) embryos. Spinal cords from 5-12 embryos were combined for each ChIP reaction with a specific antibody. Antibodies used for immunoprecipitation were rabbit (rb) anti-Isl1/2 , at HH st25. Images are representative of electroporations from multiple embryos.Lhx3-Peak-A (ms) (chr2:26194774-26194788): Fwd-CTAGAGGTAGCCAAGGCC and Rev-TGGAGAGGGCTAGCCAC. Hx-L-wt: CATTTTAACTAATGG ΔHx-L: CGCGGCCGCAGCCGG. Hx-S-wt: CTAATTAAA ΔHx-S: CGGCCGCAA. Lhx3-Peak-B (ms) (chr2:26186472-26187246): Fwd-CAATGCAGGGTGACCTGG and Rev-GTGGGATTGACTGGGGTC. Hx-L-wt: ATTTGATTAATCA. ΔHx-L: AGCGGCCGCCTCA. Isl1-Peak (hum)(chr5:51559189-51559911): Fwd-CAGATGCACCTACCTCTTAAAG. Rev-GGACATATGGCTAGAGTGTGG. (1-409) Rev-CCCTACTCTGTCTGCCACTCC. Hx-S1-wt: TTTTAATTAGCT ΔHx-S1: TTTCTAGAAGCT. H2-wt: ATATTAAAAT ΔH2: ATCTAGAAAT. A/T motif-wt: AATTTTAGCATAT ΔA/T: ACGGTTGGCGCCT. LMO4-Peak (ms)(ch3:144198960-144199257): Fwd-GACGTGGGTCCCTTTAAGATCC and Rev-GGATTCTGCCTCCTCTCCTC. Hx-L-wt: AATTTTGTTAATTAA ΔHx-L: AACCATGGTAGGTAA.The Isl1-Lhx3 ChIP-Seq peaks were constructed using the following primer sets. We have also listed the sequences for wt HxRE sequences and the sequences for mutated HxREs. Mutations were introduced using PCR. Embryos were fixed in 4% PFA/PBS for 90 min, embedded in OCT and cryosectioned at 12 µm. Embryos were incubated in primary antibody in either 0.1% fish gelatin or 0.3% bovine serum albumin (BSA) blocking buffer, overnight at 4°C. Primary antibodies used were goat (gt) anti-LacZ (Sigma 1:2000), rb anti-LacZ (Cappel 55976 1:2000), ms anti-Mnr2 (DSHB 5C10 1:250), and chicken (chk) anti-GFP (Aves Labs 1020 1:1000). Sections were imaged using a Zeiss Axio Imager.Z2 microscope.Embryos used for GFP fluorescence analysis were not immunostained for GFP. A 750-ms exposure time was used for all images that were analyzed for GFP quantification. Integrative pixel density was measured in the ventral horn of the electroporated side of the spinal cord, using ImageJ. Four to twelve embryos were analyzed for each reporter construct. And for each embryo, average fluorescence intensity was calculated from analyzing three to seven sections.in situ probes, cDNA for chick Lhx3, Isl1, and LMO4 3’ untranslated region (UTR) was cloned into pBluescript vector. Digoxigenin-labeled riboprobes were generated using T7 polymerase PCR. Probes were denatured in hybridization solution at 80°C for 5 min. Slides were incubated in probe/hybridization solution at 68°C overnight. Slides were then washed in 5× SSC (0.75 M NaCl and 75 mM sodium citrate) at 65°C for 10 min. Next, slides were incubated in 0.2× SSC (30 mM NaCl and 3 mM sodium citrate) at 65°C for 2 h and then washed in fresh 0.2× SSC at 65°C for 5 min. Slides were then blocked in buffer 1 (0.1 M Tris and 0.15 M NaCl) + 4% BSA at room temperature for 1 h, and then incubated in buffer 1 + 2% BSA + 1:5000 anti-digoxigenin antibody at 4°C overnight. Next, slides were washed three times with buffer 1 for 5 min each at room temperature, and then in buffer 2  at room temperature for 5 min. Slides were then incubated in buffer 3  at room temperature overnight, to perform the colormetric reaction. To stop the reaction, slides were then washed in TE. Prior to mounting, slides were dehydrated with serial ethanol washes  at room temperature and incubated in xylene for 10 min at room temp. Slides were then mounted using Permount.Embryos were electroporated with 1.75 µg/µL pBluescript expression vectors containing either mouse Isl1, rat Lhx3, or Isl1-Lhx3 fusion protein . EmbryosAssays were performed in cultured P19 embryonic mouse carcinoma cells. Cells were cultured in α-minimal essential media with 7.5% bovine calf serum and 2.5% fetal bovine serum. For luciferase assays, cells were seeded in 48 well plates, and transfected using Lipofectamine 2000 (Invitrogen). Cells were transfected with reporter constructs, transcription factor expression constructs, a CMV-β-galactosidase construct, to test transfection efficiency, and with empty plasmid to equalize the total amount of DNA for each condition. Luciferase and β-galactosidase activity was measured 48 h after transfection. Results are reported as activity fold change of each reporter construct when cotransfected with Isl1 plus Lhx3, compared to cotransfection with empty plasmid. Results from each experiment were obtained from the average of technical duplicates. Summarized results show the average activity fold change from at least five independent experiments.Isl1, Lhx3, and Lmo4 in the induced MN-embryonic stem cell (ESC) system  system . To test) system . These entiation .Lhx3 gene. Lhx3-Peak-A is located approximately 5.1 kb downstream of Lhx3  motif and a putative HxRE-Short (Hx-S) motif A. As botWhen coelectroporated with LacZ, the ΔHx-L reporter construct still activated GFP expression in Mnr2-positive MNs C. HoweveWe observed similar results with luciferase assays. In cells transfected with luciferase reporter constructs containing wt Lhx3-Peak-A, ΔHx-L, or ΔHx-S, transcription is activated by Isl1 plus Lhx3. However, when both response elements were mutated, cotransfection with Isl1 plus Lhx3 failed to activate transcription E.To test whether Lhx3-Peak-B acts as a MN-specific enhancer, we electroporated Lhx3-Peak-B-GFP with either LacZ or Isl1-Lhx3 in embryonic chick neural tube B. When cInterestingly, when we performed luciferase assays in P19 cells, Lhx3-Peak-B was not activated by cotransfection of Isl1 plus Lhx3, compared to control reporter construct with no enhancer E. These + MNs. Coelectroporation of Isl1-Lhx3 fusion protein also failed to activate GFP expression -ΔHx-S1, activated robust GFP in the dorsal spinal cord that colocalized with ectopic Mnr2 expression D. NotablWe also mutated the A/T-rich motif located at 470 base pairs (ΔA/T), which is only present in the full-length version of the Isl1-Peak A,B. PrevOverall, these findings support a model in which the Isl1-Lhx3 complex activates the Isl1-Peak via HxRE-S1, likely in cooperation with additional HxRE and H motifs. These results are also consistent with our findings that both Isl1 and Lhx3, bind to the Isl1-Peak in ESCs and in the mouse embryonic spinal cord A,E.+ MNs (During MN specification, LMO4 blocks the formation of the V2-interneuron specifying Lhx3 complex, and thereby inhibits the expression of V2-specific genes in MNs . In addi+ MNs C. Coelec+ MNs D.Lmo4. By blocking the formation and activity of the Lhx3 complex, LMO4 inhibits the transcription of V2-IN specific genes in MNs and increases the pool of free Lhx3 available to incorporate into the Isl1-Lhx3 complex.Luciferase assays using LMO4-Peak-LUC with Isl1, Lhx3, or Isl1 plus Lhx3, were consistent with these results E. Isl1 pThe LMO4-Peak contains one HxRE-Long motif and one HxRE-Short motif A. To tesIsl1, Lhx3, and Lmo4, in the embryonic spinal cord, we ectopically expressed mouse Isl1 and rat Lhx3, or Isl1-Lhx3 fusion protein in the embryonic chick spinal cord through neural tube electroporation. We harvested embryos at 3DPE and performed in situ hybridizations with chk-specific probes designed to recognize the 3’UTR of chick Isl1, Lhx3, or Lmo4. Because the Isl1, Lhx3, and Isl1-Lhx3 expression constructs lack 3’UTR sequences, these probes exclusively detect endogenous chick transcripts.To test whether the Isl1-Lhx3 complex can activate the transcription of Isl1, Lhx3, or Lmo4 (Lmo4, but did not affect expression of endogenous Isl1 or Lhx3 (Isl1, Lhx3, and Lmo4 throughout the dorsal spinal cord (Lhx3, Isl1, and Lmo4 in the embryonic spinal cord.Embryos that were electroporated with Isl1 alone showed no change in the expression of endogenous  or Lmo4 A--C. Lhx or Lhx3 D–F. In cnal cord G–I. ThesLhx3, Isl1, and Lmo4, both in an iMN-ESC system, and in the embryonic spinal cord. Each of these loci acts as a MN-specific enhancer and is robustly activated by the Isl1-Lhx3 complex. Additionally, we show that overexpression of the Isl1-Lhx3 complex activates the transcription of endogenous Isl1, Lhx3, and Lmo4. Together, these results show that, early in embryonic MN specification, the Isl1-Lhx3 complex is recruited to loci associated with Isl1, Lhx3, and Lmo4 to directly activate the transcription of each of these genes. This transcriptional activation generates a positive autoregulatory feedback loop where the Isl1-Lhx3 complex activates the transcription of its own components. This feedback loop contributes to the rapid induction of Isl1, Lhx3, and LMO4 expression in differentiating MNs, and to the maintenance of these factors in mature MMCm neurons.A great deal of progress has been made characterizing the activity and expression patterns of Isl1, Lhx3, and LMO4 in embryonic MNs . HoweverOnecut transcription factors, including Hnf6, activate the transcription of Isl1 in early MNs and regulate the expression of Isl1 in multiple MN subtypes . HoweverLmo4 transcription. LMO4 competes with Lhx3 to bind NLI, thereby blocking the formation of the Lhx3 complex. This action inhibits the transcription of V2-interneuron genes in MNs and increases the available pool of Lhx3, which indirectly promotes Lhx3 incorporation into the Isl1-Lhx3 complex.Early in MN specification, Hb9 is released from transcriptional repression via reduced levels of Olig2 expression . ReducedHb9 (Positive and negative transcriptional feedback loops, both direct and indirect, have been shown to contribute to the temporal regulation of gene expression in a variety of cellular contexts . In partHb9 .While each peak in this study is activated by the Isl1-Lhx3 complex, the composition of each peak varies. Thus, the genetic mechanisms utilized to recruit the Isl1-Lhx3 transcription complex also vary. Both Lhx3 peaks and the LMO4 peak contain single HxRE-Long motifs, while Lhx3-Peak-A and the LMO4-Peak also contain single HxRE-Short motifs. The Isl1-Peak contains two HxRE-Short motifs, but no obvious HxRE-Long motifs, as well as multiple H motifs that resemble HxRE-Long and HxRE-Short motifs.The enhancer activity of Lhx3-Peak-A is only completely lost when both the HxRE-Long and HxRE-Short motifs are ablated. This finding shows that these two motifs cooperate to recruit the Isl1-Lhx3 complex and induce transcriptional activation. In contrast to this observation, the HxRE-Long motifs in Lhx3-Peak-B and the LMO4-Peak are critical for the activity of these enhancers. When the HxRE-Long motifs in Lhx3-Peak-B or the LMO4-Peak are ablated, neither is responsive to even high levels of ectopic Isl1-Lhx3 complex expression. This result is consistent with Lhx3-Peak-B containing no additional HxRE motifs. However, unlike Lhx3-Peak-A, the HxRE-Short motif in the LMO4-Peak is unable to compensate for the loss of the HxRE-Long motif. Further mutational analysis of the LMO4-Peak, in which the HxRE-Short is ablated, will be necessary to determine whether this site can also contribute to the activity of this enhancer.Unlike the Lhx3 and LMO4 peaks, we found that the activity of the Isl1-Peak is mediated by cooperative action of multiple motifs. Hx-S1 contributes substantially to Isl1-Peak enhancer activity, but ablating Hx-S1 is not sufficient to completely abolish its enhancer activity. These findings indicate that other motifs, possibly HxRE-S2 and H motifs also contribute to the activity of the Isl1-Peak. As demonstrated by H2 E, some mChx10 (Chx10 . Our resIsl1 or Lhx3. LMCl neurons do not express Isl1, and Lhx3 expression is only maintained in MMCm neurons (In addition to facilitating Isl1 and Lhx3 transcription during MN specification, the Isl1-Peak and Lxh3-Peaks that we have characterized in this study likely act to maintain high levels of Isl1 and Lhx3 expression in mature MMCm neurons. Following MN specification, many MN subtypes downregulate the expression of  neurons . To halt neurons . It will


