Brief stimulation with 8-Br-cAMP was sufficient to enhance Oct4 protein levels; this enhancement was accompanied by activation of the p38 MAP kinase pathway (Figure 4A), as measured by western blotting. To test if enhanced p38 signaling was simply correlated with or actively controlled levels ofOct4 protein, P19 cells were treated with the p38 MAPK inhibitor SB202190. Inhibition of p38 activity resulted in a large and rapid decrease in Oct4 protein levels (Figure 4B). In PORE reporter assays in P19 cells, treatment with SB202190 alone caused a slight, though statistically non-significant, decrease in reporter transacti- vation; however, SB202190 was able to blunt 8-Br-cAMP-induced reporter activation by ,33% (Figure 4C; p,0.001). Furthermore, Figure 3. Specific regulationof a sub-set ofOct4 genes regulated by the PORE sequence. (A) BLAST analysis of the PORE sequence reveals hundreds of potential binding sites within the mouse genome. Sites are organized by distance from known genes: extreme, .100 kb; distal, between 10–100 kb; proximal, between 0–10 kb; intronic and exonic, within known genes. (B) Validation of fgf12 and PDE3A as bona fide Oct4 targets by chromatin immunoprecipitation with either serum (s, control) or monoclonal (1) or polyclonal (2) antibodies against Oct4. Nanog, positive control. (C) Undifferentiated P19 cells were treated with 8-Br-cAMP for the indicated times and analyzed by western blotting. (D) Treatment with 1 mM 8-Br-cAMP for four hours enhances Oct4 binding to genomic loci as determined by chromatin immunoprecipitation, followed by real-time PCR analysis at Nanog and fgf12 sites. Data is represented as the ratio of % precipitated DNA of input following precipitation with Oct4 antibody or rabbit serum control, 6SD. (E) Undifferentiated P19 cells were treated with 1 mM 8-Br-cAMP for indicated times. Expression of multiple Oct4 target genes controlled by Oct4/Sox2 heterodimers (1) or PORE-configuration homodimers (2) were assayed by RT-PCR.
miR-709 Induces Transcriptional Gene Silencing of Egr2 So far we have demonstrated that miRNAs employ a combinatorial mechanism for regulationof translation following PNS injury. This is particularly prominent for Egr2 protein the expression of which is repressed (Fig. 1A) through a complex pattern ofregulation by opposing miRNAs (Fig. 2, 3 and 4). In most cases, destabilization of mRNA usually comprises the major component of repression while some targets are repressed without detectable changes in mRNA levels . Since Egr2 is a transcription factor with a crucial role during myelination  we wanted to examine the possibility that the mRNA levels of Egr2 following sciatic nerve injury are regulated by miRNAs through transcriptional gene silencing. This would solidify the role of miRNAs as the central epigenetic regulators of the translational and transcriptional responses that characterize the cellular response to injury in the PNS. To perform this study, we first employed RT-PCR to examine the mRNA levels of Egr2 24 hours and 48 hours after sciatic nerve injury and compare this to the Egr2 mRNA expression in the uninjured nerve. This showed that expression of Egr2 mRNA is completely inhibited 48 hours after sciatic nerve injury (Fig. 5A), which agrees with the complete repression of Egr2 protein expression at the same time interval (Fig. 1A). However, RT-PCR fails to differentiate between decrease in transcription due to less efficient initiation (as in transcriptional gene silencing) versus degradation of mRNA (as in post-transcriptional silencing). As potent extinguishers of pre- existing programs [20,23], miRNAs in injury must function to suppress myelination program prior to initiating dedifferentiation of Schwann cells. To specifically address the role of miRNAs in transcriptional silencing of Egr2 we performed a STarMir search for potential miRNA binding sites on the Myelin Specific Element (MSE) of the Egr2 promoter, which controls the expression of Egr2 in Schwann cells . This showed that the MSE has efficient binding sites for miR-709, 468, 146b, 711 and 690 (Table 2). We selected miR-709 for our subsequent studies regarding the transcriptional gene silencing of Egr2 since STarMir predicted the highest total energy for miRNA 709 - MSE interaction (Table 2) indicating the possibility of a functional
Figure 1: In silico analysis of human STEAP1 and STEAP1B gene. Genomic organization (A) and transcripts (B) resulting from STEAP1 and STEAP1B gene. Exons (E), Introns (I) and their molecular sizes in bp (base pairs) are indicated. The sequence ATG and TAG/ TAA corresponds to initiation and STOP codons, respectively. White boxes indicate non-coding exons, and black or grey boxes represent regions of coding exons depending on transcript encoded by STEAP1B gene. C- Alignment of amino acids sequences of STEAP1 and putative STEAP1B isoforms. The underlined amino acids sequences correspond to predicted transmembrane regions. * indicate identical amino acids among STEAP1s proteins; “:” indicate different amino acids but with similar physical-chemistry properties. D- Prediction of transmembrane helices in STEAP1, STEAP1B1 and STEAP1B2 proteins. All sequences were retrieved from http://genome.ucsc.edu/ and the alignment was carried out using Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). The prediction of transmembrane helices was performed resorting to Center for Biological Sequence analysis (http://www.cbs.dtu.dk/services/TMHMM/).
