The complexity of regulatory networks increases throughout the phylogenetic tree, reaching a high level of complexity in mammalians. Multiple H2O2 sensors and pathways are triggered converging in the regulation of transcription factors at several levels: (1) synthesis of the transcription factor by upregulating transcription or increasing both mRNA stability and translation; (ii) stability of the transcription factor by decreasing its association with the ubiquitin E3 ligase complex or by inhibiting this complex; (iii) cytoplasm–nuclear traffic by exposing/masking nuclear localization signals, or by releasing the transcription factor from partners or from membrane anchors; and (iv) DNA binding and nuclear transactivation by modulating transcription factor affinity towards DNA, co-activators or repressors, and by targeting specific regions of chromatin to activate individual genes. We also discuss how H2O2 biological specificity results from diverse thiol protein sensors, with different reactivity of their sulfhydryl groups towards H2O2, being activated by different concentrations and times of exposure to H2O2. The specific regulation of local H2O2 concentrations is also crucial and results from H2O2 localized production and removal controlled by signals. Finally, we formulate equations to extract from typical experiments quantitative data concerning H2O2 reactivity with sensor molecules. Rate constants of 140 M−1 s−1 and ≥1.3 × 103 M−1 s−1 were estimated, respectively, for the reaction of H2O2 with KEAP1 and with an unknown target that mediates NRF2 protein synthesis. In conclusion, the multitude of H2O2 targets and mechanisms provides an opportunity for highly specific effects on gene regulation that depend on the cell type and on signals received from the cellular microenvironment.The regulatory mechanisms by which hydrogen peroxide  is mediated by sensors with high H•2O2 sensors may mediate slow (h) or localized H2O2 responses.Low reactivity H  Since its first identification in a living cell, H2O2 was considered a toxic byproduct of aerobic metabolism, something that cells had to remove , where kactivation = k2O2target + H × [H2O2]. k2O2target + H is the rate constant for the direct reaction between H2O2 and the thiol protein.For the activation step (1) •v2 = kswitchoff × [Targetoxidized] = kswitchoff × , assuming that the total concentration of the target protein is constant .For the switch-off step (2), in which the oxidized protein is regenerated back to the reduced form, For these two reactions the rate laws are defined as follows:reduced is the fraction of the target thiol protein in the reduced state:With this, the following differential equation is set up, where TargetThe analytical solution of i.e., kswitchoff = 0), • If the activation of the thiol protein is not switched-off , the steady-state fraction of protein present in the oxidized form is given by • If the thiol protein is switched-off , or if the H2O2 transient signal lasts only 30 s, only Prxs, PerR and catalase are sufficiently sensitive targets as to provide the desired response. For other targets, like PTP1B, the H2O2 signaling concentration needed to trigger the response during the 30 s of the duration of the signal would be too high, 1.2 mM. However, if cells require a slow response (1 h), or if the H2O2 transient signal lasts for 1 h, even a low reactive sensor, such as Cdc25B, will be sufficient to mediate the signaling pathway, as exposure to a 1.2 μM H2O2 concentration during 1 h would be enough to activate the response, i.e. to oxidize Cdc25B by 50%. Thus, the duration of the transient H2O2 signal is an important experimental observation that gives a hint on whether a sensor with high or low reactivity is operating. In this regard, for example, H2O2 production triggered by EGF peaks at 5 min, and returns to baseline after 20 min [2O2 transient signal excludes the possibility that a low-reactive-sensor is operating, a long transient signal is compatible with both a high and low-reactivity sensor. The same kind of information can be inferred from the time course of the signaling pathway: a very fast response is incompatible with a low-reactive sensor, while a slow response may be the result of either a low-reactive sensor that takes time to respond or, alternatively, the result of a high-reactive sensor that responds rapidly, but then oxidizes slowly an effector molecule. Using PTP1B as an example to analyze these two scenarios, analysis of experimental data where recombinant PTP1B inactivation was studied as a function of H2O2 concentration in vitro [k2O2target + H = 22 M−1 s−1 iredoxin  Fig. 4)2O2, the iredoxin ,50 showe process . Recentlbit Prx1 . Both th2O2 signaling. GSH is several orders of magnitude more abundant than a low-reactive thiol protein such as PTP1B, and has a rate constant for the reaction with H2O2, 0.87 M−1 s−1 at pH 7.4, that is about 20–40 times lower than that of PTP1B. Taking into account these data, can GSH be considered a sensor molecule for H2O2 or can it inhibit H2O2 signaling mediated by PTP1B? The answer in both cases is no. In terms of reaction with H2O2, GSH certainly outcompetes PTP1B  residing within and near the promoter sequences of PerR-controlled genes. In Bacillus subtilis, PerR, when bound to DNA, represses the genes coding for proteins involved in the oxidative stress response  [hemAXCDBL, fur, and zosA) [perR) [2O2 (8 µM) [The Peroxide Regulon Repressor (PerR) is a metal-dependent TF and a major regulator of the peroxide inducible stress response in bacteria . PerR wa2O2 . In fact2O2 . Howevernd mrgA) ,68, metand zosA)  and its ) [perR) . Most Pe2 (8 µM)  or cells2 (8 µM) .2O2 at the level of cytoplasm/nucleus traffic, which creates a new layer on the regulatory mechanisms when compared with OxyR and PerR. Thus, though Yap1, like OxyR and PerR, essentially allows cells to deal with oxidative stress response, the complexity of its regulatory mechanism already reflects cell compartmentalization. This regulatory layer is also found in Maf1, but the traffic regulator partners are now replaced by post-translational modifications (PTMs) that create different intracellular pools of the protein and determine its subcellular localization. In the case of Hsf1 and Msn2/4, both TFs allow cells to respond to a variety of different environmental stresses from heat shock to starvation and oxidative stress. Thus, in yeast, the response to oxidative stress is also part of a more general cellular response to stress, probably making this response to oxidative damage more robust.In yeast, a eukaryote but still a unicellular organism, we will focus on the analysis of four TFs, namely, Yap1, Maf1, Hsf1, and Msn2/4. In this group Yap1 is regulated by H2O2 gradients between distinct compartments [2O2 toxic and regulatory responses, thus facilitating evolution of H2O2 as a regulatory molecule. Also, endogenous sources of H2O2, such as dismutation of superoxide produced by NADPH oxidase (NOX) enzymes began to be established. Up until recently, it was believed that the genomes of Saccharomyces cerevisiae (S. cerevisiae) and Schizosaccharomyces pombe (S. pombe) did not contain genes encoding NOX enzymes. Recently, Rinnerthaler et al. [S. cerevisiae ORF encodes an authentic NOX, which is located in the endoplasmic reticulum (ER) membrane and produces superoxide in a NADPH-dependent fashion. Interestingly, most NOX enzymes are plasma membrane bound but, NOX4 has been localized in endomembranes of ER, the nucleus and, in spite of some contradictory data, in the mitochondria [2O2.Compartmentalization creates new opportunities to generate new levels of regulation and confine certain pathways and metabolic pathways to specific compartments. Moreover, yeast cells are larger than bacteria and this may have contributed to create endogenous Hartments . In the r et al.  showed tchondria . This clS. cerevisiae activator protein (Yap) family that comprises eight members [Yap1 (yeast AP-1) is one of the members of the yeast  members . All mem members . Structu members . Under o members . Yap1 haS. cerevisiae [Maf1 is a transcriptional repressor of RNA polymerase III (Pol III) that was originally discovered in revisiae . Howeverrevisiae . In yeasrevisiae . Maf1 dorevisiae . In thisMaf1 is a hydrophilic protein conserved from yeast to human and it contains three signature domains not found in any other polypeptide: A, B and C boxes . Yeast Mcis-acting promoter elements in target genes, called heat shock elements (HSE) [In eukaryotic cells, the heat shock response is primarily mediated by a homotrimeric DNA-binding TF, the heat shock factor 1 (HSF1), a member of the heat shock TF family that binds to ts (HSE) . Each HSts (HSE) .S. cerevisiae HSF1 homologue, Hsf1, is constitutively a trimer, and is localized in the nucleus where it associates with high-affinity HSEs under normal conditions and additional HSEs during stress [The g stress ,84. The g stress . Besidesg stress . Target g stress .2 zinc finger yeast TFs [S. cerevisiae, disruption of both MSN2 and MSN4 genes results in a higher sensitivity to different environmental stresses, including carbon source starvation, heat shock and severe osmotic and oxidative stresses. Msn2/4 are required for activation of several