CAT, OMIM: 115500) is an endogenous antioxidant enzyme and genetic variations in the regulatory regions of the CAT gene may alter the CAT enzyme activity and subsequently may alter the risk of oxidative stress related disease. In this study, potential influence(s) of the A-21T (rs7943316) and C-262T (rs1001179) genetic polymorphisms in the CAT promoter region, using the ALGGEN-PROMO.v8.3 online software were analyzed. Our findings show that the A allele at the -21 position creates a new potential binding site for PAX-6 and the T allele at the -262 position changes the TFII-I binding site into STAT4 response element. The PAX-6 and STAT4 are the multifunctional and enhancing transcription factors.Catalase ( Oxidative stress may play a significant role in the risk of chronic diseases . AlthougCAT, OMIM; 115500), an endogenous antioxidant enzyme, plays a major role in controlling hydrogen peroxide concentration in human cells. It decomposes H2O2 into H2O and O2, thereby protecting the cells from oxidative stress. It has been suggested that functional polymorphism in the gene encoding catalase enzyme affects the enzyme activity, thereby altering the protection against oxidative stress [Catalase  was used. To identify transcription factor area, wild type allele and variant alleles were analyzed separately. In all analysis the maximum matrix dissimilarity rate was assumed 3%.First, the catalase gene promoter sequence was obtained from the NCBI. Then both polymorphic sites (rs1001179) and (rs7943316) were identified on sequence. For identification of transcription factor binding site the ALGGEN-PROMO.v8.3 online software  (CTATCCC) into STAT4 (CTATTCC) responsive element (The result shows that the -21A>T substitution in the promoter region of o GTCTC) . Moreove element .CAT at the -844 position (rs769214) might create a binding site for PAX-6 [This is the first report showing that the A-21T and C-262T polymorphisms create new putative PAX-6 and STAT4 binding sites, respectively. The PAX-6 is a highly conserved multifunctional transcription factor and has been proposed to bind to promoter sequences of catalase gene. Also PAX-6 is a key regulatory gene of eye and brain development . Previouor PAX-6 . The A aor PAX-6 .CAT promoter only if the T allele was present at the -21 positions. To our knowledge, the T allele at the -21 position may induce the CAT promoter activity. The signal transducer and activator of transcription (STAT) family of molecules is localized to the cytoplasm. STAT4 regulates various genes expression as a transcription factor and is involved in T helper cell (Th1) cell development [Our present findings also show that PAX-6 bind to the elopment .CAT promoter only if the T allele is present at the -262 position; however the C allele is the putative binding site for TFII- 1 transcription factor. Oxidative stress induces catalase activity. In addition, studies have shown that reactive oxygen species activate the STAT family protein via serine/threonine kinases [CAT promoter activity. The influence of the CAT A-21T and C-262T polymorphisms on the CAT mRNA levels should be further researched.The present results show that STAT4 binds to the  kinases . It may


This family is diverse, ranging from the housekeeping Group I (RpoDs), to Group IV (ECF) σ factors, that transcribe smaller regulons under more stringent conditions. RpoDs employ a kinetic mix-and-match mechanism, where promoter elements complement each other binding strengths in achieving sufficient transcription activity. On the other hand, it is assumed that ECF σs, which are the most distant from the housekeeping σ factors, cannot exhibit mix-and-matching. However, mix-and-matching for ECF σ factors was not quantitatively checked before, and recent results show a much larger flexibility in the promoter recognition by the members of this group.Transcription initiation is in bacteria exhibited by different σ factors, most of which fall within σE and σW), for which we use a biophysics based model of transcription initiation. For σE, we perform a separate analysis for in-vitro active and in-vitro inactive promoters, which allows us investigating how mix-and-matching depends on the external factors that may control transcription activity in the in-vitro inactive set. We show that the promoter elements of canonical ECF σs significantly complement each other strengths, where such mix-and-matching is in the in-vitro active set even stronger compared to the correlations observed for the housekeeping σs. This complementation however significantly decreases for the in-vitro inactive set, which we propose is due to mix-and-matching with regulatory sequences outside of the canonical promoter elements. In line with this proposition, we show that a conserved spacer element, which appears in the in-vitro inactive promoter set, significantly increases the promoter element complementation. While RpoD promoter elements mix-and-match to achieve sufficient total transcription activity, for σE they complement each other to achieve sufficiently strong total binding affinity, which we relate to differences in physiological responses between the two groups of σ factors.To this end, we quantitatively investigate mix-and-matching in two canonical ECF σ family members  contains supplementary material, which is available to authorized users. Promoters which are transcribed by this family share the same general structure , [P], [RNAP-P]c and [RNAP-P]o, respectively. The on and off rates of RNAP binding to the promoter are denoted as kon and koff, the transition rate from closed to open complex as kf, while the rate of RNAP escape from the promoter as ke. Thus, the first step in the scheme denotes reversible binding of RNAP to the promoter, which is followed by the opening of the two DNA strands and forming the open complex, as illustrated by the second step in the scheme. The last step is the irreversible RNAP promoter escape, followed by RNAP transition to the elongation.where RNA polymerase, promoter DNA, closed and open RNAP-promoter complexes, are denoted as [As RNAP binds and unbinds on a much faster timescale (~1 s) compared to subsequent transition from closed to open complex ~100 s) [0 s [10],RNAP] denotes the concentration of free RNAP in the cell. Note here that transcription activity, is in Eq. (1.2) directly proportional to KBkf, whereby this product (of the binding affinity and the transition rate) corresponds to the usual measure of the promoter strength [where [strength .The kinetic parameters are directly related to the interaction energies between σ factor and the promoter DNA. First, we start with the binding affinity of RNAP to dsDNA, which depends on the interaction energies of σ factor with −35 element, with dsDNA segment of −10 element, and with the length of the spacer sequence between −35 and −10 elements , 13:1.3\S(−35), S(−10)ds)), ΔGds(S(−10)ds) and ΔG(γ) are, respectively, the interaction energies of σ factor with −35 element, dsDNA segment of −10 element, and the differences of the interaction energies due to the variable spacer length.where kf with the interaction energies, we use the mechanistic model of the open complex formation [Moreover, for relating ormation :1.4\docuS(−10)ss)( denotes the −10 element segment which is melted during the open complex formation (interacts with the σ factor in ssDNA form). ΔGm(S(−10)ss) denotes the energy of opening S(−10)ss)( in the absence of RNAP (the DNA melting energy), while ΔGss(S(−10)ss) denotes the interaction energy of σ factor with ssDNA sequence S(−10)ss):From the expressions given above, the transcription activity of the promoter sequence KB(S) and φ(S) can be expressed as follows:To parameterize Eqs. (1.3)-(1.5), we use the independent nucleotide approximation –30, wherwiα denotes the weight matrices, and the superscripts ((−35), (−10) or (γ)) indicate that these matrices correspond, respectively, to −35 element, −10 element or the spacer length. Different positions within −35 and −10 promoter elements are marked with the index i, whereas the index j denotes five possible spacer lengths.where KB(S) ). Similarly, from Eq. (1.7), we see that the log transcription activity log(φ(S) ), is obtained by summing the weight matrix scores of the promoter elements which interact with σ factor in ssDNA form. These relations between the weight matrix scores and the relevant kinetic parameters were used for analyzing how the promoter elements complement each other strengths.In summary, from Eq. (1.6), we see that the weight matrix scores of the promoter elements that interact with σ factor in dsDNA form contribute additively to the log binding affinity - log omitted, due to the difficulty in aligning its −35 element (bearing at least five mismatches from the consensus). These promoters were retrieved from the DBTBS database, that contains information on Bacillus subtilis promoters and σ factors [σnditions . σW prom factors . RpoD prde novo aligned through Gibbs Motif Sampler, in the Motif Sampler mode, by searching only the direct strand, setting the number of motifs to 1 and the total number of sites to the number of query sequences. The motif length was set to several different values for each promoter set (to verify the robustness of the detected motif), while the remaining parameters were at their default values.All the sequences were wα,i, defining weights for base α at position i in the motif are calculated as [The weight matrix elements- lated as , 31:\docn is the number of motifs in the alignment, va,i is the frequency with which the base α appears in the alignment at the position i, and pa represents the base background frequency; adding of pa in the numerator corresponds to the pseudocounts.where Weights, corresponding to different spacer lengths, are calculated according to :\documenwi represents the weight of a spacer of length i, and vi is the spacer frequency.where Also, from each column of a weight matrix, we subtracted the value that corresponds to the consensus base, so that the score of the consensus motif becomes zero.E and σW promoters (datasets described above) – for σE we used the alignments of its −35 elements, extended −10 elements, short −10 elements, and the alignment of the spacer motif identified in this study  routine. The same MATLAB function allows calculating