STAT3 is a transcription factor involved in many biological functions, such as cell proliferation, differentiation and survival, development and immunity, among others. STAT3 is functional when it dimerizes with itself or with STAT1. These dimers can be phosphorylated and become active, but inactive STAT3 dimers can influence its activity as well. Dysregulation of STAT3 activation initiates, contributes and sustains a variety of human diseases, including cancer. The aim of this project was to study specific residues/domains that had been described to influence STAT3 activity in the literature: K49, L78, K140, R609, K685, Y705, S727 and the SH2 domain. A Venus-STAT3 BiFC system was used in order to study the dimerization and intracellular distribution of STAT3 dimers in unstimulated cells. Asymmetric post-translational modifications change the intracellular distribution of STAT3 homodimers more strikingly than symmetric ones. The symmetric combinations carrying the L78R, R609Q and Y705F mutations were the only ones to affect intracellular distribution. Meanwhile, combinations carrying one or more K-R substitutions affected the nucleocytoplasmic shuttling. The L78 residue is also important for dimerization, mostly when combined with K49 and R609, as well as C-terminal of STAT3. This could mean a new level ofregulationof STAT3 activity, and therefore a new possible therapeutic target. These results could be highly relevant for other protein complexes regulated by post- translational modifications beyond STAT3. Given the essential roles of STAT3 in development, immunity, tissue stress and cancer, our findings could have important implications for the diagnosis, treatment and understanding of a wide spectrum of human pathologies.
Besides ubiquitin, eukaryotic cells also express a group of ubiquitin-like proteins, such as SUMO that is also conjugated to the lysine residues of targeted substrates by an isopeptidic bond similar to that observed for the ubiquitin system . SUMO regulates many aspects of cellular physiology to maintain cell homeostasis, both under normal and during stress conditions . In contrast to ubiquitination, the covalent attachment of Ub-like proteins, SUMOylation- has critical roles in diverse cellular processes including genomic stability, cell cycle progression, intracellular trafficking, and transcription . In most cases, SUMO conjugation affects localization, stability and/or activityof specific transcription factors, therefore emerging as an important post-translationalregulationoftranscriptional function . Up to date, some studies have revealed that SUMO serves to repress transcription, while others have discussed that SUMO can also have profound positive effects on transcription . As a dynamic and reversible process, substrates can be rapidly SUMOylated and deSUMOylated by specific proteases in the cell, termed Ub-like protein-specific proteases (Ulps). S. cerevisiae cells express a single SUMO paralogue, called Smt3, and two SUMO proteases- Ulp1 and Ulp2, that cleave Smt3 from distinct sets of substrates. Ulp1 is responsible for both removing SUMO/Smt3 from specific target proteins, and processing precursor SUMO into its conjugation- competent form (which turns Ulp1 essential for viability); whereas Ulp2 only appears associated to the disassembly of polySUMO chains [15, 16]. Moreover, Ulp1 localizes to the nuclear envelope and is encoded by an essential gene .