yeast genes, whose induction is mediated through the presence of a stress responsive element (STRE) consisting of a pentameric core of CCCCT, such as CTT1, coding for cytosolic catalase, and HSP12 [Msn2 and Msn4 (Msn2/4) are homologous and functionally redundant Cys2Hiseast TFs . In S. cnd HSP12 .via receptors in the plasma membrane.The complexity of the next step in eukaryotes evolution is attained by acquisition of multicellularity and, therefore, the appearance of different cell types and tissues. Cells are now able to differentiate and gain very specialized functions, their proliferative organization is diverse and cells must integrate cell–cell and cell–matrix connections. Some cells maintain their ability to be totipotents, and multicellularity is now upstream of cell compartmentalization. Cells are now exposed to a new environment and information is received from this environment mainly 2O2, despite still having the capacity to cause damage, acquires a prominent role as a regulatory molecule. Now, cells do not only respond to environmental H2O2, but H2O2 evolved as a second messenger necessary for many signaling pathways. Therefore, although the stress-response strategies found in yeast for the regulation of transcriptional factors by H2O2 can be detected in multicellular organisms , now evolution expanded this to an incredible complexity and invented new regulatory mechanisms and combinations between the pre-existent mechanisms allowing each protein to have different code bars and, therefore, integrate different signaling pathways and compartments. In a few cases, the TF is even part of a membrane receptor and is released upon activation, playing membranes a critical role.Interestingly, in this new scenario, He.g. TP53, NF-κB, CREB and SCREB) that are relevant to metazoan multicellularity evolved prior to the emergence of the metazoan stem lineage, and they can be found in some transition organisms, such as choanoflagellates or Capsaspora owczarzaki, but not in other lower unicellular eukaryotes such as yeast [2 levels at the cellular and systemic level. Like it happens in yeast, HSF1 still orchestrates the cellular response to a variety of cellular stresses, and H2O2 is still under the surveillance of general cell protecting mechanisms. The ability of certain TFs for transactivating response genes still recalls OxyR since they directly sense H2O2 as is the case of human HSF1.In multicellular organisms we focused our attention in nine different transcriptional factors, namely AP-1, NRF2, CREB, HSF1, HIF-1, TP53, NF-κB, NOTCH, SP1 and SCREB-1. It should be noted that some of these TFs  is a TF that regulates several cellular processes, including cell proliferation, apoptosis, survival, and differentiation. Such functional diversity derives primarily from its structural and regulatory complexity . The ter2O2 also activates NRF2 [The NRF protein family constituted by NRF1, NRF2, and NRF3, which are also bZIP proteins, regulate electrophilic xenobiotic detoxification and oxidative stress response. The main activators of this TF are electrophile agents but Htes NRF2  by a multes NRF2 ,93. In tThe cAMP response element-binding protein (CREB) is one of the three members of the cAMP responsive TFs family occurring in mammals . These TF play important roles in the nuclear responses to a variety of external signals, by binding different promoters of genes encoding proteins involved in transcription, metabolism, cell proliferation, differentiation, apoptosis, and the secretory pathway . CREB biTTP53 gene that commonly has lost its function by mutations in the majority (75% of the cases) of human cancers.TP53  has been studied for nearly three decades, and is best known for its potent ability to be a tumor suppressor . In factTP53 is a DNA-binding TF that both activates and represses a broad range of target genes constituting a critical hub that integrates a huge variety of signals and allows a complex set of cellular responses to DNA damaging agents, oxidative stress, oncogene activation , mitotic spindle damage, hypoxia, nutrient deprivation, telomere erosion, ribosomal stress and is involved in cellular senescence . The TP5The receptors and ligands in the NOTCH signaling pathway are membrane proteins that imply cell–cell contact for their activation, and they constitute the basis of NOTCH-dependent transcription activation. Mammals express four receptors, NOTCH1, NOTCH2, NOTCH3 and NOTCH4 and two families of ligands, Jagged (Jagged1 and Jagged2) and Delta-like  . The canThe NF-κB/REL family of TFs has key regulatory roles in inflammation, innate and adaptive immune response, proliferation and apoptosis . It consThe IκB proteins bind to NF-κB in the cytoplasm preventing NF-κB translocation to the nucleus and its binding to DNA. Therefore, complexation with IκBs has to be removed for NF-κB activation . NF-κB a2 – type zinc finger), which are required for recognizing GC-rich promoter sequences [SP1 (Specificity protein 1) was the first TF to be identified, purified and cloned from mammalian cells . SP1 is equences ,119. SP1equences ,121. SP1equences .SP1 directly interacts with TATA-binding protein associated factors  and otheHIF-1 (hypoxia-inducible factor) is a TF that has an essential role in the response to hypoxia at systemic and cellular level. This TF has been implicated in the activation of angiogenesis and erythropoiesis ,130 and 2, this TF can also be activated by a hypoxia-independent mechanism that is mediated by the superoxide radical and by H2O2 [HIF-1α contains two hypoxia-dependent degradation domains with two conserved prolyl residues. The hydroxylation of these residues catalyzed by PHD promotes the interaction between HIF-1α and the Hippel–Lindau tumor suppressor (pVHL), targeting the former for proteasomal degradation ,133 . Al by H2O2 ,135.2O2 has a strong influence on SREBP1 activity in cells with a high sensitivity to insulin, promoting lipid accumulation [via the interaction with the protein INSIG (Insulin-induced gene), which binds directly the protein SCAP [Sterol regulatory element binding proteins (SREBPs) are a family of critical TFs that bind the sterol regulatory element (SRE), activating genes encoding the enzymes that regulate the synthesis of cholesterol, lipids and fatty acids and cellular uptake of lipoproteins . H2O2 hamulation . There amulation . SREBP pmulation ,140. Themulation ,142. Theein SCAP . Despiteein SCAP ,145. In ein SCAP  where Inein SCAP . Once inein SCAP , in ordeein SCAP .The mammalian heat shock factor (HSF) family has four members HSF, HSF2, HSF3 and HSF4 but HSF1 is the key stress-responsive regulator of the heat shock response . HSF1 ha, elevated levels of both Hsp70 and Hsp90 regulate HSF1 through a negative feedback preventing trimer formation during heat shock [HSF1 is constitutively expressed in most tissues and cell lines . In the at shock . Also, Hat shock .de novo or their stability increases, and (ii) by controlling the activity of a pre-existent TF. There are different mechanisms by which TFs can be activated from an inactive to an active form, most of them meditated by PTMs: state of oligomerization, by binding to a ligand, dissociation from an inhibitory protein, cleavage of a larger precursor, cellular relocation, and access to promoter regions. In principle, activation of a pre-existent TF allows for a faster response to stimuli in order to alter the activity of cellular TFs and produce alterations in gene expression, but, for example, mobilization of a pre-existent mRNA for de novo synthesis is also a rapid process [2O2 regulates TFs at each of these levels – synthesis of TF, degradation of TF, cytoplasm-nuclear trafficking, and DNA binding and transactivation – illustrating with representative examples of known regulatory mechanisms.The activity of TF can be regulated by two mechanisms: (i) by synthesis/degradation, where upon activation they are synthesized  process . In what2O2 regulates several TFs by upregulating their synthesis at the transcriptional, post-transcriptional and translational levels  subgroups c-JUN amino-terminal kinase (JNK), p38 MAPK and extracellular signal-regulated protein kinase (ERK) [AP-1, when upregulated, spontaneously concentrates in the nucleus to activate gene expression ,153. Uprse (ERK) . c-JUN ise (ERK) . JNK, p3se (ERK)   respectively, and are required for efficient transcription of TP53 in human cells [2O2 synergistically increases TNF-α-induced activation of NF-κB [2O2 would also affect NF-κB-dependent TP53 transcription activation.Several studies have suggested that TP53 protein levels increase in response to a rise of intracellular H2O2 . This matrengths . One sucan cells . TNF-α-ian cells . Since Hof NF-κB ,116,164 2O2-dependent activation of the phosphatidylinositol 3-kinase (PI3K) pathway [2O2 role in the induction of HIF-1α transcription might also be mediated by NF-κB in vascular smooth muscle cells [2O2 (10–100 µM) and NOX4 overexpression increase HIF-1α mRNA levels [2O2 acts as a general second messenger for HIF-1-dependent gene expression under normoxia conditions.Another TF that is activated at the transcriptional level is HIF-1α. Activation of HIF-1α transcription by Angiotensin II, in vascular smooth muscle cells, involves