The transcriptional activation of CXCL10 and CXCL11 by ZNF395 was abolished after inhibition of IKK by BMS-345541, which increased the stability of ZNF395. ZNF395 encodes at least two motifs that mediate the enhanced degradation of ZNF395 in response to IKK activation. Thus, IKK is required for ZNF395-mediated activation of transcription and enhances its turn-over to keep the activity of ZNF395 low. Our results support a previously unrecognized role of ZNF395 in the innate immune response and inflammation. Activation of the interferon (IFN) pathway in response to infection with pathogens results in the induction of IFN-stimulated genes (ISGs) including proinflammatory cytokines, which mount the proper antiviral immune response. However, aberrant expression of these genes is pathogenic to the host. In addition to IFN-induced transcription factors non-IFN-regulated factors contribute to the transcriptional control of ISGs. Here, we show by genome wide expression analysis, siRNA-mediated suppression and Doxycycline-induced overexpression that the cellular transcription factor ZNF395 activates a subset of ISGs including the chemokines CXCL10 and CXCL11 in keratinocytes. We found that ZNF395 acts independently of IFN but enhances the IFN-induced expression of CXCL10 and CXCL11. Luciferase reporter assays revealed a requirement of intact NF Secreted type I IFNs bind to the ubiquitously expressed IFNA receptor (IFNAR) on the same cells as well as on neighboring cells and initiate a signaling cascade finally leading to the activation and phosphorylation of the signal transducers and activators of transcription (STAT) STAT1 and STAT2. This enables complex formation with IRF9 to build the trimeric transcription factor ISGF3 which binds to the IFN-stimulated response element (ISRE), present in the upstream region of ISGs. ISGF3 activates the transcription of a set of several hundred ISRE driven ISGs which have antiviral and proinflammatory activity. The products of ISGs counteract viral replication, transcription, and translation and thus establish an antiviral status in the infected and uninfected bystander cells and stimulate the adaptive immune response. Shortly after the IFN exposure cells acquire an IFN-desensitized state allowing recovery from the effects of IFN. Dysregulation of IFN production and signaling results in autoimmune disorders such as systemic lupus erythematosus (reviewed in [The interferon- (IFN-) mediated innate immune response is the first line defense against invading pathogens. IFNs limit the spread of infectious agents and stimulate the adaptive immune system by promoting the development of high affinity specific T and B cell responses and immunological memory (reviewed in ). The deγ, which signals through the IFNG receptor (IFNGR), is largely restricted to the cells of the immune system. However, since the IFNGR is widely expressed nearly every cell can respond to IFNγ. The binding of IFNγ to the IFNGR results in the tyrosine phosphorylation of STAT1 monomers and the formation of STAT1:STAT1 homodimers, which translocate into the nucleus and bind to γ-activated sequence (GAS) elements present in the promoters of IFNγ responsive genes . IFNγ h+ T cell subsets and thus contributes to the regulation of the inflammatory process [Types I and II IFN-mediated induction of proinflammatory mediators, including the chemokines CXCL9, CXCL10, and CXCL11, plays a key role in establishing local inflammation. The three chemokines are ligands of the CXCR3 receptor that is expressed on different leucocyte subsets including monocytes, natural killer (NK) cells, and activated T lymphocytes and attract these leucocytes to local sites of inflammation [ process . The CXC process ). Here t process  and they process , 8.κB binding motifs within their upstream regulatory region and STAT factors were found to cooperate with NFκB. The transcriptional activity of NFκB is controlled by the IκB kinase (IKK) complex, which is activated upon signaling induced by multiple pattern recognition receptors and proinflammatory cytokines .α-mediated induction of IFIT1, IFIT2, IFI16, and IFI44 [Previously, we could show that the barely characterized transcription factor ZNF395  containing medium (1 μg/ml) for 24 h. Pooled primary human fibroblasts, isolated from foreskin (HFF), were cultivated in DMEM supplemented with 10% FCS and the antibiotics Streptomycin/Penicillin. PolyI:C (from Invivogen) was used at 10 ng/ml for 24 h, TNFα  at 1 ng/ml for 24 h, and BMS-345541 (from Sigma Aldrich) at 5 μM for 24 h [β and IKKα in vitro [Primary normal human keratinocytes (NHEK) were obtained from Promocell and cultivated in KGM2 medium. The immortalized keratinocyte cell line RTS3b  and the for 24 h , a concein vitro .β (Addgene Plasmid 15470) and HA-IKKα (Addgene Plasmid 15469) are published [The CXCL10 promoter-luciferase constructs have been described in . The expublished .γ (Biomol) or 1000 U/ml IFNα (Biomol) was added 24 h or 42 h after transfection. The cells were harvested 48 h after transfection and total cellular RNA was isolated. The supernatant was assayed for CXCL10 using an CXCL10 ELISA kit from BioLegend.Small interfering RNAs (siRNAs) were obtained as a pool of four annealed double stranded RNA oligonucleotides from Dharmacon (siZNF395: M-020387 and siControl: D-0012061420). Cells were seeded in six wells and transfected with 50 pmol siRNA using Lipofectamine RNAiMax (Invitrogen) one day later. As indicated, 850 U/ml IFN4, pH 7.8, 0.1% NP40, and 1 mM DTT and the luciferase activity was determined with Promega's GloMax® Multi Reader as described previously [Cotransfections of siRNA and CXCL10-Luc promoter constructs were done in 24 wells using 7.5 pmol siRNA, 62.5 ng luciferase reporter plasmid and Lipofectamine 2000 (Invitrogen) according to the manufacturer's instruction. 48 h later the cells were harvested in 100 mM KPOeviously . RTS3b ceviously .μg of RNA was reverse transcribed using random primer and the Go-Script Reverse Transcriptase (Promega) or the Maxima reverse transcriptase (Invitrogen). QRT-PCR was performed with the Go-Taq qPCR mastermix including Sybr green (Promega) and a Roche Light Cycler 480 (Roche Diagnostics). The sequences of the primers used for PCR are provided in Total RNA was isolated by the NucleoSpin® RNA Mini Kit from Macherey-Nagel . C-DNA synthesis and hybridization to Affymetrix Exon 2.0 ST array was performed by the group of Professor Nürnberg . The raw data were processed with the help of the Affymetrix Expression and Transcriptome analysis console. For quantitative RT-PCR, 2 t-test for paired samples.Significance of the differences was determined by the α-mediated gene expression. Since we initially cloned ZNF395 from a keratinocyte cell line [α in keratinocytes. Type I IFNs comprise IFNα, IFNβ, IFNε, IFNκ, and IFNω. While nearly every cell is capable of producing IFNα/β the other type I IFNs display tissue specific expression. Primary keratinocytes constitutively express IFNκ [κ and the induction of ISGs by exogenously added IFNα we chose the keratinocyte cell line RTS3b that does not have constitutive expressed IFNκ, since it is epigenetically silenced by methylation . We performed a microarray to analyze the ZNF395-dependent gene expression profile of IFNα treated RTS3b keratinocytes. RTS3b cells were transfected with control siRNA or with a pool of four siRNAs targeting ZNF395 to suppress the endogenous level of ZNF395. 18 h later the cells were incubated in medium containing IFNα for 6 h and total cellular RNA was isolated and transcribed to c-DNA. The expression of ZNF395 was reduced by 80% (data not shown). C-DNAs were hybridized to Affymetrix HuGene 2.0 ST Arrays. We identified 304 transcript clusters/terms that were differentially expressed by more than 2-fold (p < 0.01) due to the lack of ZNF395 (p < 0.05 and fold change > 2) validated IFN-regulated genes. This revealed that our gene list contained 25 known human IFN-regulated genes that were activated and 29 that were repressed by ZNF395. As depicted by the Venn diagram in p = 0.019 and SAMD9, p = 0.0002) and 1.49-fold  upon the knock-down of ZNF395. This congruency of the data sets indicated the reliability and reproducibility of the results of the two microarrays, identifying differentially expressed genes due to ZNF395 overexpression, as reported in [α treated cells, as reported here.We systematically addressed the impact of ZNF395 in the IFNell line  we invesess IFNκ . In ordef ZNF395 . These iorted in , and dueα. We visualized the impact of ZNF395 by analyzing their IFNα-mediated induction after the suppression of ZNF395 by siRNA on the one hand , and primary human foreskin fibroblasts (HFF). As before, all cell types were transiently transfected with a pool of siRNAs against ZNF395 to suppress ZNF395. To compare the expression of CXCL10/CXCL11 and ZNF395 among the different cell types, the gene specific qRT-PCR crossing points (CP) were normalized with those for the house keeping gene HPRT and the resulting values from untreated, siControl transfected NHEK were set as 1. This approach revealed that siControl transfected RTS3b cells had lower basal level of CXCL10 with 0.07 compared to primary keratinocytes . Thus, it seems that ZNF395 is not significantly involved in the IFN-controlled transcription of CXCL9 and CXCL11 in HFF but in the IFNα and IFNγ-induced activations of CXCL10, while in keratinocytes it is required for CXCL11 as well. In addition, CXCL10 and CXCL11 are more efficiently activated by IFNs in HFF, which do not express IFNκ [We also analyzed the expression of CXCL11 under these conditions in the keratinocytes and fibroblasts. Although the net values differed, the si-RNA mediated suppression of ZNF395 significantly reduced the effects of IFNinocytes . Surprisess IFNκ  and the α and IKKβ, the catalytic subunits of IKK [α. This activation was completely lost in the presence of BMS-345541. This clearly demonstrates that ZNF395-mediated activation of CXCL10 is not dependent on the IFN-induced transcription factors but on active IKK. In line with the observation shown in α induced activation of CXCL10  and the AP1 site, respectively, the promoter activity decreased by about 50%, and, again, the suppression of ZNF395 led to a clear further drop of activity. The lower activities with these mutated promoter constructs as a result of the suppression of ZNF395 were significant. The observation that the suppression of ZNF395 further significantly reduced the promoter activities independent of the mutations within ISRE, STAT, and AP1 motif, indicates that endogenous ZNF395 still activates the promoter even in the absence of these factors. In agreement with previous publications, also the mutation of either of the two NFκB motifs had a drastic effect on the CXCL10 promoter. Although the suppression of ZNF395 slightly further reduced the luciferase activities of the NFκB mutated CXCL10 promoters, these effects turned out to be not significant S/T. The WD40 repeats of β-TRCP bind to the DpSGXX(X)pS/T diphosphorylated sequence in its substrates and initiate the degradation. The amino acids from positions 211–216 of ZNF395 with the sequence DSGSSTT are similar to such a motif. We addressed the role of these amino acids in the IKK-regulated turnover of ZNF395. We initially used two fragments of ZNF395, encoding either amino acids 189–378 (Δ3), containing the putative phosphodegron motif, or amino acids 223–378 (Δ4), lacking this element, and overexpressed them in RTS3b cells. To enhance IKK activation and, consequently, degradation of ZNF395 the cells were treated with either TNFα or poly I:C (pI:C) [α-induced degradation and the BMS-345541-mediated stabilization of the mutated ZNF395Δ3 4xST/A fragment  was still able to induce the degradation of ZNF395  induced the degradation of all fragments. The degradation of ZNF395Δ10, encoding the C-terminal 133 amino acids illustrates that there is one additional sequence mediating IKK-dependent destabilization present in ZNF395  . A westef ZNF395 , indicatn ZNF395 . Previouand IKKα . Figure α-mediated control of specific well characterized antiviral ISGs, we have identified here, is supported by several reports correlating reduced ZNF395 expression in patient in the context of an infection with HIV1, CMV, and EBV [Here, we report that the endogenous level of ZNF395 determines the IFN-mediated induction of a subset of ISGs including CXCL10 and CXCL11. We visualized the impact of ZNF395 on the expression of these ISGs in the keratinocyte cell line RTS3b in which we modulated the level of ZNF395 over a wide range by siRNA-mediated suppression and by Dox-induced overexpression. The functional relevance of the contribution of ZNF395 to the IFN and EBV –31. Our α- but also to the IFNγ-mediated induction of CXCL10 in skin fibroblasts representing the main cell source of this chemokine in the skin [α and IFNγ. Maybe the IFN-regulated transcription factors are more efficient or other cofactors are available in HFF making the contribution of ZNF395 dispensable, especially in light of the lower affinity of ZNF395 to the CXCL11 promoter compared to the CXCL10 promoter.We confirm the relevance of ZNF395 for the transcription of CXCL10 and CXCL11 