The sections were pretreated at 65 uC for 2 h, followed by graded deparaffinization. Antigen retrieval was performed prior to incubation with the primary antibodies of cyclin D2 (1:300 dilution; Millipore), cyclin E2 (1:300 dilution; Millipore), and proliferating cell nuclear antigen (PCNA) (1:300 dilution; Dako), overnight at 4 uC, with normal IgG as a negative control. Thereafter, slides were incubated for 2 h at room temperature with the HRP-conjugated secondary antibody (1:100; DAKO). HRP activity was detected using a Liquid DAB+ Substrate– Chromogen System (DAKO). Finally, sections were counter- stained with hematoxylin and photographed. The immunohisto- chemical results were assessed using a semiquantitative method, with the final scores based on the intensity and percentage of positive epithelial cells determined by their immunochemistry; these scores were classified as ‘‘negative’’ (,3) or ‘‘positive’’ ($3) . The PCNA levels were scored according to the percentage of positive epithelial cells as ‘‘0’’ for #5%; ‘‘1’’ for 5%–25%; ‘‘2’’ for 25%–50%; ‘‘3’’ for $50%. The samples were classified into groups with a ‘‘low proliferative index’’ (,2) or ‘‘high proliferative index’’ ($2) [13,17]. The immunohistochemical results were then further analyzed with follow-up data.
Post-transcriptionalregulation is involved in several processes of circadian timing in plants. CCA1 and LHY are phosphorylated by casein kinase II (CK2) in a manner simi- lar to the pace-control mechanism of the mammalian circa- dian clock (Sugano et al., 1998, Sugano et al., 1999). The robustness of LHY oscillations has been attributed to post- translational modifications and proteasome-mediated deg- radation (Kim et al., 2003; Song and Carré, 2005). A second type of posttranslational modification, implied in clock function, is protein acetylation; mutants lacking the N-acetylglucosamine transferase activityof SPINDLY (SPY) exhibit altered leaf movement rhythms (Tseng et al., 2004). Recent evidence has demonstrated the crucial role of light- and clock-controlled proteolysis for the plant endog- enous clock. The novel family of putative photoreceptors ZEITLUPE (ZTL), LOV KELCH PROTEIN 2 (LKP2) and FLAVIN-BINDING KELCH REPEAT F-BOX 1 (FKF1) provides a direct link between the central oscillator and ubiquitin-mediated protein degradation (Nelson et al., 2000; Somers et al., 2000; Schultz et al., 2001). ZTL is a component of the Skp1-Cullin-F-box (SCF) complex that recruits TOC1 for proteasomal degradation (Somers et al., 2000; Más et al., 2003b; Han et al., 2004).
spatio-temporal pattern formation in the early embryo of the vinegar fly Drosophila melanogaster. The biological question we are addressing is the importance ofpost-transcriptionalregulation in animal development. While many studies of pattern formation focus on differential transcriptionalregulationof genes (e.g. [41,42]), other levels of expression control—such as regulated RNA splicing, processing, translationalregulation, or regulated stability and degradation of gene products—cannot be ignored . There is increasing evidence that protein levels do not generally match those of their respective mRNAs [44–46], and many protein expression patterns do not even coincide with the timing and localisation of mRNA transcription [47,48]. These discrepancies are due (at least in part) to control at the level of protein translation. Indeed, some of the earliest studies oftranslational control were carried out in Drosophila (reviewed in ). A number of pioneering studies examined the effect oftranslational repression on maternal morphogen gradients, such as those formed by the protein products of the maternal genes hunchback (hb) and caudal (cad). mRNAs derived from those genes are distributed uniformly while their proteins form steep concen- tration gradients with antero-posterior polarity [49–54]. More recently, systems-level studies indicate that such post-transcrip- tional regulation is widespread and of general importance. Protein expression levels in yeast cannot be predicted from mRNA concentrations alone , and a similar lack of correlation between mRNA and protein is observed in systems as different as the minimal bacterium Mycoplasma pneumoniae  and mamma- lian cell lines . Therefore, post-transcriptional regulatory mechanisms must be incorporated in a systems-level understand- ing of cellular and organismal function.