the H pathway ,166. The2O2 leads to an increase in cytoplasmic HUR levels [2O2 may also regulate TP53 mRNA stability through HUR.There are several studies indicating that TP53 mRNA stability increases in response to cell stress conditions. The 3′- UTR of TP53 mRNA is a target for the RNA-binding protein HUR, which binds and stabilizes TP53 mRNA in response to short-wavelength UV light (UVC) . ExposurR levels  and HUR R levels . Consequvia CAP-dependent mechanisms while protein synthesis of stress response factors is done via IRESs, which can induce cells to rapidly produce sufficient amounts of protein in response to the stress [2O2 upregulates both CAP-dependent and CAP-independent translation of TFs  CAP-dependent ribosome scanning, in which the eukaryotic initiation factor 4F complex (eIF4F), recruits the 40S ribosome that scans the 5′-untranslated region (UTR) until it finds the initiation codon AUG; and, (ii) CAP-independent internal ribosome entry mediated by internal ribosomal entry sites (IRESs). Normal physiological protein synthesis is mostly done e stress . H2O2 upn of TFs .2O2 promotes the activation of a specific kinase for the S6 ribosomal protein, which is a component of 40S ribosomal subunit, p70S6k, increasing HIF-1α translational rate. This mechanism is induced by insulin and mediated by H2O2-dependent activation of MEK/ERK signaling [In addition to the transcriptional activation discussed previously, HIF-1α expression is also regulated at the translational level. Hignaling .2O2 [2O2 steady-state of 12.5 μM [2 M−1 s−1, for the reaction between H2O2 and the target(s) mediating this response. However, if a gradient of 6.8 between extracellular and intracellular H2O2 concentrations is considered [3 M−1 s−1 is obtained instead. As this estimate is based on NRF2 protein levels, the sensor that triggers this pathway should actually have a higher rate constant, and thus we may speculate that it is a highly reactive H2O2 sensor.Concerning NRF2, upregulation of its translation is an important regulatory control exerted by H2O2 , as a ne 12.5 μM . After ansidered , a rate 2O2 stimulates NRF2 translation is both cap-dependent and independent [2O2 [2O2 in HeLa cells; a treatment with 100 μM H2O2 for 10 min was sufficient to trigger nuclear export of this ITAF, followed by binding to the NRF2 5′ UTR and subsequent translation [2O2 stimulates translocation of La Autoantigen to the cytoplasm is still unknown. Either dephosphorylation of Ser366 [2O2 [The mechanism by which Hependent  -proteasome pathway, which consists in the degradation of poly-ubiquitinated proteins by a multicatalytic protease called proteasome. Ub-protein ligase (E3) enzymes transfer the activated Ub from an Ub-conjugating protein E2, first to a lysine residue of the target protein and then to lysine residues present in the last added ubiquitin, yielding an Ub chain [Degradation of key modulators of TF function is an important regulatory mechanism in many signaling pathways. The majority of intracellular proteins are degraded Ub chain .2O2 is able to modulate the degradation of specific proteins mainly by two mechanisms. In the first mechanism H2O2 targets the E3 ligase complex, as are the cases for NRF2 and TP53, and in the second, PTM of the TF either increases (acetylation of AP-1 protein Jra) or decreases (e.g. c-JUN and NRF2 phosphorylation and HIF-1 hydroxylation) its association with the E3 ligase complex . KEAP1 serves as the substrate adaptor subunit in the E3 holoenzyme in the ubiquitination pathway, leading to NRF2 ubiquitination and degradation [de novo  [Like it happens for NRF2, the cellular TP53 levels are mainly regulated by its ubiquitin-mediated proteasomal degradation ,191. In ize TP53 . The H2Oed (ATM)  and c-ABed (ATM)  phosphored (ATM) ,199.2O2 through the formation of disulfide bonds between the catalytic cysteine residues of the SUMO E1 subunit Uba2 and the E2-conjugating enzyme Ubc9 [A PTM that stabilizes MDM2 and promotes its ability to ubiquitinate TP53 is sumoylation . Sumoylayme Ubc9 ,202.2O2. MDMX is essential for the MDM2-mediated TP53 polyubiquitination [2O2 because H2O2 activates the kinases c-ABL [2O2 promote the rapid dephosphorylation of CK1αLS, a nuclear splice form of CK1α, and enhances the kinase activity [The protein MDMX is an additional key partner to the couple TP53/MDM2 and a potential target for Htination  as it entination . Phosphotination  and Ser3tination  or CHK2 tination , activates c-ABL , AKT [24es c-ABL . On the activity   . Active s GSK-3β ,214 acti-3β [2O2 . Other k-3β [2O2 , and it -3β [2O2 . All of  of NRF2 . It shous GSK-3β ,221,222,•2O2 levels (15 μM) in HeLa cells [Phosphorylation catalyzed by protein kinase RNA (PKR)-like ER kinase (PERK), a transmembrane protein kinase that is required for the cellular response to ER stress , and thaLa cells .•2O2.Phosphorylation at Ser40 catalyzed by PKC-δ , a kinasTwo additional mechanisms that involve NRF2 phosphorylation causing dissociation of the NRF2/KEAP1 complex, NRF2 stabilization and, ultimately, leading to its nuclear translocation have been identified:2O2 also controls the poly-ubiquitination of the AP-1 members c-JUN and CREB by modulating their PTMs [2O2 by activating both the apoptosis signal-regulating kinase 1 (ASK1), which activates JNK (see section Modulation of DNA binding by PTM), and c-ABL [2O2 increases CREB-Ser133 phosphorylation and decreases CREB protein abundance via a proteasomal mechanism, but Ser133 phosphorylation is not necessary for CREB degradation [Heir PTMs ,227. c-Jeir PTMs , or by ceir PTMs  stabiliznd c-ABL  kinases,nd c-ABL . Concern2 to catalyze the hydroxylation of the sub-unit HIF-1α, which is subsequently tagged with ubiquitin for degradation [2 sensing mechanism. Many other factors were shown to play a role in the regulation of HIF-1α expression, particularly reactive oxygen species, which have been the center of intense investigation. The molecular mechanism underlining H2O2 effect in HIF stabilization has been attributed mainly to an inhibition of PHD activity  or masks a NES ; release/association from/with a partner . In other cases the TFs precursors are kept in the cytosol associated to membrane anchors; for their activation to occur these factors must be processed, which enables the TFs to enter the nucleus, where the nuclear form activates target genes (SREBP and NOTCH). Below we describe in detail each of these cases where H2O2 activates redox-dependent TFs  conditions. Maf1 phosphorylation, some of which occurs in the vicinity of an NLS, is mediated by Sch 9 [2O2-dependent Maf1 nuclear translocation in yeast [2O2. What has been established so far is that the activity of protein phosphatase 2A (PP2A) is essential for Maf1 dephosphorylation and nuclear accumulation in cells treated with H2O2 [trx1Δtrx2Δ cells treated with 0.8 mM extracellular H2O2, Maf1 remains in the cytosol and its dephosphorylation is impaired [The negative regulator Maf1 represses RNA polymerase III activity under carbon source starvation, ER stress (5 mM DTT), and oxidative stress  is added to trr1Δ cells, which only lack thioredoxin reductase, are higher than in wild-type cells [2O2-dependent activation of Msn2 involves reaction of H2O2 with Tsa1 which is then reduced by the thioredoxin system forming oxidized thioredoxins. How the oxidized thioredoxins transmit the signal to Msn2/4 has not yet been established. However, site directed mutagenesis of the unique Cys residue present in a truncated Msn2 that still exhibits the behavior of the full protein established that there was still thioredoxin-dependent translocation to the nucleus [2O2-dependent Msn2 translocation does not involve a direct oxidation of a cysteine residue by the oxidized thioredoxins.Msn2 is a direct substrate of protein kinases PKA  and Snf1activity . Therefoast Prxs . Howeverpe cells . These r nucleus . Therefo2O2 and we will describe the process in detail.Yap1 and NRF2 are two TFs that undergo nuclear export mediated by CRM1 (chromosome maintenance region 1). For Yap1 this export is absolutely central for the control of its activation by H2O2 requires the multistep formation of disulfide bonds in the CRD and/or the cCRD domains [2O2 exposure masks the NES present in the cCRD domain, resulting in structural changes in the NES [2O2 cysteine residues in Yap1 are not oxidized by a direct reaction with this agent but require GPx3/Orp1, which acts as sensor protein for H2O2 [in vitro peroxidase activity by a mechanism involving a Cys36–Cys82 catalytic disulfide, which is distinct from those of classical glutathione peroxidases. Also, Orp1 is recycled by thioredoxin and not by GSH. Gpx3/Orp1 becomes oxidized at Cys36 which forms a heterodimeric disulfide bond with Cys598 in Yap1 upon H2O2 exposure [in vivo and influences the nuclear localization of Yap1 in response to H2O2 [in vivo to more rapidly prepare a defensive response to an H2O2 challenge  induces c-REL nuclear translocation to values similar to those obtained with physiological levels of TNF-α [2O2 promotes degradation of IκB, thus exposing the NLS of NF-κB and subsequent NF-κB activation are complex and are not well defined. IκB is signaled for proteasome degradation following its phosphorylation catalyzed by the IKKs. The