in primary keratinocytes. In addition, ZNF395 not only contributes to the IFNthe skin . We cannκB motifs. Two possibilities concerning the role of ZNF395 can be envisioned. Firstly, the loss of the activating function of ZNF395 is due to the absence of NFκB factors bound at their specific DNA motifs. Here, ZNF395 might be recruited to the CXCL10 promoter via a direct interaction with NFκB subunits. Alternatively, ZNF395 might directly bind to a DNA motif overlapping the NFκB consensus motif. In such a scenario, ZNF395 acts independently of NFκB. Several mutational analysis revealed that the C-clamp DNA binding domains of ZNF395 and its closely related factor SLC2A4RG/GLUT4EF are sufficient for binding to helper site like sequences which have been mapped to CC/GGCC/G [κB motifs within the CXCL10 promoter  do not contain such a motif, stretches of C/G are present, which have been mutated to A or T nucleotides in the CXCL10 promoter construct we used here [κB site [β, IL-6, IL-8, and LIF under hypoxia [κB sites within their regulatory regions as well. It will be tempting to analyze the role of NFκB recognition motifs in the ZNF395-mediated activation of these factors.Our results in keratinocytes are in line with the assumption that ZNF395 acts independently of the IFN-induced STAT transcription factors but affects basal promoter activity mainly through the NFC/GGCC/G , 14, 16.sed here . It may [κB site . We prev hypoxia . These gκB signaling pathway. IKK enables ZNF395 to activate transcription and enhances its turn-over to limit its activity. The observation reported by Loveless et al. that ZNF395 was stabilized by MLN4924, an inhibitor of the cullin-RING E3 ubiquitin ligases, including β-TRCP, demonstrates that β-TRCP is responsible for the ubiquitin-dependent degradation of ZNF395 [β-TRCP. ZNF395 encodes more than one independent acting IKK and β-TRCP dependent degradation motif. Beyond the one in the central part, whose functionality we demonstrated here, there is at least one additional degradation motif present in the C-terminal 133 amino acids, although no canonical phosphodegron motif is obvious. It has been observed that several bona fideβ-TRCP substrates contain highly degenerated or noncanonical degrons, for instance, DSG/DDG/EEG/SSGXXS/E/D motifs [κB subunit p65. Phosphorylation of p65 induces a conformational change which impacts p65 ubiquitination, stability as well as protein-protein interactions. In addition, the phosphorylation of p65 within its transactivation domain leads to enhanced recruitment of CBP/p300 [κB-independent functions of IKK [κB independent mechanism to activate CXCL10 expression. IKK was found to be required for the release of CXCR3 ligands from IFNγ stimulated bronchial epithelial cells, independent of NFκB activation. The release of CXCL10 was very sensitive to inhibition of IKKβ. It has been shown that IκBα did not undergo phosphorylation and NFκB was not activated in these cells after stimulation with IFNγ [Strikingly, our results support the connection of ZNF395 with the NFf ZNF395 . Our obsD motifs . IKK notCBP/p300 , 57. Thes of IKK . Severalith IFNγ . Moreoveith IFNγ .α-mediated activation of both promoters in keratinocytes (Figures α. Thus, ZNF395 may have a higher affinity to the CXCL10 promoter and lower affinity to the CXCL11 promoter, which therefore requires high ZNF395 level for efficient activation. The regulation of the expression of the two CXCR3 ligands in a concentration dependent manner by ZNF395 may also have biological consequences. Despite binding to the same receptor, CXCL10 and CXCL11 were shown to stimulate distinct intracellular signaling pathways. CXCL9 and CXCL10 are so-called driver chemokines that support inflammation. CXCL10 induces Th1/Th17 cells to promote inflammation. In contrast, CXCL11 binding to the CXCR3 results in the development of IL-10 high T regulatory 1 subsets, leading to dampening of inflammation [The CXCL10 and CXCL11 promoters seem to differ in their sensitivity towards ZNF395. The suppression of ZNF395 reduced the IFN Figures , demonstammation . Taken i+ T cells to the epidermis. Vitiligo is an autoimmune disease of the skin that leads to depigmentation. It is initiated by autoimmune T cell mediated killing of melanocytes. These autoreactive T cells are homed to the skin by CXCL10 produced from keratinocytes to kill the melanocytes [CXCL10 expression by keratinocytes drives vitiligo pathogenesis through the recruitment of autoreactive CD8anocytes . In the anocytes . RegardiOur results demonstrate that the barely characterized transcription factor ZNF395 is required for the maximal IFN-mediated induction of a subset of ISGs. ZNF395 acts independently of IFN. As depicted in


The epigenetic events imposed during germline reprogramming and affected by harmful exposure can be inherited and transferred to subsequent generations via gametes inheritance. In this study, we examine the transgenerational effects promoted by widely used herbicide atrazine (ATZ). We exposed pregnant outbred CD1 female mice and the male progeny was crossed for three generations with untreated females. We demonstrate here that exposure to ATZ affects meiosis, spermiogenesis and reduces the spermatozoa number in the third generation (F3) male mice. We suggest that changes in testis cell types originate from modified transcriptional network in undifferentiated spermatogonia. Importantly, exposure to ATZ dramatically increases the number of transcripts with novel transcription initiation sites, spliced variants and alternative polyadenylation sites. We found the global decrease in H3K4me3 occupancy in the third generation males. The regions with altered H3K4me3 occupancy in F3 ATZ-derived males correspond to altered H3K4me3 occupancy of F1 generation and 74% of changed peaks in F3 generation are associated with enhancers. The regions with altered H3K4me3 occupancy are enriched in SP family and WT1 transcription factor binding sites. Our data suggest that the embryonic exposure to ATZ affects the development and the changes induced by ATZ are transferred up to three generations. Epigenetic factors, such as DNA methylation, non-coding RNAs and histone modifications are involved in the regulation of many cellular functions and developmental reprogramming . EpigeneRecently, a series of studies have shown that exposing the gestating mammalian female F0 to environmental factors during embryonic gonadal sex determination causes changes in DNA methylation that are retained in adult F1–3 male progeny, which produce sperm with altered DNA methylation –12. The de novo after replication. During replication, parental histones re-associate locally with newly synthesized DNA in budding yeast  on transgenerational inheritance. ATZ is the most common contaminant found in underground waters in the USA  and manyer genes ,36. A seer genes . Recent er genes . In mammer genes –42, ovarer genes –47, braier genes ,49, liveer genes –54 and ter genes .We hypothesized that embryonic exposure to ATZ during the E6.5 to E15.5 developmental window will cause heritable epigenetic reprograming (modifications) and affect reproduction in subsequent generations. To test this hypothesis, we treated gestating outbred CD1 mice with ATZ and studied subsequent generations, F1 and F3. We examined H3K4me3 modifications in the testes and RNA expression in several tissues. We show here that embryonic exposure to ATZ globally changes tissue-specific RNA expression patterns, which become deregulated concomitant with the alterations in H3K4me3 marks. We suggest that H3K4me3 occupancy pattern in the F3 generation of ATZ-lineage is derived from embryonic exposure of F1 males and that the pattern of altered H3K4me3 peaks in F3 is preserved from the previous generations, however, the mechanism of this inheritance is not known. We believe this work to be the first comprehensive study that integrates genome-wide ChIP-seq and RNA-seq approaches in two generations (F1 and F3) of animals with the goal of understanding transgenerational inheritance upon toxicant exposure. Moreover, this study generates a large number of novel sequencing data for an outbred CD1 mouse strain that is commonly used in many studies.Total RNA was prepared from fresh or frozen tissue using TRIzol reagent (Life Technologies) and RNA easy plus kit (Qiagen)t-test. The qPCR-quantification of ChIP-seq experiments was performed as previously described  according to the manufacturer's instructions. Applied Biosystems (ABI) 7500 real-time polymerase chain reaction (PCR) system was used for quantitative PCR. Duplicates of at least four independent experiments were performed. Statistical significance was calculated by Student's escribed . Equal aChromatin immunoprecipitation was conducted as previously described . An Illuhttps://github.com/najoshi/sickle) with –q33 and mapped back to the mouse mm9/Ensembl genome using Bowtie 1.0.0 with seed length 20. Only tags that passed the quality filter and mapped uniquely to the genome were used. ChIP enrichment was further verified using CHANCE  term analyses were performed using DAVID v6.7  with enrFunctional annotation was performed by GREAT version 3.0.0  with defFor this analysis, we used the summits of 500-bp sequences from the differential peaks. Motif finding was performed using MEME-ChIP  with theA strand-specific library preparation protocol for RNA sequencing was performed using the sequencing technological platform at IGBMC, Strasbourg, France. We used three biological replicates for each tissue, testis, liver and hypothalamus. The sequencing was performed in massive parallel sequencing paired end mode, and the size of the sequencing tag was 50 bp.http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). For the analysis of differentially expressed genes (DEGs), the quality-checked reads for each condition were processed using the TopHat version 2.0.12 package (Mus musculus Ensembl mm9 sequence), and the alignment files were generated as binary sequence alignment map (BAM) files. These files were used as the input for Cufflinks, a complementary method used to generate assembled transcripts for each condition and the abundance was evaluated using read data. The assembled transcripts were compared and annotated using Cuffmerge against the Ensembl (mm9) gene annotations  and Coding Potential Calculator (CPC) ,73. Any It has been suggested that the ratio between H3K4me3 and H3K4me1 indicates the tendency of the region to act as either a promoter or an enhancer . To clasThe additional methods are described in SI in the ‘Methods’ section.To evaluate the effects of environmental chemicals on epigenetic inheritance, gestating outbred CD1 strain mice were treated with ATZ during embryonic period E6.5 to E15.5. This exposure window corresponds to reprogramming in the germline when the genome is undergoing global demethylation and subsequent remethylation. The progeny of F1-exposed males were crossed with unrelated females for three generations. F1 (exposed) and F3 (unexposed) males were used for transgenerational inheritance analysis. We used ATZ at 100 mg/kg/day. This dose is commonly used in toxicological studies with ATZ ,77. AccoThe morphological analysis of H&E stained sections did not reveal notable differences in testis architecture between ATZ-derived and vehicle-treated F3 males . To evaluate whether the relative proportions of germ and Sertoli cells were altered, we immunostained the testis sections with antibodies against proteins specific to these cell types ). Marker analysis did not reveal any notable differences between the ATZ-derived and control samples in F3 males . To assay the relative contributions of all cell types in the testes, we sorted the cells using FACS. Our data showed that ATZ treatment