We showed, by systematic knockdown of cyclin D1, p53 and p21, that p21was necessary for the stability of cyclin D1 protein, independent of p53. Suppression of p21, by downregulating p21 or p53, restored the mitogenic effects of cyclin D1 suggesting an epistatic relationship between p14 ARF /p53/p21 and cyclin D1. These results are consistent with other reports showing, in co- immunoprecipitation experiments, p21 interacts with the cyclin D1/CDK4 complex to inhibit cell proliferation [47,48,49] and cyclin D1 requires p21 to maintain protein stability . It has been suggested that p21 protein stabilisation acts by inhibiting cyclin D1 nuclear export thus preventing ubiquitination and degradation . Again our findings are consistent with these reports whereby we showed nuclear retention of cyclin D1 upon p14ARF expression is a prerequisite for rapid cell cycle-inhibition. Others have shown that cyclin D1 is necessary to prevent p21 degradation , however cyclin D1 did not regulate p21 at the translational or transcriptional level in our experiments invalidat- ing any suggestion of a feedback mechanism by cyclin D1 in the context of p21 cell cycle control in our model system. Further- more, repression of p21 had no effect on p53 levels, and cells expressing high p53 protein post-p14ARF induction continued to proliferate. Again this suggests that p21 does not directly feedback and suppress p53 expression in our system (see Table 1 and 2).
12-myristate 13-acetate (PMA), treatments that initiate stem cell differentiation, compared to the pre-treated samples. The three sites in the region of interest, which are in the concentrated region of mutations found in Noonan and LEOPARD syndrome samples, follow the same pat- tern and exhibit no relative change until after 24 hours of treatment, where they decrease in phosphorylation. We were surprised the quantitative data amongst the three sites were identical. Upon revisiting the original supplementary information on this dataset in Rigbolt et al., the data is faithfully represented in ProteomeScout, but the assignment score suggests the specific site of modification was not accurately identified. However, given multiple sources of identification for these phosphorylation sites [23, 27, 35–43] it is likely that this experiment indicates that at least in hESCs, phosphorylation occurs on some subset of these sites and that they demonstrate a dynamic response to initiation of stem cell differentiation. Phosphorylation on these alternate sites is not currently appreciated as playing a role in regulating 14-3-3 activity, yet this may rep- resent a process by which traditional S259/14-3-3 recognition is altered. It also expands the pos- sibility of mechanisms by which these mutations affect protein function and regulation, and may help lead to hypotheses of how V263A plays a role in development of Noonan Syndrome, despite having no measurable effect on 14-3-3 binding .
MicroRNAs are small non-coding RNAs containing about 21 nucleotides that have been implicated in the post- transcriptionalregulationof gene expression (35). Early studies established these RNAs as promoters of mRNA degradation; however, more recent evidence indicates a role in translational control as well. There are at least 800 genes encoding microRNAs in the mammalian genome, suggest- ing that these molecules affect a wide range of gene expres- sion. MicroRNAs interact with a complementary sequence in an mRNA, usually in its 3’-UTR. If the complementarity is perfect, the mRNA is degraded, sometimes through a deadenylation mechanism. However, if the complementar- ity is imperfect, inhibition of translation may occur instead. There are reports that either the initiation phase is inhibited through a mechanism involving eIF4E or that the elonga- tion phase is inhibited. To further complicate the situation, there are reports that microRNAs can stimulate translation of specific mRNAs, whereas a recent paper questions whether microRNAs affect protein synthesis at all (36). Clearly, a detailed understanding of how microRNAs affect protein synthesis is not yet available. Nevertheless, it can be anticipated that future studies will rapidly elucidate such mechanisms, as microRNA regulationof gene expression appears to be important in many disease states.
One of the puzzles that has so far been unresolved concerning DNA mechanics at short scales is whether in vivo and in vitro experiments tell a different story. In particular, in vivo experiments, in which repression of a given gene is measured as a function of the interoperator spacing [11,12], have the provocative feature that the maximum in repression (or equivalently the minimum in looping free energy) correspond to interoperator spacings that are shorter than the persistence length. Some speculate that this in vivo behavior results from the binding of helper proteins such as the architectural proteins HU, H-NS or IHF [1,12,13] or the control of DNA topology through the accumulation of twist. In the TPM measurements reported here, there are neither architectural proteins nor proteins that control the twist of the DNA. As a result, these experimental results serve as a jumping off point for a quantitative investigation of whether DNA at length scales shorter than the persistence length behaves more flexibly than expected on the basis of the wormlike chain model. To address this question, we performed a series of simulations of the probability of DNA looping for short, tethered DNAs like those described here using, a variant of the wormlike chain model to investigate the looping probability. Our theoretical model used no fitting parameters; the few parameters defining the model were obtained from other, non- TPM, experiments.