IKK complex consists of a dimer of IKKγ subunits linked through a disulfide bond forming the NF-κB essential modulator (NEMO) to which IKKα and IKKβ bind in the resting state. Activation of IKK is made by phosphorylation catalyzed by an IKK kinase, either TGFβ-activated kinase (TAK1) or AKT, in the canonical pathway of activation, or NF-κB inducing kinase-1 (NIK1) in the alternative pathway, which can all be modulated by H2O2 [2O2 effects. In the reduced form LC8 binds to IκB and inhibits its phosphorylation by IKKs and subsequent degradation, and thus H2O2 by forming LC8 dimers linked via a disulfide bond [2O2 could be mediated by KEAP1-dependent degradation of IKKβ [NF-κB is a TF whose central activation step involves its release from an inhibitory protein, IκB, that masks an NLS in NF-κB. Concerning the most abundant form of NF-κB, the dimer p65–p50, Hr per se ,115,263.of TNF-α . The mec by H2O2 . Alternaide bond , promoteide bond . On the  of IKKβ .Concerning NRF2, in addition to the mechanism previously described, its cellular localization is also mediated by interaction with its partner, KEAP1. NRF2 contains three NLS, one of which is present in the NEH2 domain of NRF2, the domain that mediates interaction of NRF2 with KEAP1 ,268. So,2O2 promotes TP53 nuclear localization [2O2 and, therefore, the nuclear localization of TP53 induced by H2O2 may be mediated by MDM2.In the case of TP53, there are several evidences showing that Hlization ,274 but lization ,275. Conlization ,276,277.lization . Interes2O2 may affect TP53 microtubule binding and nuclear translocation through alterations on TP53 phosphorylation status since several reports describe the phosphorylation of Ser15 in TP53 upon H2O2 exposure  or, inversely, with extracellular catalase added to the cultures [2O2. ADAM17/TACE, the metalloproteinase responsible for the NECD release, has been demonstrated to be activated by H2O2 through the oxidation of critical sulfhydryls of the protein, suggesting its involvement in the redox regulation of NOTCH signaling [In the case of NOTCH signaling, it was observed that colonic tissues of NOX1et genes . The inccultures . These aignaling ,285.2O2  stimulates expression of SREBP-1 gene as well as its target genes involved in lipid accumulation [2O2 suppresses SREBP-1c transcriptional activity. The stimulatory effect of H2O2 may be mediated by the insulin signaling pathway at least in part through the oxidative inhibition of protein tyrosine phosphatases [2O2 activates SREBP by inducing ER stress [2O2 observed between the two types of cells, HepG2 and COS-7 cells. Such differences may be due to a difference in nutrient metabolic function, which could cause differences in SREBP-1c needs, highlighting the specialized role of this TF.Concerning SREBP1c, this TF is predominantly produced in the liver and white adipocytes, and controls fatty acid biosynthesis under glucose/insulin stimulation ,286. In mulation , while iR stress , produci2O2, changes its conformation, and as such it increases  or decreases (PerR) its affinity towards DNA. Many mammalian TFs contain cysteine residues in their DNA binding domain whose oxidation impairs DNA binding, constituting an additional layer of control in a complex signaling pathway. In addition, H2O2 modulation mediated by PTM of the TF can modulate the affinity of the TF either towards DNA , towards a co-activator (HIF-1) or towards a repressor . The state of chromatin adds another layer of control and, actually, H2O2 is able to target specific regions of chromatin to selectively activate individual genes, as exemplified for SP1 , and S-glutathionylation (Cys199-S-S-G) forming a modified OxyR able to activate the transcription of target genes [2O2 [in vivo [In directly ,67. Diffdirectly ). Two baet genes ; althougnes [2O2 , which s2O2 [in vitro by heat shock temperatures and by extracellular H2O2 (0.1 µM–200 µM) [in vivo activation of Hsf1 by extracellular H2O2  requires the formation of a disulfide bond between the two cysteine residues (Cys35 and Cys105) present in the Hsf1 DNA-binding domain. Similarly to what happens for murine Hsf1, H2O2, diamide and heat stress also induce the formation in vitro of an intramolecular disulfide bond between the Cys36 and Cys103 residues in the DNA-binding domain of human HSF1, which leads to HSF1 trimerization and DNA binding [Mammalian HSF1 can be activated directly by H2O2 . The ini2O2 . Multime–200 µM) . Ahn and–200 µM)  were abl binding . However binding .2O2 sensing is mediated by Fenton chemistry and not cysteine oxidation. Because this is a less common mechanism for H2O2 signaling effects, we will discuss in some detail the mechanistic and structural aspects of the modification of this TF by H2O2. This TF is a transcription repressor and its oxidation releases it from the promoter regions of the target genes, thus activating gene expression. The crystallographic structures of PerR from B. subtilis [Streptococcus pyogenes [2O2 sensor and also of how Fe2+ binding to the protein leads to activation of PerR binding to DNA. Just like the other proteins of the Fur family, PerR is a homodimer and each monomer has two functional domains, an N-terminal DNA binding domain that contains a winged helix interaction motif and a C-terminal domain that is involved in the dimerization of the protein [2+ coordinated by four cysteine residues [2+  or Mn2+ , and that is required for DNA binding [4 site gives structural stability to the dimer by locking together three β-strands present in the dimerization domain [PerR provides a very interesting example in which Hsubtilis  and Strepyogenes  have hel protein . Each moresidues  and a re binding . The Zn forming 2-oxo-histidine [Lee and Helmann  estimate by H2O2 , and shoistidine . The foristidine  and in mistidine . Oxidatiistidine . Oxidatiistidine ,297  . REF-1 wThioredoxin also plays a role in keeping critical Cys residues in TFs reduced. In fact, overexpression of thioredoxin reverses the oxidation of the Cys62 residue in p50 NF-κB subunit , and end(WAF1/CIP1) responding element [Finally, cysteine oxidation allows discriminating between target sequences in distinct genes. Oxidation of Cys277 in TP53 caused a decrease in TP53 binding to the GADD45, involved in DNA-repair, but not to the p21 element .2O2 modulation of these PTMs further adds a layer of complexity and can contribute for selective gene expression by H2O2.TP53 activation is the most representative example of how a complex network of PTMs, namely phosphorylation, acetylation, ADP-ribosylation, ubiquitylation, sumoylation and neddylation  modulatee.g. Thr55, Ser376 and Ser378) are constitutively phosphorylated in cells and dephosphorylated in response to stress conditions [2O2 it seems that H2O2 is able to induce different pathways that lead to diverse TP53 phosphorylation patterns, which is most probably related with used dosages and cell types as summarized in Concerning TP53, upon stresses the majority of the available sites are rapidly phosphorylated, although some sites  [2O2 may influence the apoptosis responses in cells.Finally, concerning CREB, this TF is regulated stimulus . H2O2 ca of CREB . In immo(150 µM) . These o2O2 increases acetylation of TP53 [2O2 were found, a simultaneous decrease in the activity of the deacetylase Sirtuin 1 (SIRT1) also occurs, but the upregulation of target genes can vary in different cell types [2O2 inhibition, since extracellular concentrations as low as 1 μM inhibit SIRT1 by oxidizing critical cysteine residues in the SIRT1 active center [2O2 triggers the dissociation of HUR from the HUR-SIRT1 mRNA complex, promoting SIRT1 mRNA decay, reducing SIRT1 abundance, a process that seems to be regulated by Chk2 kinase [2O2. SIRT1 may function as a termination pathway because REF-1 was found to reduce SIRT1 cysteine residues, stimulating its activity [In general, H of TP53   enzymes . This hydroxylation inhibits the association with p300, a transcriptional co-activator. It has been shown that FIH is more sensitive to inhibition by H2O2 than PHD and actually FIH may represent the preferential target for H2O2-dependent regulation of HIF [Binding to a co-activator is a common mechanism of regulation. Regulation at this level by Hn of HIF . In the n of HIF .2O2 control is done not by promoting the binding of the TF with a co-activator, but instead it promotes the release of the TF from an inhibitor. Induction of c-JUN is mostly dependent on its upstream kinase, JNK. Once activated, JNK translocates to the nucleus [2O2 is at the center of these events because JNK is activated by ASK1 [2O2 by several mechanisms. ASK1 contains 5–6 cysteine residues that, upon exposure to H2O2, form intermolecular disulfide bonds that mediate ASK1 homo-oligomerization and kinase activity [2O2, with its oxidation being mediated by Prx1, which acts as the direct sensor of H2O2 [2O2, thioredoxin is oxidized dissociating the complex, allowing for ASK1 activation [2O2 has been proposed to mediate JNK activation [2O2 [2O2 causes oxidation and inhibition of JNK-inactivating phosphatases by oxidizing their catalytic cysteine, thus contributing to JNK activation [c-JUN provides a very interesting example in which H nucleus  where it nucleus ,350, and nucleus . After r nucleus   