does not affect the relative proportion of cell types in the testes . Furthermore, we did not observe any significant changes in body or organ weights  or any decrease in testosterone and Follicle-stimulating hormone (FSH) levels , which are the expected outcomes of ATZ treatment in adult males . We cannThe number of spermatozoa is an important physiological parameter of normal testis function, so we counted the number of spermatozoa in the epididymis in each group of males. We found that the number of spermatozoa was significantly decreased in ATZ-derived F1 and F3 generation males compared to vehicle-treated control mice, with a decrease of nearly 30% in F3, suggesting disrupted meiosis or spermiogenesis . In summary, the embryonic exposure of F1 males to ATZ does not obviously affect the morphology or cell types in F3 testis tissues but results in a lower spermatozoa count.n = 199 in control and n = 195 in ATZ derived cells) showed the significant increase of meiotic defects in F3 male progeny of ATZ derived males . To confirm this phenotype we immunostained the spreads with antibody against TERF1 . TERF1 protein binds to repeats in telomeres and protects them against the degradation  formation. SCs are zipper-like structures, which are assembled between homologous chromosomes during the prophase of the meiosis I. Formation of SCs is essential for proper parental chromosome segregation before meiotic division . We immuradation . We founTo elucidate an ATZ effect on spermiogenesis we analyzed the protein level of protamine 2 from whole testis extract in F3 males. We found the protein levels of protamine 2 decreased 2.6 times in ATZ-derived testis . Recent study demonstrated that acetylation at histone H4 (H4K5ac and H4K8ac) is essential for bromodomain-containing protein (BRDT) binding, and that binding is, in turn, critical for histone-to-protamine transition . We analIn summary, our results demonstrate that meiosis and spermiogenesis are affected in F3 generation of ATZ lineage males and that could contribute to spermatozoa decline that we observe.Nbn, Rif1, Sun1, Ctc1) are also known to be involved in the regulation of telomere function  that have altered function in ATZ-lineage males were also identified in other ATZ studies. The large numbers of these genes are associated with mitochondrial function. Specifically, these are genes encoding oxidative reduction enzymes , genes of mitochondrial homeostasis . Many of ATZ-responsive DEGs are encoding ATP-binding proteins . Importantly, the expression level of Atpaf2  encoding the ATP synthase assembly protein has dramatically (eight times) decreased. This data suggests that mitochondrial metabolism and ATP synthesis could be an important part of ATZ lineage phenotype.To identify the cellular origin for the DEGs we used recently published datasets of genes that are expressed in different testicular cell types . We idenase.org/  of protease.org/  is basedP = 5.40E-07, Fisher exact test) and genes associated with meiotic cell cycle (P = 7.55E-05) among the others. The genes without H4K5Ac marks are associated with sensory perception of chemical stimulus function (P = 3.83E-82) and with G-protein coupled receptor signaling pathway (P = 7.94E-68). These data provide further support for the possibility that histone H4 acetylation is altered at regions of DEGs in ATZ progeny male mice.This data and the work of others  have sugMe1, Cyp2c44, Akr1b3, Glud1, Pgd, Cat, Dus1l, Hadha, Mtrf1, Slc25a22, Mtscp1, Timm23) and to mitochondrion function  pathways (Supplementary Table S3). Similarly, among very few DEGs that were identified in brain, we noted genes associated with embryonic development (B9d1), oxidation-reduction  and imprinted brain development (Nnat) (Supplementary Table S4).We noted that genes associated with mitochondrial function are also affected in the third generation in liver. The analysis of transcripts from liver revealed that many DEGs belong to the oxidoreductase . We acknowledge the possibility that LincRNAs does not necessarily regulate genes located in cis positions.Among differentially expressed transcripts in the testes, 41% are non-coding RNAs . Long non-coding RNAs play essential roles in many developmental processes, diseases and cancer –91. To cEmbryonic exposure to ATZ is associated with increases in the appearance of the new isoforms of RNA initiated from alternative TSSs, with increase in expression of alternatively spliced RNA isoforms and alternative polyadenylation (APA) transcriptsSlfn5 gene in ATZ-derived F3 testes  changes the RNA expression level in liver only in F3 ATZ-derived males. We identified a change in the H3K4me3 occupancy at the Adat1 promoter in the testes of F1 ATZ-derived males , suggesting that the changes in the testes of F1 males could contribute to the alteration in expression of RNA in other, non-testis, tissues in F3 generation males.In addition to characterized transcripts, we identified 241 previously un-annotated differentially expressed transcripts in the testes of F3 males from ATZ-derived progeny. These new isoforms encode proteins with transcription factor activities and well-established developmental roles (Supplementary Table S5). Alternative transcription is often used for the tissue-specific production of new proteins. Therefore, we asked whether these novel testis transcripts are being expressed elsewhere in other tissues. We determined that out of 241 alternative new transcripts found in the testes, 61 are expressed in both the liver and the brain, 16 only in the liver and 7 only in the brain, while 157 new isoform transcripts are specific for the testes only Figure . For exas Figure  that ares Figure  and C. Tferation . H3K4me3feration . At leasAlternative splicing is another important tissue-specific mechanism for regulation of gene expression. We asked whether the alternative splicing mechanisms are also affected in the ATZ F3 progeny of males. We found transcripts with alternative TSSs that also have fewer exons or more exons, as well as transcripts with the same number of exons but using different donor–acceptor splicing sites (Supplementary Table S5).Cd68 APA transcript has altered 3′UTR. The change of 3′UTR in Cd68 APA transcript occurs during spermatogenesis. The Cd68 APA transcript in F3 ATZ-derived testes has an altered APA site, which in the control testes is detected only in a small fraction of leptotene spermatocytes and at later stages it becomes shorter. However, in F3 ATZ-derived testes, the transcript does not change in length . These data suggest that Cd68 transcript acquires the altered APA site specifically in the ATZ lineage, however, the role of this transcript or Cd68 gene in meiosis is unknown.In addition to alternative TSS isoforms and alternatively spliced transcripts, we identified 50 differentially expressed transcripts with APA sites (Supplementary Table S6). Among differentially expressed APA transcripts, 21 are found in the testes, 18 in the liver and 11 in the brain. Recently, it has been shown that APA regulation is important for RNA processing during spermatogenesis . We compIn summary, ATZ exposure during embryonic development in F1 generation associated with the appearance of transcripts with novel transcriptional initiation sites, differentially spliced and APA transcripts in the third generation after treatment.P-value of 10−5, Poisson distribution test, generated by MACS 2.0.1) . We found the differential peaks to be distributed throughout the genome  to be significantly decreased. In contrast, the expression levels of histone H3 lysine demethylase, Kdm5b is increased in the testes of F3 ATZ-lineage males . These data suggest that the altered activities of histone modifying enzymes in the F3 generation may contribute to the global changes in H3K4me3 mark occupancy. To investigate the genomic context of regions with altered H3K4me3 occupancy, we inspected the genes located in the vicinity of differential peaks using GREAT, v3.0.0  signaling pathway, show decreased H3K4me3 occupancy; in contrast, the region of Rxra have increased H3K4me3 occupancy. RA plays key roles in SG differentiation and is essential for meiosis initiation  and post-meiotic cells (spermatids) using cell sedimentation in bovine serum albumin gradient method as described in SI ‘Methods’ section and performed ChIP-seq using H3K4me3 from recovered fractions. We identified 53 974 and 52 201 peaks in spermatocyte and spermatid fractions, respectively. We compared the differential peaks with normalized peak intensity of each fraction. We identified 273 differential peaks that have high signal in spermatocytes (meiotic fraction) and 200 differential peaks with high signal in spermatid fractions (postmeiotic fraction). H3K4me3 peaks in both spermatocytes and spermatids are generally located distally from promoters .Mei1, Trip13, Spin1, Spdya and Wrn . Differential peaks of spermatid fraction are enriched in genes associated with mitosis and chromatin organization functions, includingAkap9, Asz1, Cdc7, Cdk1, Pabpc1l, Rad21, Trip13 and Vrk1 .The identified peaks were functionally annotated with GREAT. We found that differential peaks that belong to spermatocyte fraction are enriched in epithelial cell development genes and meiosis-specific genes, including (H3K9me demethylase) and BRD4 (chromatin remodeling factor). These enzymes maintain the pluripotent state of the cell . They are also required for mesenchymal to epithelial transition during induced reprogramming of human fibroblasts into stem-like cells  harbor altered H3K4me3 marks (FC > 1.2) within 5 kb of the TSS. Because H3K4me3 marks are also known to associate with meiotic double strand break (DSB) hotspots, we excluded H3K4me3 peaks that could be DSB-associated, namely the intergenic distally located weak signal peaks. A previous work determined that peaks associated with DSBs normally comprise ∼17% of the total number of H3K4me3 peaks .This current work suggests that, in addition to alterations in the H3K4me3 occupancy in proximal locations, a large number of testis differential peaks in the ATZ lineage are located distally and correspond to the enhancers and promoters of non-coding RNAs. We identified 121 out of 473 differential peaks (25.6%) as presumptive promoters; the majority of peaks correspond to presumptive enhancers (74.4%). To correlate the promoter peaks intensity with altered RNA transcript expression level, we analysis the altered promoter peaks corresponding to alterations in RNA expression levels and found 56 out of 121 promoter peaks correlates with altered RNA expression (FC > 1.2) . However, we found that only a few regions gained or lost peaks in ATZ males , with most of them located in intergenic regions.Dmrt1, Cux1, Runx1, Zfpm2, Foxc2, Foxk1, Sox6, Sox14, Sp5, Asxl2 among others. For example, Dmrt1 is an essential gene regulating pluripotency during development . We found that 71 enhancer-associated genes (out of 558) are genes encoding for transcription factors. These are genes essential for embryogenesis, such as elopment . This daIn summary, while a small fraction of DEGs harbor altered H3K4me3 pattern at their TSS, the majority of modified H3K4me3 peaks are localized to enhancers.P-value of 10−5. Using a cut-off fold change value of 2 and FDR < 10%, we identified 270 differential peaks between control and F1 ATZ-lineage males. To reveal the regions in the genome that become altered in F1 in ATZ-derived males and could contribute to transgenerational change in the F3 generation males, we performed functional annotation using GREAT v3.0.0. We identified the promoters of 375 genes with altered H3K4me3 occupancy. We found the highest enrichment of altered H3K4me3 occupancy at genes associated with stem cell differentiation and