Cellular differentiation during development involves the coor- dinated change in expression of many thousands of genes in appropriate spatial and temporal contexts. A principal mechanism by which this occurs is through modification of the core histones (H3, H4, H2A, and H2B) that comprise nucleosomes, which are the fundamental units of chromatin. There are at least 8 distinct types of histone modifications, of which the most critical for transcriptional repression is lysine methylation, the enzymatic transfer of one or more methyl groups from the donor S- Adenosylmethionine (SAM) onto the e-nitrogen of lysine [1,2]. Genome-wide mapping studies using ChIP-Chip and ChIP-Seq have shown that di-methylation of histone H3 at lysine 9 (H3K9me2) is widely found at repressed genes during develop- ment and in embryonic stem cells [3,4] while perturbation of the H3K9me2 mark results in a profound change in the repression status [5,6]. The relevant histone lysine methyltransferase enzyme is G9a, a member of the highly conserved SET domain family, which, as part of a complex containing GLP, is responsible for catalyzing the H3K9me2 mark [7–11]. Thus, mutant mice lacking G9a are seriously impaired in the H3K9me2 mark and display embryonic lethality reflecting the consequences of global pertur- bation of gene repression . Notably, the H3K9me2 mark, which is associated with transcriptional repression (regulated inhibition of gene expression), is distinct from tri-methylation of H3K9, which is associated with transcriptional silencing (perma-
Methodology/Principal Findings: In this study, we investigate the relationship between long-range regulationof nuclear receptor family and their known functionality. Towards this goal, we identify the nuclear receptor genes that are potential targets based on counts of highly conserved non-coding elements. We validate our results using publicly available expression (RNA-seq) and histone modification (ChIP-seq) data from the ENCODE project. We find that nuclear receptor genes involved in developmental roles show strong evidence of long-range mechanism of transcription regulation with distinct cis-regulatory content they feature clusters of highly conserved non-coding elements distributed in regions spanning several Megabases, long and multiple CpG islands, bivalent promoter marks and statistically significant higher enrichment of enhancer mark around their gene loci. On the other hand nuclear receptor genes that are involved in tissue- specific roles lack these features, having simple transcriptional controls and a greater variety of mechanisms for producing paralogs. We further examine the combinatorial patterns of histone maps associated with dynamic functional elements in order to explore the regulatory landscape of the gene family. The results show that our proposed classification capturing long-range regulation is strongly indicative of the functional roles of the nuclear receptors compared to existing classifications.
Like it was previously mentioned, after a synaptic stimulus, CtBP1 shuttles between pre- synaptic terminals and nucleus, (71). Curiously, a cell morphogenesis gene ANGUSTIFOLIA, which encodes a CtBP1-like protein, is involved in the control of the microtubule cytoskeleton by interaction with a kinesin motor molecule (99). Also, upon synaptic activity, mitogen activated protein kinase shuttles along axons toward the nucleus and the repair of nerve injury requires retrograde axonal transport of importin subunits (72). Interestingly, nucleocytoplasmic shuttling of CtBP1 was observed several hours following synaptic stimulation. This time frame is at odds with the rapid expression of Arc, Fos, and BDNF observed in postsynaptic neurons (71). Moreover, the CtBP1 may influence microtubules stability through binding the cytoskeletal-associated PDZ-containing proteins found in the presynaptic compartment (73). Probably, in dopaminergic neurons, CtBP1 may also moves from the nucleus towards the axonal terminal to controls microtubule cytoskeleton dynamics. Moreover, after sensing a neurotoxic stimuli, this protein possibly goes to the nucleus and represses pro-apoptotic genes. However, the possibility of CtBP1 play a role similar to the ANGUSTIFOLIA resulting protein in dopaminergic neuronal function remains to be investigated.