3–9 homologue 1), which allows SP1 to recruit HDAC1 to the promoter region of its target genes. After SP1 methylation associated with Suv39H1 and HDAC1, the chromatin is remodeled with histone deacetylation and histone methylation, culminating in repression of SP1 target genes expression. The authors suggest that the recruitment of HDAC1 is essential for SP1 methylation; however, the enzyme that provides the methyltransferase activity and the residue(s) that are methylated are still unknown [An illustration on how Hactivity . In fact unknown .2O2 based on the affinity of promoter regions towards the TF. In the case of NF-κB, simple association–dissociation equilibriums helps to understand why genes with lower affinity towards NF-κB are preferentially modulated by H2O2 rather than those with higher affinity [2O2 not just regulates a TF, but also how it selectively discriminates between genes responding to the same TF is essential to have a complete view of regulation of gene expression by H2O2.Selective gene expression of individual genes whose promoter regions are exposed may also be accomplished by Haffinity . Underst2O2 increases from bacteria to unicellular eukaryotes, and from these to multicellular organisms. One consequence is that while the regulatory mechanisms in bacteria and yeast are well defined, a lot of uncertainties still remain in mammalian cells. As noted in a recent review, while in lower organisms a few publications were sufficient to establish the regulatory pathways, in mammalian systems thousands of publications have failed to do so [2O2 were identified [de novo, DNA binding and transactivation and nuclear cytoplasm trafficking. To overcome the problems caused by the higher complexity of mammalian systems, experimental approaches with a more solid quantitative basis are needed. The concept of operating mechanism should be in place. When a new mechanism is discovered, it is imperative to know if it is operating in vivo and to assess its relative importance compared with alternative competing pathways. Otherwise the newly discovered mechanism will certainly add new information but its biological meaning is uncertain. Here we deduce a set of simple equations that help to extract kinetic quantitative data from typical experiments. Although, these equations are simplifications and may lack the rigor of a true kinetic approach, they, hopefully, can be easily applied to typical experimental data present in most signaling papers. We exemplified this with a couple of calculations arriving to a conclusion that in HeLa cells KEAP1 reactivity with H2O2 is weak, while a highly reactive sensor is probably mediating stimulation of NRF2 synthesis de novo by H2O2.The complexity of the regulation of TFs by Hto do so . One exaentified . If NRF22O2 activates SP1 translation only in neurons and not in glia cells. In spite of this cellular specialization, it is tempting to identify a set of players that recurrently appear as intermediates in H2O2 signaling pathways and that may function as hubs, namely ASK1/MAPK; GSK3β, ATM, c-ABL, SIRT1, REF-1, as well as several phosphatases. Many of these molecules are kinases and phosphatases, underlying the well-known fact that H2O2 affects the phosphorylation state of signaling pathways. Recent advances with the discovery of the inhibition of Prxs by phosphorylation, show that this is a two-way relationship where signaling by phosphorylation/dephosphorylation affect the levels of H2O2 by decreasing its removal. If other thiol proteins have their SH groups reactivity with H2O2 also affected by its phosphorylation state, a cross-talk between protein thiol oxidation state and protein phosphorylation state would be established.Part of the complexity mentioned above may be artificial because there is a tendency to integrate information coming from different types of cells. One of the consequences of multicellularity is cell specialization and so different signaling mechanisms operate in different cells. For example, H2O2 signaling can be specific. Depending on the time of exposure and on H2O2 concentration different sensors may be activated. This chemical specificity combined with the localized production of H2O2, where only a handful of molecular sensors are available, yields a high degree of biological specificity. We have proposed that H2O2 is not only a generic modulator of TFs and signaling molecules, but has the potential of being a specific regulator of individual genes [2O2 exerts its effects described here supports such view. To know which genes are activated by H2O2 in each type of tissue and how this is affected by the presence of single nucleotide polymorphisms in the promoter regions of genes is essential to have a complete view of the regulation of gene expression by H2O2. Ultimately, such understanding will be essential for redox biology to be an active player in the emerging area of personalized medicine.Our calculations shown in al genes , and the


Identification of diffuse signals from the chromatin immunoprecipitation and high-throughput massively parallel sequencing (ChIP-Seq) technology poses significant computational challenges, and there are few methods currently available. We present a novel global clustering approach to enrich diffuse CHIP-Seq signals of RNA polymerase II and histone 3 lysine 4 trimethylation (H3K4Me3) and apply it to identify putative long intergenic non-coding RNAs (lincRNAs) in macrophage cells. Our global clustering method compares favorably to the local clustering method SICER that was also designed to identify diffuse CHIP-Seq signals. The validity of the algorithm is confirmed at several levels. First, 8 out of a total of 11 selected putative lincRNA regions in primary macrophages respond to lipopolysaccharides (LPS) treatment as predicted by our computational method. Second, the genes nearest to lincRNAs are enriched with biological functions related to metabolic processes under resting conditions but with developmental and immune-related functions under LPS treatment. Third, the putative lincRNAs have conserved promoters, modestly conserved exons, and expected secondary structures by prediction. Last, they are enriched with motifs of transcription factors such as PU.1 and AP.1, previously shown to be important lineage determining factors in macrophages, and 83% of them overlap with distal enhancers markers. In summary, GCLS based on RNA polymerase II and H3K4Me3 CHIP-Seq method can effectively detect putative lincRNAs that exhibit expected characteristics, as exemplified by macrophages in the study. Unlike messenger RNA, non-coding RNAs (ncRNAs) are a class of RNAs that are not intermediates between DNA and protein products. Rather than being regarded as “transcriptional noise”, there is emerging recognition and appreciation of the functional importance of these ncRNAs in health and diseases, such as cancer RNA polymerase II (Pol II) plays a central role in transcribing both coding and non-coding RNAs. The ability of Pol II to initiate transcription is dependent upon the combinatorial functions of general and sequence–specific transcription factors that establish an open chromatin template and mark the site of transcriptional initiation. This process is dependent on nucleosome remodeling factors and histone modifying enzymes that mark histone tails with specific post-translational modifications serving as docking sites for transcriptional co-regulatory proteins. Trimethylation of histone H3 lysine 4 (H3K4me3) is a well-known marker on promoter regions correlated to gene activation, and trimethylation of histone H3 lysine 36 (H3K36me3) is another marker along a given transcribed region High resolution snap shots of active transcription are recently enabled by chromatin immunoprecipitation (ChIP) combined with ultra high-throughput massively parallel sequencing technologies, also known as ChIP-Seq technology The CHIP-Seq signatures of DNA associated proteins such as of Pol II and histone markers are diffuse and span a wide range from several nucleosomes to thousands of bps. Such signals are not easily detected by existing peak-finding algorithms for transcription factor binding, such as FindPeaks The data in this study are obtained from primary macrophage cells under resting and activated conditions. Deriving from circulating monocytes in the blood stream, macrophages are terminally differentiated cells with important innate and adaptive immune functions Unlike the local-clustering approach of Zang et. al D). We carried out several computations in each step, including determining the potential binding sites, signal amplification upon binding, and calculating the simulated Pol II expression levels. We used the first 300 Mbps of Chromosome 1 for the representative gene data and show a resulting distribution after a time evolution of these signal peaks in Motivated by this observation, we constructed a simplified simulation of the Pol II signals modeled over time assuming a simple one-dimensional reaction-diffusion system, in order to demonstrate that the above hypothesis is plausible. . We assume the distribution of Pol II signal to be uniform and the diffusion of the signal followed Fick's second law with a selected diffusion coefficient  algorithm to reconstruct