stem cell development and maintenance  (Supplementary Tables S9 and 10). For example, two conservative blocks are present within the promoter of Sox18, which contain binding sites for the SP1/SP3/WT1 and SP4 factors  located within the sequences of differential peaks Figure . The inss Figure . The anade novo DNA methyltransferases; the significant increase of Dnmt3a expression was found . Changes in DNA methylation in embryonic germ cells can be associated with changes in histone epigenetic landscape since DNA methylation and histone modifications are functionally linked and can affect gene expression ; the expression of SINE-B1 was also higher in F3 adult males (data not shown). Our data suggest that exposure to ATZ causes changes in embryonic germ cells, which could result in transcriptional network deregulation and transmit these changes to subsequent generations.To assess whether embryonic ATZ exposure directly affects the germ line cells, we performed the analysis of the number of germ cells using the paraffin testis sections at E15.5 and E18.5 of F1 generation males immunostained with the antibody against specific germ cell marker, DDX4 . The quantitation of DDX4-positive cells revealed that germline population is significantly diminished at E18.5 . The somatic to germ line reprogramming is also associated with global DNA demethylation and remethylation. We analyzed the expression level of pression . It is apression . To explSun1 gene to be differentially expressed and observed an increase in TERF1 at fused telomeres. This is important as the recent paper showed that the increase in TERF1 leads to formation of chromosome fusions  . These are key regulators of germ cell development  and genes that are important for spermatogenesis  and cell cycle . RFC1 and RFC2 are also ATP-binding proteins. The SG derived from affected cells have modified transcriptional network that impacts both meiotic and post-meiotic testis fractions. The defective meiotic and post-meiotic cells could be eliminated either by Sertoli cell phagocytosis   and publ3.4.139) . We idenocytosis  or in epocytosis  and ultiocytosis  and SHH ocytosis  are esseocytosis  and FOXIocytosis . ATZ-indDclk1 expression in the brain is initiated from an upstream promoter during early postnatal development, and is switched to the downstream promoter in adult tissue . Genetit tissue . This prt tissue . We suggNanog gene expression . However, whether the differences we observed are sufficient to impact the zygote development remains to be understood. Our model predicts that the changes in germ cells induced by ATZ will be preserved at limited genomic regions. Specifically, we propose that the genes, which are expressed from both paternal and maternal alleles in the very early zygote  are required to be in an open chromatin state , cell growth- and differentiation-  and cell adhesion- (Itgb3) associated genes. Combined, these data hint at the existence of ‘sensitive’ regions in the mouse genome that are involved in transgenerational inheritance, albeit mediated through different mechanisms. We suggest that these regions possess vulnerable chromatin structure, which makes them sensitive to exposure. Additionally, the modified epigenetic state could reflect the involvement of these regions in responding to toxic conditions, for example, to augment protein synthesis, which requires a new chromatin state. Both DNA methylation and histone modifications must cooperate in generating a defined epigenetic environment to respond to toxic stress. A functional connection between histone H3 methylation and DNA methylation state is known to exist, and histone H3K4me3 marks instruct DNA methylation by inhibiting the binding of de novo methylases (To determine whether exposure to different toxicants targets different or similar genomic regions, we compared the H3K4me3 regions affected by ATZ and the previously published data on differentially methylated regions in a vinclozolin study . In thatthylases ,132. We Our data suggest that transgenerational inheritance in ATZ-lineage males is mediated via altered epigenetic state of defined regions in the genome.


CRH gene expression in the placenta is epigenetically controlled setting gestational trajectories characteristic of normal and pathological pregnancies. Here we determined histone modification and DNA methylation levels and DNA methylation patterns at the CRH promoter in primary trophoblast cultures by chromatin immunoprecipitation combined with clonal bisulfite sequencing and identified the transcriptionally active epialleles that associate with particular histone modifications and transcription factors during syncytialisation and cAMP-stimulation. CRH gene expression increased during syncytial differentiation and cAMP stimulation, which was associated with increased activating and decreased repressive histone modification levels at the promoter. DNA methylation levels remained unchanged. The nine CpGs of the CRH proximal promoter were partially and allele-independently methylated displaying many (>100) epialleles. RNA-polymerase-II (Pol-II) bound only to three particular epialleles in cAMP-stimulated cells, while phospho-cAMP response element-binding protein (pCREB) bound to only one epiallele, which was different from those selected by Pol-II. Binding of TATA-binding protein increased during syncytial differentiation preferentially at epialleles compatible with Pol-II and pCREB binding. Histone-3 acetylation was detected only at epialleles targeted by Pol-II and pCREB, while gene activating histone-4 acetylation and histone-3-lysine-4 trimethylation occurred at CRH epialleles not associated with Pol-II or pCREB. The suppressive histone-3-lysine-27 trimethyl and–lysine-9 trimethyl modifications showed little or no epiallele preference. The epiallele selectivity of activating histone modifications and transcription factor binding demonstrates the epigenetic and functional diversity of the CRH gene in trophoblasts, which is controlled predominantly by the patterns, not the overall extent, of promoter methylation. We propose that conditions impacting on epiallele distribution influence the number of transcriptionally active CRH gene copies in the trophoblast cell population determining the gestational trajectory of placental CRH production in normal and pathological pregnancies.Placental CRH production increases with advancing pregnancy in women and its course predicts gestational length. We hypothesized that  Corticotropin releasing hormone (CRH) concentration increases exponentially in the maternal plasma during the third trimester of pregnancy. Spontaneous preterm birth is associated with an accelerated rise, while post-term pregnancies are characterised by a significantly retarded increase of CRH level in the maternal circulation . The traCRH gene expression in trophoblasts [CRH gene activity in cultured trophoblast cells supporting the possibility that trophoblast-derived hormonal factors acting via cAMP, such as hCG, may also drive CRH expression in an autocrine feed-forward manner [CRH promoter-reporter constructs have indicated that CRH gene regulation in trophoblast cells depends on complex molecular interactions centered around a canonical cAMP response element (CRE) in the proximal promoter [CRH expression control under various basal, glucocorticoid- and cAMP-stimulated conditions [The source of plasma CRH in pregnancy is the syncytial layer of the placenta . It has hoblasts . Furtherd manner , 5. Studpromoter . A seconnditions –9.CRH gene regulation involving epigenetic chromatin modifications in the promoter region. In the central nervous system of rats and mice, CRH gene activity is inhibited by DNA methylation and histone deacetylation in the CRH promoter region, while in human trophoblasts, dynamic histone-3, lysine-9 acetylation at the CRH promoter has been shown to underpin the glucocorticoid stimulation of CRH expression [CRH gene in a highly methylated domain of the trophoblast genome containing genes with tissue specific expression [CRH proximal promoter is located within a CpG island shore region (relative to the intragenic CpG island), which is typically associated with tissue-specific differential methylation and gene expression [CRH gene contains 9 CpG dinucleotides, which can be methylated. These CpGs are conserved in the mouse, rat and the human including their positions within the promoter sequence [CRH proximal promoter is located within the CRE. Methylation of the CpG within the CRE has been shown to have major effects on the transcription factor binding properties of the sequence, diminishing its affinity to pCREB (phosphorylated cAMP response element binding protein) and enhancing its activity to bind CEBPA  with profound functional consequences on gene expression control [CRH gene activity in human trophoblasts by influencing transcription factor binding and the deposition of histone modifications that control chromatin structure coordinately with DNA methylation. We used primary human trophoblast cells in the experiments, which differentiate to syncytia in culture and respond to cAMP stimulation with robust increase of CRH expression [CRH promoters in subpopulations of CRH gene copies bound to transcription factors or marked with various histone modifications. The results uncovered the epigenetic and functional heterogeneity of the CRH promoter in the trophoblasts. The results have also suggested that only a minority of CRH gene copies is configured epigenetically to be transcriptionally active in response to 8-Br-cAMP. The size of this subpopulation, potentially influenced by the uterine environment, may define the gestational trajectory of CRH expression in normal and pathological pregnancies.Recent studies revealed an additional level of pression –12. A repression . Furtherpression . The prosequence . Notably control . Based opression . CombiniCRH mRNA, CRH peptide and CRH promoter methylation were determined in cultures from Placentae No. 1, No. 2 and No. 3. Chromatin isolated from one primary trophoblast culture was sufficient to perform 5 combined chromatin immunoprecipitation (ChIP)–bisulfite-sequencing procedures. For this reason, cultures from Placentae No. 1, No. 2 and no. 3 were used for pCREB, TBP  and acetylated-histone 3 (acH3) ChIP-bisulfite sequencing, and Placentae No. 4, No. 5 and No. 6 were used for acetylated histone 4 (acH4), trimethyl histone 3-lysine 4 (H3K4me3), trimethyl histone 3-lysine 27 (H3K27me3) and trimethyl histone 3-lysine 9 (H3K9me3) ChIP-bisulfite sequencing. Cultures from all 6 placentae were processed for RNA polymerase II (Pol-II) ChIP-bisulfite sequencing analysis. The placenta numbers are indicated in the respective figures.The study was approved by the Hunter New England Health Human Ethics Committee and the University of Newcastle Human Ethics Committee . All participants provided written informed consent to participate in the study. Placentae were obtained from uncomplicated singleton pregnancies after spontaneous labour and delivery at the John Hunter Hospital, Newcastle NSW, Australia. Participants delivered at term (37–41 weeks of pregnancy) as confirmed by early gestation ultrasound dating. Labour was uninduced and unaugmented, and no maternal or fetal morbidities were encountered during pregnancy and labour. Cytotrophoblasts were isolated, purified and cultured as described by Kliman et al., [CRH mRNA relative abundance has been described in detail [The procedures of extracting and purifying RNA from cultured trophoblasts, removing contaminating DNA, reverse transcription and measuring n detail  includinn detail   with thermal cycle conditions following the manufacturer’s protocol for small input DNA (between 50 pg and 100 ng). The  in Ref.  and