KIF14 (kinesin family member 14) is a mitotic kinesin and an important oncogene in several cancers. Tumor KIF14 expression levels are independently predictive of poor outcome, and in cancer cells KIF14 can modulate metastatic behavior by maintaining appropriate levels of cell adhesion and migration proteins at the cell membrane. Thus KIF14 is an exciting potential therapeutic target. Understanding KIF14’s regulation in cancer cells is crucial to the development of effective and selective therapies to block its tumorigenic function(s). We previously determined that close to 30% of serous ovarian cancers (OvCa tumors) exhibit low-level genomic gain, indicating one mechanism of KIF14 overexpression in tumors. We now report on transcriptional and epigenetic regulationof KIF14. Through promoter deletion analyses, we identified one cis- regulatory region containing binding sites for Sp1, HSF1 and YY1. siRNA-mediated knockdown of these transcription factors demonstrated endogenous regulationof KIF14 overexpression by Sp1 and YY1, but not HSF1. ChIP experiments confirmed an enrichment of both Sp1 and YY1 binding to the endogenous KIF14 promoter in OvCa cell lines with high KIF14 expression. A strong correlation was seen in primary serous OvCa tumors between Sp1, YY1 and KIF14 expression, further evidence that these transcription factors are important players in KIF14 overexpression. Hypomethylation patterns were observed in primary serous OvCa tumors, suggesting a minor role for promoter methylation in the control of KIF14 gene expression. miRNA expression analysis determined that miR-93, miR-144 and miR-382 had significantly lower levels of expression in primary serous OvCa tumors than normal tissues; treatment of an OvCa cell line with miRNA mimics and inhibitors specifically modulated KIF14 mRNA levels, pointing to potential novel mechanisms of KIF14 overexpression in primary tumors. Our findings reveal multiple mechanisms of KIF14 upregulation in cancer cells, offering new targets for therapeutic interventions to reduce KIF14 in tumors, aiming at improved prognosis.
The HTRIdb offers several mechanisms of data query and extraction, such as download in spreadsheet or text format and the visualization of TF-TG interactions. There is an update mechanism that allows scientists to send new data. The HTRIdb currently holds a collection
We used two methods to verify that activation of the transcriptional reporter correlates with the formation of MCPs. First, we applied another fluorescence- based system in which the first 18 amino acids of the MCP-encapsulated enzyme PduP are fused to GFP to create an MCP-encapsulated fluorescent reporter (Pdu 1-18 -GFP). As previously reported, punctate fluorescence was observed by microscopy when S. enterica concurrently express this encapsulation reporter and Pdu MCPs [20, 35] (Fig. 2A, B), indicating localization of the reporter fusion to Pdu MCPs. In a microscopy time course, we first observed cells with one or more fluorescent puncta within two hours after addition of 1,2-PD (2.5% of cells, n 579), showing agreement with flow cytometry results (see Fig. S2 in Material S1). The proportion of cells with fluorescent puncta increased over time until we observed that nearly all cells contained at least one fluorescent puncta after six hours (95.2% of cells, n 5104).
The pore forming first transmembrane domain of MscL was found to be a hot mutational spot in a random mutagenesis study where many mutations, almost all to more hydrophilic residues, led to more sensitive or GOF channels . A subsequent study mutated a single resi- due, G22, to all other amino acids and confirmed that at least for this site, G22, the GOF phe- notype correlated with the hydrophilicity of the residue . There are several lines of data that suggest this domain twists in a clockwise (as seen from the periplasm) corkscrew fashion upon channel opening [5, 19, 40, 41]. Furthermore, it has been proposed that a “hydrophobic lock” at the constriction point of the pore stabilizes the closed state of MscL and it is the tran- sient exposure of these residues to an aqueous environment upon channel gating that is the pri- mary energy barrier . Cysteine substitutions in the cytoplasmic part of TM1 effected only GOF phenotypes (S1B Fig), ; for residue V23C, the proposed constriction point, post-trans- lational modification with hydrophobic probes reverted this phenotype (Fig 4A). A similar effect has been described for residue G26C [19, 20, 42], for which a longer time of treatment with decyl-MTS leads to a non-functional channel, probably due to an unbreakable hydropho- bic lock when more subunits are substituted. As shown for the V23C MscL (S2 Fig), the behav- ior in patch of this mutated channel is complicated, presumably due to disulfide bridging in the in vitro redox environment. This is true for other TM1 mutants including R13C and G26C, presumably due to their close proximity in the pentameric structure. It is interesting to point out that, in the case V23C, treatment with MTS reagents to cross-linked channels is indeed effective as seen for the reversion of the channel phenotypes, implying that the probes are able to break these cysteine bonds (S2 Fig).