the most correct transcription units from these peak components see . BrieflyBefore applying the GCLS method to discover lincRNAs, we first tested it on Pol II peaks located within RefSeq genes, and compared the result to that of the comparison method SICER, in three different gap parameter settings: 600 bp (SICER600), 1200 bp (SICER1200), and 2200 bp (SICER2200). We show the summary of comparison in To further evaluate GCLS vs. SICER on the ORF of RefSeq genes, we investigated the coverage of these two methods in the 5′-upstream of TSS (transcription starting site), TSS-TES (transcription ending site) and 3′-downstream of TES from the identified genes . InteresWe found a total of 374 putative lincRNAs in macrophages under the no treatment condition and 189 lincRNAs under LPS treatment . We compAs Toll-like receptor (TLR) activation dramatically alters the transcription program of protein-coding genes in macrophages We selected 11 regions from the putative lincRNAs for experimental validation . Within The correlation between lincRNA Pol II tag fold change upon LPS and its nearest RefSeq gene expression fold change upon LPS treatment was reasonably good . Based on the idea that lincRNA would most likely regulate their nearest genes To investigate the evolutionary conservation among the putative lincRNAs, we carried out analysis in the predicted exons by GENSCAN as well as their respective predicted promoter regions labeled by H3K4Me3 signatures. By comparing the cumulative distribution of PhastCon scores of the predicted exons of the lincRNA to those of the introns and exons of protein coding genes, it is clear that overall the predicted exons of lincRNAs are slightly more conserved than the collective introns of the protein coding genes on average. They are, however, much less conserved than the exons of the protein coding genes . This rend under the LPS treatment condition  and low free energy of the thermodynamic −38.03 kcal/mol at position 43006516– 43006636  are highly correlated with the interpeak distance, whereas others (Type 2 peaks) are not. Type 1 peaks reflect that a limited amount of enzymes compete for specific genomic loci, assuming the total loading capacity of the enzymes within a particular actively transcribed locus is relatively constant. Due to the stochastic nature, the more likely one sees some enzymes aggregating closely as one single, wide peak, the farther apart one would expect to see the other competing enzymes near that particular locus. These peaks should therefore be merged to create a single transcription unit. Type 2 peaks appear on the boundaries of transcription units where there is a larger separation between successive signals. These peaks do not need to be merged, as that would be more likely to merge different transcription units. The simulation results based on the diffusion-reaction mechanism further confirmed the plausibility of our reasoning. Based on this initial observation, GCLS algorithm aims to reconstruct the most correct transcription units from these peak components.GCLS proved effective through validation at several levels. It displayed superior performance to SICER, which relies on a rigid cut-off threshold of gap distance, on the known protein coding genes. GCLS method has the least discrepancy between the total number of Refseq regions and the actual Refseq gene counts, compared to the results of SICER. Additionally, The flexibility of allowing varying inter-peak gaps in GCLS enables the best coverage in the protein coding regions of RefSeq genes, yet much lower noise in the 5′ upstream of TSSs and 3′ downstream of TESs. This advantage is further manifested by the larger number of putative lincRNAs and larger length variation found by GCLS, when compared to SICER. The fact that 8 out 11 putative lincRNAs identified by GCLS were responsive to LPS from QPCR results increases our confidence in GCLS.Additionally, we carried out extensive computational characterization of GCLS with its application to lincRNA. GO analysis and KEGG analysis on the nearest RefSeq genes associated with these putative lincRNAs show that they are associated with fundamental cellular processes without LPS treatment, but upon LPS treatment, they are shifted to be associated with immune cell specific processes. Also consistent with other studies, the predicted exons of the putative lincRNAs have modest conservation over introns of protein coding genes, but appear to be much less conserved than the exons of protein coding genes The number of putative lincRNAs identified in our report is comparable to those from Guttman et al. ChIP-Seq experiments were performed on Genome Analyzers I and II according to protocols of the manufacturer (Illumina). The first 23 bps for each sequence tags were used for alignment to mouse mm8 assembly using ELAND allowing up to 2 mismatches. Only tags that were uniquely mapped to the genome were considered for peak analysis. Two lanes of Pol II ChIP-Seq data were pulled together, under both the Kdo2 lipid A treatment (a purified LPS lipid) for one hour or no treatment conditions, so were the H3K4Me3 ChIP-Seq experiments. One lane of input genomic DNAs (without antibody) was also sequenced as the control sample.The data in this study were obtained from primary macrophage cells under resting and activated conditions To identify intergenic ncRNAs, peaks that overlapped with all RefSeq genes and known pre-microRNA regions were excluded. The mm8 homologs for human (hg18), dog (canFam2), and rat (Rn4) protein coding genes also were excluded, using the liftOver tool in UCSC Genome Browser, similar to We implemented an algorithm to reconstruct the most correct full transcription unit from these peak components. We first separated out the Type 1 peaks from the remaining peaks by determining the best linear separator that went through the point of minimum density between the two groups. The Type 1 peaks are located upper-left to the linear seperator. The density in this case is determined by the number of data points in a region where each data point is plotted based on the log of the base pair width of the tag versus the log of separation in base pairs of successive tags.x-axis. Also, let Assume Using these parameters, the linear separator can be computed as the array  Once we determined the separator, we merged the Type 1 peaks with their nearest neighbors that were also classified as Type 1 peaks in a given iteration and were located upper-left to the separation line. After merging, we replotted the peak data and found there may still be some Type 1 peaks and so we continued to iterate on merging Type 1 peaks together until the final Pol II units were constructed. As shown in the pseudocode, the stopping criteria for the algorithm were selected to be when the total percentage of peaks merged at each iteration was large enough  and the total number of merged peaks was large enough (greater than five). We clustered the H3K4Me3 units in the same way. Finally, we ensured that all the lincRNA units have the overlapping signatures of Pol II units and H3K4Me3 units. The pseudo-code for forming the clusters is presented in the nbp). In each time step, several computations were carried out. First, a number of potential binding locations (nb) were randomly selected. If a binding activity occurred because a gene was present at the location, the signal was amplified in that location by a specified factor (fa). Directionality of the signal amplification was randomly selected and occurred over a fixed length of the array (fl). Noise was also simulated by allowing a fixed percentage (fp) of binding activities not falling on a gene to also result in a reaction event. The resulting signal expression was renormalized so that the total signal added up to unity. The diffusion of the signal followed Fick's second law with a selected diffusion coefficient (D). Finally, the simulated Pol II expression levels are found by taking regions of the signal array that are above the mean value of the signal array plus a factor (fstd) times the standard deviation of the signal. To verify the distribution observations, we constructed a simplified simulation of the Pol II signals modeled over time assuming one-dimensional reaction-diffusion with the Pol II enzyme  and the binding sites. Initially, the distribution of signal was assumed to be uniform and represented by an array of floating point values where each array element represented the signal over a fixed number of base pairs  as the minimum span for positive Pol II islands. We did similar clustering with the H3K4Me3 data using SICER. We used the whole mouse genome microarray gene expression as testing examples to compare the performance of GCLS vs. SICER, the results were shown in a Receiver Operator Curve (ROC) plot. All data are MIAME compliant.The software SICER was downloaded from We could not get access to the code of Guttman et al., and relied on their published coordinates of ∼1600 lincRNAs for comparison. These lincRNAs were found in mouse embryonic stem cells (ESCs), mouse embryonic fibroblasts (MEF), mouse lung fibroblasts (MLF) and neural precursor cells (NPC), rather than macrophages http://genes.mit.edu/GENSCAN.html) as a fast approach to predict the exons