SuppCRH mRNA relative abundance and peptide production values were logarithmically transformed to approach normal distribution and analysed by ANOVA  with time , treatments (± 8-Br-cAMP), time-treatments interaction and placenta ID as independent variables. Time, treatments and their interaction were entered in the model as repeated measures variables. Variance on placenta ID was significant. The ANOVA was followed by postestimation where contrasts were tested between time and treatment at each experimental time point by F-tests. The P-values were corrected for multiple testing according to Sidak.The Methylation was considered a binary dependent variable at each CpG site being either unmethylated or methylated. Data were entered in a generalized estimating equations (GEE) model using the logit link function as recommended for repeated measures analysis for binary outcomes . PlacentCRH promoter region . Data obtained with different placentae were pooled for these tests. Correlations were established using Spearman’s non-parametric method. All statistical calculations (except for the Berger test) were performed using the STATA software package . P-values < 0.05 were considered significant.Methylation frequencies between ChIP-selected and unselected promoter epialleles shown in  were comCRH mRNA abundance between 24 h and 72 h of culture (−4 moles/L) stimulated CRH mRNA levels relative to vehicle at all time points, which was expected [CRH mRNA abundance was 52-fold higher after 72 h of 8-Br-cAMP treatment than in untreated cells at 0 h showing robust time dependent upregulation of CRH gene activity .Cytotrophoblast cells spontaneously syncytialised during 72 h in culture as reported previously , 25. Syn culture . Treatmeexpected , 26–28 wactivity . CRH pepactivity . In lineCRH proximal promoter contains 9 CpG dinucleotides as illustrated in CRH promoter was partial and differential. Furthermore, methylation was stable under our experimental conditions with frequencies exhibiting no significant change during culture, with syncytial differentiation, or in response to 8-Br-cAMP.The The methylation patterns of the cloned individual copies of the promoter are presented in CRH promoter and explore their involvement in the epigenetic control of CRH expression. For the ChIP analyses, four PCR primer pairs (CRH2-5) were used to scan the proximal promoter, while CRH1 served as the upstream control. The positions of the amplicons are shown in Posttranslational histone modifications alter chromatin structure and influence transcription factor binding to chromatin. We have used chromatin immunoprecipitation (ChIP) to determine the level of key histone modifications at the CRH promoter area including the upstream control, CHR1. The level of acH3 increased significantly during 72h of culture in the region covered by CRH2 –CRH5. There was no additional increase in 8-Br-cAMP-treated cells. Histone-4 acetylation acetyl-histone-4 antibody) and histone-3, lysine-4 trimethylation (H3K4me3) are also associated in general with transcriptionally active genes [CRH promoter, increase significantly during culture and are enhanced further by 8-Br-cAMP treatment. We have also determined whether the repressive histone modifications histone-3, lysine 27-trimethylation (H3K27me3) and histone-3, lysine 9-trimethylation (H3K9me3) [CRH proximal promoter. Both modifications were found throughout the ChIP-scanned region acetyl-histone-3 (acH3) antibody are shown in ve genes , 30. ChIH3K9me3) , 30 are CRH proximal promoter contains two TATA box sequence elements upstream of the two major transcription initiation sites, respectively  using an antibody against RNA-polymerase-II  with CpGs 1, 2, 3, and 4 unmethylated and CpGs 5, 6, 7, 8, and 9 methylated . Thus, CCRH promoter DNA immunoprecipitated by the TBP antibody from vehicle treated and 8-Br-cAMP treated cultures was bisulfite sequenced as well. The TBP-bound CRH promoters had significantly higher methylation levels than promoter sequences in control (not ChIP-selected) DNA in both vehicle treated and 8-Br-cAMP treated cells acH4 and a H3K4me3 antibody with 8-Br-cAMP-treated cells, was significantly lower than the non-immunoprecipitated control  and decreasing levels of suppressive  histone modifications at the CRH proximal promoter. Treatment with 8-Br-cAMP robustly stimulated CRH gene expression and augmented the changes in histone modifications with the exception of acH3, which appeared unresponsive to cAMP ; rather, they revealed that a variety of epialleles is present in the trophoblast cell population. These attributes suggest that the CRH proximal promoter represents an “intermediate methylation region” (IM) in the genome as determined by recent comparative epigenomic studies involving a number of human tissues and cell types [CRH expression in rodents [The analysis of CpG methylation radically reshaped the picture of epigenetic ll types . The IM ll types  in line  rodents , 36.CRH promoter epialleles were functionally different. We addressed this possibility using ChIP combined with bisulfite sequencing of the immunoprecipitated DNA [CRH promoter copies were significantly more methylated than the promoter population overall, and only 3 specific methylation patterns were detected in the Pol-II-bound fraction: (1) all 9 CpGs methylated, (2) only CpG4 unmethylated and (3) CpGs 4 and 6 unmethylated. During the study we have encountered 117 various CRH promoter methylation patterns in the primary trophoblasts CRH promoCRH promoter copies were engaged in transcription in 8-Br-cAMP-stimulated primary trophoblasts as only 2 of 120 sequenced CRH promoter copies showed methylation patterns compatible with Pol-II binding in DNA not subjected to ChIP , not providing enough DNA for bisulfite sequencing after Pol-II ChIP. The function of the CRH promoter sub-population activated by H3K4me3 and acH4 modifications, but not involved in the 8-Br-cAMP-response, also remains to be determined. Finally, it will be critical to explore how is the epiallele distribution of CRH promoters established in the trophoblast cell population. Methylated CpG levels were unchanging with no indication of significant turnover in our culture system, which contained non-dividing term trophoblasts  and CRH peptide concentration in the culture medium was determined by radioimmunoassay. Bars show the average and SEM of 3 independent experiments. *, significant pairwise differences between time points as indicated by the arrows; #, significant stimulation by 8-Br-cAMP at each time point (p<0.05).(PDF)Click here for additional data file.S2 FigCRH proximal promoter was determined by chromatin immunoprecipitation (ChIP). Significant treatment effects at each promoter region (CRH promoter copies marked by H2K27me3 were determined by bisulfite sequencing of ChIP-isolated DNA from 8-Br-cAMP-treated cultures. Arrows (←) mark promoter epialleles compatible with Pol-II binding, while (x) indicates epialleles that can bind pCREB. Placenta numbers are on the left; filled and open circles indicate methylated and unmethylated CpGs, respectively. The positions of CpG Sites are as shown in (A) H3K27me3 marking at the r region  are deno(PDF)Click here for additional data file.S3 FigCRH promoter copies in chromatin marked by H3K9me3 were determined by bisulfite sequencing of DNA isolated by ChIP from 8-Br-cAMP-treated cultures. The arrow (←) marks a promoter epiallele capable of Pol-II binding. Placenta numbers are on the left. Filled and open circles indicate methylated and unmethylated CpGs, respectively. The positions of CpG Sites are as shown in (A) H3K9me3 marking of the promoter was determined by chromatin immunoprecipitation (ChIP). Significant treatment effects at each promoter region  are deno(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.


IGF1R gene constitutes a typical feature of most human cancers. Consistent with these biological roles, cells expressing high levels of IGF1R are expected not to die, a quintessential feature of cancer cells. Tumor specific chromosomal translocations that disrupt the architecture of transcription factors are a common theme in carcinogenesis. Increasing evidence gathered over the past fifteen years demonstrate that this type of genomic rearrangements is common not only among pediatric and hematological malignancies, as classically thought, but may also provide a molecular and cytogenetic foundation for an ever-increasing portion of adult epithelial tumors. In this review article we provide evidence that the mechanism of action of oncogenic fusion proteins associated with both pediatric and adult malignancies involves transactivation of the IGF1R gene, with ensuing increases in IGF1R levels and ligand-mediated receptor phosphorylation. Disrupted transcription factors adopt the IGF1R signaling pathway and elicit their oncogenic activities via activation of this critical regulatory network. Combined targeting of oncogenic fusion proteins along with the IGF1R may constitute a promising therapeutic approach.The insulin-like growth factor-1 receptor (IGF1R) has been identified as a potent anti-apoptotic, pro-survival tyrosine kinase-containing receptor. Overexpression of the  IGF2 constitutes a typical example of an epigenetically regulated gene. IGF2 is monoallelically expressed in somatic tissues, whereas it is usually expressed from both alleles in malignantly transformed cells [IGF2 is focally expressed early in tumorigenesis, providing critical oncogenic signals [The insulin-like growth factors (IGFs) constitute a family of cellular and secreted factors with important biochemical, cellular and physiological roles , 2. Sinced cells . Finally signals .ras-raf-MAPK and PI3K signaling cascades. The IGF1R is vital for cell survival, as illustrated by the lethal phenotype of mice in which the IGF1R gene was disrupted by homologous recombination. During normal ontogenesis, the IGF1R gene is expressed at every developmental period, including the oocyte stage [IGF1R gene resulted in small (more than 50% reduction in weight) animals that died in the immediate postnatal period from respiratory failure [IGF1 and IGF2 activate a common, ubiquitously expressed receptor, the IGF1 receptor (IGF1R), which signals mitogenic, antiapoptotic and transforming activities , 11. Thete stage . Late emte stage . Disrupt failure . These a failure .IGF1R expression and ligand-mediated activation of the cell-surface IGF1R are regarded as fundamental prerequisites for acquisition of a malignant phenotype [IGF1R-null embryos do not undergo transformation when exposed to most oncogenes [Clinical and experimental studies provide evidence that most tumors and transformed cells display augmented cell-surface IGF1R levels (leading to enhanced IGF binding) and express high IGF1R mRNA levels compared to non-transformed cells –18. In thenotype . Consistncogenes . It is incogenes .every type of cancer. Thus, whereas IGF1R overexpression is a common feature of most pediatric tumors, often associated with recurrent chromosomal translocations (see below) and other solid tumors such as brain and renal cancers, the situation in epithelial tumors, which are more widespread in adults , is more intricate and involves a tight interplay with additional cellular factors, including steroid hormones [IGF1R expression and action. In parallel, it is imperative to identify biomarkers that can predict responsiveness to IGF1R-directed therapies [The above paradigm, however, is not necessarily true for hormones –26. The hormones –30. Spechormones . In ordeherapies , 32, 33.IGF1R gene in both normal and malignant cells is determined, to a large extent, at the transcriptional level [IGF1R transcription is also dependent on the presence of inhibitory transcriptional