computationally (P>0.10) We randomly selected 11 regions , to valiWe predicted the functions of the lincRNA by assigning its membership to the nearest RefSeq gene. We then used Database for Annotation, Visualization and Integrated Discovery (DAVID) to perform GO analysis and KEGG pathway analysis to find the terms that are significant To show the enrichment of H3K4Me3 signals on promoter regions of the lincRNA, we first determined their +/− strand orientation by comparing the mass center of H3K4Me3 on each Pol II transcription unit, then aligned all sequences  bp around the putative transcription starting sites (TSS) that are determined by H3K4Me3 termination position. We counted the tag frequency in this interval. We also used this interval to find the positional phastCons17way conservation scores of the lincRNAs. We averaged the phastCon scores by the total sequence counts.de novo motif analysis by the HOMER software To examine the motifs of promoter regions, we took the regions of the lincRNAs that have H3K4Me3 signatures and subdivided them into 200 bp segments. These segments were then subject to both known motif and http://www.tbi.univie.ac.at/~wash/RNAz/), a program that predicts structurally conserved and thermodynamically stable RNA secondary structures in multiple sequence alignments To find the secondary structures of the lincRNAs, we first obtained the putative “transcription regions” from the Pol II units that are free of H3K4Me3 markers. We downloaded RNAz  of the Pol II tags in the same lincRNA regions from LPS treatment vs. no treatment, where n is the normalized tag sum from LPS treatment and no treatment, and p = 0.5, similar to Figure S1Density heat-map plot of the emerging genome-wide patterns of Pol II CHIP-seq peaks in RefSeq genes and intergenic regions. Data are displayed as log10 transformation of the width of peaks vs. log10 transformation of the distance between two successive peak centers. A: pattern of Pol II CHIP-Seq peaks in RefSeq genes. B: pattern of Pol II CHIP-Seq peaks in intergenic regions.(TIF)Click here for additional data file.Figure S2Evidence of two clusters with K-mean clustering methods (K = 2). The ‘cosine’ metric was used for clustering. Data are displayed as log10 transformation of the width of peaks vs. log10 transformation of the distance between two successive peak centers. Red data points (type 1) denote peaks that appear to be linearly correlated between peak width and inter-peak distance. Blue data points (type II) denote peaks that lack the linear correlation between peak width and inter-peak distance.(TIF)Click here for additional data file.Figure S3Reaction-diffusion simulation creates similar two-cluster CHIP-Seq pattern. The first 300 Mbps of Chromosome 1 were selected for the representative gene data for this simulation. We used the following parameters Click here for additional data file.Figure S4The RefSeq genes and regions in GCLS overlap those in SICER. SICER is parameterized over 3 different gap distances: 600 bp, 1200 bp and 2200 bp. A region is defined as the maximum contig of several overlapping genes if there are any, or the locus of one single gene if there are no other overlapping genes.(TIF)Click here for additional data file.Figure S5The Receiver Operator Curve (ROC) plot based on the binary classification of known genes identified by Pol II vs. measured by microarray gene expression. GCLS and SICER can extract clusters that overlap with known genes with very similar trends of true positive rate vs. false positive rate. To make binary classification, a gene is classified as “truly expressed” when a cluster overlaps the transcription region of that gene; otherwise, it is classified as “falsely expressed”. To measure the two algorithms, the log2 transformation of the microarray intensities of genes is further normalized to represent the probability of gene expression, ranging on .(TIF)Click here for additional data file.Figure S6PCR validation of lincRNAs in, using bone marrow derived macrophage primary cells under control or LPS treatment. The notation “c2a” denotes “exon a” from the lincRNA in (TIF)Click here for additional data file.Table S1The coordinates of lincRNAs under LPS vs. no treatment conditions predicted by GCLS.(XLS)Click here for additional data file.Table S2lincRNA overlap among three different computational methods: GCLS, SICER600 and Guttman's method in .(XLS)Click here for additional data file.Text S1Exon sequences that are predicted by GENSCAN from the 11 regions of lincRNAs as shown in , as well as primer sets from these exons that were used to validate the effects of LPS treatment. The strand information and chromosome coordinates are shown in the header of each sequence. The specific primer set is listed right before each exon The predicted exon sequences are in bold and located between two arrow signs. Other regions irrelevant to the exons are omitted. The indices in front of each line in the sequence are relative to the start position of each range.(DOC)Click here for additional data file.Text S2The pseudo code to separate the clusters.(DOC)Click here for additional data file.


Trypanosoma brucei variant surface glycoprotein (VSG) expression is a classic example of allelic exclusion. While the genome of T. brucei contains >2,000 VSG genes and VSG pseudogenes, only one allele is expressed at the surface of each infectious trypanosome and the others are repressed. Along with recombinatorial VSG switching, allelic exclusion provides a major host evasion mechanism for trypanosomes, a phenomenon known as antigenic variation. To extend our understanding of how trypanosomes escape host immunity by differential expression of VSGs, we attempted to identify genes that contribute to VSG silencing, by performing a loss-of-silencing screen in T. brucei using a transposon-mediated random insertional mutagenesis. One identified gene, which we initially named LOS1, encodes a T. brucei MCM-Binding Protein (TbMCM-BP). Here we show that TbMCM-BP is essential for viability of infectious bloodstream-form (BF) trypanosome and is required for proper cell-cycle progression. Tandem affinity purification of TbMCM-BP followed by mass spectrometry identified four subunits (MCM4-MCM7) of the T. brucei MCM complex, a replicative helicase, and MCM8, a subunit that is uniquely co-purified with TbMCM-BP. TbMCM-BP is required not only for repression of subtelomeric VSGs but also for silencing of life-cycle specific, insect-stage genes, procyclin and procyclin-associated genes (PAGs), that are normally repressed in BF trypanosomes and are transcribed by RNA polymerase I. Our study uncovers a functional link between chromosome maintenance and RNA pol I-mediated gene silencing in T. brucei. Trypanosoma brucei is a protozoan parasite that causes African sleeping sickness in humans and a similar disease in livestock, in sub-Saharan Africa. T. brucei cycles between the insect vector (tsetse) and mammalian hosts. Two proliferating life-cycle forms, the insect-midgut procyclic form (PF) and the mammalian bloodstream form (BF), can be cultured and genetically manipulated. A single species of VSG is expressed at any time in each BF parasite, where VSG homogenously coats the parasite surface. All VSGs are repressed in PF, whose surface is coated with members of a small family of proteins called procyclins.VSGs and VSG peudogenes VSGs reside in minichromosomes and telomere-distal  arrays, but they appear to lack promoters. VSGs in BF are transcribed by RNA polymerase I (Pol I) from about 15 subtelomeric polycistronic ‘Bloodstream-form VSGExpression Sites’ (BES) VSGs are located adjacent to telomere repeats and ∼50 kb downstream of their promoters. Only one BES is transcriptionally active and expresses one VSG in each bloodstream form cell whereas the remaining VSGs are repressed VSGs are transcribed from monocistronic subtelomeric expression sites that are distinct from BESs Four distinct loci contain VSGs appears to be regulated by factors involved in telomere dynamics and chromatin modification. Loss of TbSIR2-rp1 (a T. brucei SIR2 homologue) or TbRAP1 (a telomere-binding protein) led to derepression of silent VSGsVSG silencing; TbISWI, a member of the ISWI family of chromatin remodeling complexes VSGs was also observed in BF cells depleted of chromatin assembly factors, TbASF1A and TbCAF-1b, and a linker histone, H1 Silencing of BES promoters and T. brucei antigenic variation have been largely relied on searching for homologues that have been characterized in other organisms. However, because T. brucei proteins are highly divergent from those of other model organisms, due to early separation during evolution, homologue searches have limitations. To better understand the molecular mechanisms underlying VSG silencing and to isolate components relevant to VSG gene silencing, we performed a large-scale forward genetic screen in T. brucei: a ‘loss-of-silencing’ (LOS) screen. We isolated 19 los clones that had impaired ability to repress a silent BES. One of these genes, LOS1, encodes a T. brucei MCM-Binding Protein (MCM-BP). MCM-BP is a component of a replication complex that consists of subunits of the Mini-Chromosome Maintenance (MCM) complex and of MCM-BP Thus far, studies of in vitro study with Xenopus egg extracts showed that MCM-BP is required for removal of the MCM complex from DNA at the end of the S-phase to ensure replication licensing for the next round DNA replication initiates with binding of the Origin Recognition Complex (ORC) to replication origins and recruitment of members of the pre-replication complex (pre-RC), including CDC6, CDT1, CDC45/GINS complex and MCM complex. This activates the replication origins and proceeds to replication by DNA polymerases  was targeted with a triple-reporter cassette containing puromycin-resistance (PUR), luciferase (LUC), and emerald-GFP (emGFP) genes, adjacent to its promoter  flanked by mariner inverted repeats (IRs) and a neomycin-resistance gene (NEO) for the selection of cells transformed by the plasmid. Neomycin-resistant cells (RNEO) were expanded to a population of ∼5×108 cells, prior to selection with hygromycin. Cells can only become resistant to hygromycin once the donor cassette is transposed into a transcribed chromosomal orientation.To generate a reporter cell line for a loss-of-silencing (LOS) screen, BES11 (containing promoter . ReportePUR marker (‘los’ clones) were isolated by simultaneously selecting with 40 µg/ml of hygromycin and 100 µg/ml of puromycin. Parental cells cannot grow at this concentration of puromycin while los mutants that lost the ability to repress expression of PUR gene should. 