regulators, including p53 [loss-of-function mutation of tumor suppressor genes is linked to the inability of mutated (inactive) tumor suppressor proteins to repress their downstream targets, including the IGF1R gene [Gain-of-function mutations of oncogenes, on the other hand, lead to enhanced transactivation of the IGF1R promoter. Increased cell-surface IGF1R concentrations are usually correlated with augmented receptor activation by circulating and locally produced IGF1/IGF2, with ensuing mitogenic responses [IGF1R gene expression is described below.The net level of expression of the al level , 19. Basal level , 35, E2Fal level , high moal level , and othding p53 –41, breading p53 , 42–44, ding p53 , etc. Itesponses . Interplc-myc gene in Burkitt’s lymphoma. In this pathology, the c-myc proto-oncogene is juxtaposed to an immunoglobulin gene by chromosomal fusion, thereby activating the oncogene. Extensive research on the biology of these (and other) aberrant transcription factors over the past decades had a huge impact on our current understanding of the mechanistic events associated with chromosomal translocations and their roles in cancer etiology [Tumor specific chromosomal translocations that disrupt the genomic organization of transcription factors are a common theme in oncogenesis , 48. As etiology . FurtherEWSR1 gene on chromosome 22 to the 3′ end of the FLI1 gene on chromosome 11 [EWSR1 gene is a member of the TET gene family [EWSR1 gene is involved in a number of translocation events that are hallmarks of a variety of specific cancers [As referred to above, several pediatric and adolescent tumors are characterized by recurrent chromosomal translocations, frequently resulting in the fusion of unrelated genes, most commonly transcription factors and nucleic acid-binding proteins. An example of this growing family of dislocated transcription factors is the t chromosomal translocation found in nearly 90% of Ewing’s sarcomas (EWS) and primitive neuroectodermal tumor of childhood (PNET). This specific translocation results in the fusion of the 5′ end of the ubiquitously expressed osome 11 –55. The e family , a classe family . The EWS cancers . The chiEWSR1 gene. The distinctive hallmark of DSRCT is the t translocation [EWSR1 to the C-terminal DNA-binding domain of Wilm’s tumor-1 (WT1) [IGF1R promoter in transient transfection assays (see below) [Desmoplastic small round cell tumor (DSRCT), an aggressive primitive tumor of children and young adults, particularly males , is alsolocation  that fus-1 (WT1) –63. WT1 -1 (WT1) . Consiste below) , 66.EWSR1 and ATF-1 genes is characteristic of soft tissue clear cell sarcoma or malignant melanoma of soft parts [EWSR1 gene fuses to an orphan nuclear receptor gene, initially named TEC [Fusion of the ft parts . A recuramed TEC . Althougamed TEC , 70. Antras-raf-MAPK and PI3K networks. Following its initial identification in congenital fibrosarcoma, as mentioned above, the ETV6-NTRK3 chimera was also detected in tumors derived from different cell lineages, including secretory breast carcinoma [The ETV6-NTRK3 chimera was discovered by breakpoint analysis of the t translocation characteristic of congenital fibrosarcoma, a pediatric soft tissue malignancy. The fusion protein includes the sterile alpha motif oligomerization domain of the ETV6 transcription factor linked to the tyrosine kinase domain of the neurotrophin-3 receptor NTRK3 . This abarcinoma  and inflarcinoma .The oncogenic activity of ETV6-NTRK3 is tightly dependent on IGF1R action and, furthermore, insulin receptor substrate-1 (IRS1) is constitutively phosphorylated in chimera-transformed cells . Of inteTMPRSS2 gene to the ERG or ETV1 genes in prostate cancer. The TMPRSS2 gene is located on chromosome 21 and is highly expressed in prostate epithelium [TMPRSS2 has been identified as an androgen-sensitive gene. Fusion of this gene to members of the ETS family of transcription factors, in particular oncogenes ERG or ETV1, leads to over-expression of these transforming agents in a significant portion of prostate cancers, but not benign prostate tissue, in an androgen-dependent manner [Cytogenetic profiling of all major cancers led to the widespread dogma that recurrent translocations underlie the development of sarcomas and hematological malignancies whereas, on the other hand, adult epithelial tumors are rarely correlated with this type of rearrangements . In receithelium . The genithelium . While tt manner .gain-of-function  activities that abrogate the intrinsic biological role of each of the parental genes. In the specific case of TMPRSS2-ERG, the androgen responsiveness of the TMPRSS2 promoter is responsible for the steroid-dependent expression of oncogene ERG in prostate gland epithelium, a key event in prostate carcinogenesis. In the clinical context, reports identified a statistically significant association between TMPRSS2-ERG fusion and prostate cancer specific death [The identification of TMPRSS2-ERG as an important player in prostate cancer etiology had a major impact in basic and translational oncology . Recurreic death . Hence, BCAM, a membrane adhesion molecule, and AKT2, a member of the PI3K signaling pathway, was recently described in high-grade serous ovarian cancer cases [A novel, cancer-specific gene fusion between er cases . This reIGF1R gene. The rationale for this postulate is the fact that IGF1R is frequently overexpressed in tumors associated with chromosomal translocations. To examine the hypothesis that the prostate-specific TMPRSS2-ERG fusion is able to transactivate the IGF1R gene, M12 prostate cancer cells were infected with an ERG-encoding retroviral vector, followed by IGF1R expression measurements. Western blots and qRT-PCR indicate that ERG expression led to a marked increase in IGF1R protein and mRNA levels compared to uninfected cells IGF1R genrylation .Fig. 2ReIGF1R gene is the EWSR1-WT1 oncogene, the typical hallmark of DSCRT. As alluded to above, fusion of EWSR1 to WT1 in this pediatric malignancy abrogates the tumor suppressor role of WT1 as well as the RNA-binding capacity of EWSR1, and generates an oncogenic molecule capable of binding and transactivating WT1 target genes, including the IGF1R promoter [IGF1R transcription, the pathogenic EWSR1-WT1 fusion molecule leads to enhanced IGF1R transcription, with ensuing activation of cell-surface receptors by circulating and locally produced IGF1 and/or IGF2.An additional chimeric transcription factor whose mechanism of action involves transactivation of the promoter , 85. HenPAX3 gene with the 3′ sequences of the FKHR gene to generate a PAX3-FKHR chimera [PAX3 expression [IGF1R promoter construct, whereas PAX3 exhibited a reduced potency in comparison to the chimera [A further malignancy characterized by a recurrent chromosomal translocation is alveolar rhabdomyosarcoma, a pediatric soft tissue tumor. The t translocation characteristic of the disease juxtaposes the 5’-DNA binding domain-encoding sequences of the  chimera –89. PAX3pression . Transfe chimera . These r chimera .IGF1R, it has been postulated that the opposite scenario, i.e. that the presence of an intact IGF1R is critical for the oncogenic action of the chimeras, is also biologically plausible. To evaluate the hypothesis that the transforming activity of EWSR1-FLI1 requires the presence of IGF1R, W and R- mouse fibroblasts  were transfected with a chimera-expressing vector, after which anchorage-independent growth was assessed [While studies described above support the idea that the mechanism of action of oncogenic fusion proteins involves transactivation of assessed . ResultsBCR-ABL+ leukemia cells and supports the self-renewal of CML cells [BCR-ABL+ CML cells. Therefore, IGF1R directs BCR-ABL+ leukemia cells toward the myeloid fate. Given the fact that IGF1R is dispensable for the activity of hematopoietic cells but regulates BCR-ABL leukemia cell fate and supports self-renewal of CML cells, targeting IGF1R has been suggested to constitute an ideal anti-leukemia approach.Further support for a crucial role of IGF1R in leukemia was provided by studies showing that IGF1R regulates the cell fate determination of ML cells . IGF1R eFinally, the MYB-NFIB gene fusion has been identified as the characteristic hallmark of adenoid cystic carcinoma (ACC), an aggressive type of cancer that most often occurs in the salivary gland. Recent studies performed on cultured cells or tumors from ACC patients provided evidence that MYB-NFIB fusion drives proliferation and is crucial for spherogenesis of these cells. Furthermore, the fusion was shown to be regulated through AKT-dependent signaling induced by IGF1R overexpression. IGF1R inhibition, on the other hand, led to downregulation of MYB-NFIB action. Given the potential role of the chimera as a therapeutic target, its interaction with the IGF1R is of major translational relevance .cascade’ fashion  is regarded as a fundamental requirement in transformation. As mentioned above, both viral and cellular oncogenes require an intact IGF1R signaling network in order to elicit their transforming activities. This phenomenon is commonly regarded as ‘adoption’ of the IGF1R pathway by oncogenic agents [Activation of the IGF1R signaling pathway in a ‘c agents .In mechanistic terms, the modes of action of oncogenic fusion proteins depend on their ability to phosphorylate the IGF1R and downstream cytoplasmic elements. In the specific case of the prostate cancer-specific TMPRSS2-ERG aberrant transcription factor, we have demonstrated that the chimera was able to phosphorylate the IGF1R tyrosine kinase domain and downstream target Akt. ERG silencing in TMPRSS2-ERG-containing VCaP prostate cells led to a reduction in IGF1R expression. Decreased IGF1R levels were associated with a reduction in IGF1R activation as well as diminished Akt phosphorylation , 84. In adoption of the IGF1R signaling pathway, seems to be a universal mechanism of action of oncogenes. Thus, transformation by pp60src, the protein encoded by the src oncogene of Rous sarcoma virus, results in the constitutive phosphorylation of the IGF1R β subunit [src-transformed cell while addition of IGF1 synergistically increased the extent of phosphorylation. In a similar fashion, the hepatitis B virus X protein was shown to enhance IGF1R mRNA levels in hepatocellular cancer cell lines. These results suggest that the oncogenic role of hepatitis B virus X is mediated via transactivation of the IGF1R gene [Finally, activation of IGF1R target elements by oncogenic chimeras, i.e.,  subunit . It has F1R gene . In summto survive, a hallmark of cancer cells. Overexpression of the IGF1R gene constitutes a typical feature of most human cancers. IGF1R gene expression is determined, to a significant extent, at the transcriptional level. Evidence has been presented showing that the IGF1R promoter constitutes a target to a number of aberrant transcription factors that result from recurrent, cancer-specific chromosomal translocations. The classical dogma that disrupted transcription factors are mainly associated with pediatric and hematological malignancies has been challenged in recent years by landmark studies showing that the etiology of a number of adult epithelial cancers is also linked to oncogenic chimeric proteins. Oncogenes adopt the IGF1R signaling pathway and elicit their transforming activities via activation of this critical network. Combined targeting of oncogenic fusion proteins along with the IGF1R may constitute a promising therapeutic approach in oncology and may overcome the difficulties seen with IGF1R-directed monotherapy.The IGF1R has been identified as a potent anti-apoptotic, pro-survival tyrosine kinase-containing receptor. Cells expressing high levels of cell-surface IGF1R are expected