19 RPUR clones were obtained  and affect expression levels of neighboring genes.Transpositions that led to increased expression of obtained . LuciferTb927.7.1770 (gene ID number in http://www.genedb.org and http://tritrypdb.org) open reading frame (ORF), so were not independent events, causing an about 3–6-fold increase in luciferase activity near the reporter-tagged silent BES promoter .Five clones had an identical insertion in the middle of one allele of the mcm-BP null mutant los1 allele may express an N-terminal portion of TbMCM-BP, which may act as a dominant negative allele in los1 heterozygous clones, resulting in the loss of BES promoter silencing. LOS1 will be referred to as TbMCM-BP henceforth.BLAST searches with LOS1 identified sequence similarities to the MCM-Binding Protein (MCM-BP) that interacts with subunits of the MCM complex VSG silencing in BF, where antigenic variation occurs, we attempted to make a homozygous TbMCM-BP deletion mutant in BF but we were not able to delete both alleles, suggesting that TbMCM-BP is an essential gene. To deplete the TbMCM-BP transcript by RNAi, the wild-type TbMCM-BP allele in heterozygous −/+TbMCM-BP was first epitope-tagged in situ with myc. The −/MYCTbMCM-BP cells did not show any growth defect, verifying that the tagged version is functional. The −/MYCTbMCM-BP strain was then transfected with a tetracycline-inducible construct for TbMCM-BP dsRNA expression To investigate whether TbMCM-BP is required for TbMCM-BP gene took 96 hr (about 16 generations) and cells seemed to escape from RNAi silencing after 144 hr. To have a definite answer for the essentiality of TbMCM-BP in BF T. brucei, and to generate a more reliable cell line to examine cellular functions of TbMCM-BP, we constructed a conditional-knock-out (cKO) cell line . We quantified derepression of three silent VSGs: two silent BES-linked subtelomeric VSGs, VSG3  and VSG13 (VSG113), and a minichromosomal VSG, VSG24 BSD and VSG2 from the active BES1 and of two chromosome-internal housekeeping control genes (TbTUB and TbURA3). Cre-expression was induced to knock out TbMCM-BP-myc and cells were harvested 24 and 48 hr after Cre induction. Levels of mRNA were quantified by RT-qPCR and values were normalized to the level of TbURA3 mRNA. TbMCM-BP wild type cells (0 hr). Expression of silent BES-linked VSG3 and VSG13 increased 10- and 18-fold after 24 hr and 30- and 35-fold after 48 hr upon TbMCM-BP removal. Interestingly, expression of minichromosomal VSG (VSG24) increased 2- and 12-fold after 24 and 48 hrs after TbMCM-BP removal. T. brucei contains a large number of small chromosomes called minichromosomes (MCs), which are 30–150 kb in size. According to genome-wide high-throughput sequencing analysis, it appears that there are ∼60 MC VSGs in the genome of this strain of T. brucei . Mapping of 17 MCs showed a core structure consisting of a large central palindromic 177-bp repeat region and telomeric ends, some of which contain VSGs VSGs do not seem to have their own Pol I promoter VSGs and a minichromosomal subtelomeric VSG, but did not significantly change the expression level of the active VSG2 and BSD, or of another chromosome-internal housekeeping gene, TbTUB. Our data suggest that TbMCM-BP may have roles in silencing subtelomeric VSGs.To examine whether TbMCM-BP removal  and anti-VSG3  or VSG13  antibodies. We observed cells co-expressing the active VSG2 and the silent VSG3 (data not shown) and cells co-expressing the active VSG2 and the silent VSG13 . Expression levels of PUR and LUC genes  and procyclin-associated genes (PAGs) that are also transcribed from an RNA Pol I promoter, which is less active in BF than in PF PAGs are located downstream of procyclin genes under the RNA Pol I promoter  precede those of nuclear DNA (N) In Interestingly, the number of abnormal cells greatly increased over time, reaching up to 58% 36 hr post-induction , some ofCYC6 increased the appearance of zoids in PF, but caused the accumulation of multinucleated endoreduplicated cells in BF T. brucei cell cycle and mitotic checkpoint genes encoding Polo-like kinase 1, and cdc2-related kinases PF and BF trypanosomes have different mitotic checkpoints. Deficiency of the mitotic-cyclin T. brucei MCM-BP complex, we first examined interaction of TbMCM-BP with two MCM subunits, MCM2 (present in plant but not in human and yeast MCM-BP purification) and MCM5 . We carried out reciprocal co-immunoprecipitations (co-IPs) using epitope-tagged proteins expressed from their endogenous loci, TbMCM-BP-myc, MCM2-flag, or MCM5-flag in BF cells. The epitopes were inserted in heterozygous backgrounds  to ensure functionalities of tagged versions. Lysates were immunoprecipitated and analyzed by immunoblotting using anti-myc or anti-flag antibodies. TbMCM-BP-myc pulled down only MCM5 but not MCM2 and TbMCM-BP-myc was consistently present only in MCM5-flag IP but not in MCM2-flag IP  protease site, and tandem protein A (ProtA) domains. As it is difficult to grow BF cells in a large quantity due to their sensitivity to cell density, TbMCM-BP was purified from PF trypanosomes, which grow to a 10–20-fold higher density.To further characterize the TbMCM-BP complex, we isolated TbMCM-BP by tandem affinity purification (TAP) employing an approach that was developed in trypanosomes TbMCM-BP allele in −/+TbMCM-BP PF cells by targeted insertion of plasmid pTbMCM-BP-PTP-NEO, thereby generating a cell line that expressed only C-terminally tagged TbMCM-BP  from the first targeted allele to generate a pool of homozygous mutants, which is particularly critical for isolation of ‘loss-of-function’ alleles. It is therefore important that a screen should start with a sufficiently large pool of workable number of cells for the success of large-scale searches. Because VSGs are also repressed in PFs, which can grow to a 10–20 fold higher density than BF cells, PFs offered better chances for success of our initial screen.We performed our loss-of-silencing screen in PF, due to technical problems in BF and also due to some of advantages that the PF system offers, although monoallelic HYG gene flanked by mariner inverted repeats (IRs) and NEO for the selection of cells transformed by the plasmid. NEO-resistant cells were expanded to a population of ∼5×108 cells, prior to selection with hygromycin. Cells can only become resistant to hygromycin once the donor cassette is transposed into a transcribed chromosomal orientation. HYG transposition appears to be about 5% 7 of 5×108NEO-resistant cells should be HYG-resistant, having transposon insertion somewhere in the genome. Previously, two independent homozygous glycosylation mutants were isolated from a large population of trypanosomes mutagenized with a mariner-transposon and the second allele seemed to have been disrupted by gene conversion by a mariner-targeted allele with an apparently significantly elevated GC rate, ∼2.5×10−3TbURA3 locus (∼1×10−5) 4 of 2.5×107HYG-resistant cells should be homozygous mutants, which is more than enough to cover the entire genome of T. brucei (∼8×103 genes).Transposon insertional mutagenesis was induced by transfecting a transposon-donor plasmid los clones  7R HYGRNEO cells should represent about 250 homozygous los mutants, covering only 3.1% of the genome.However, we were unable to identify any known genes that affect BES promoter silencing in PF, such as TbISWI, TbNLP, or TbDAC3 One of the differences between the previous proof-of-principle transposon screen that isolated two homozygous glycosylation mutants and our LOS screen is that, in the former, a pool of transposon-mutagenized trypanosomes was strongly selected using eight sequential treatments with increasing amount of concanavalin A , which may have allowed further enrichment of homozygous mutants. On the other hand, a trypanosome pool was treated one time with 100 µg/ml puromycin in our LOS screen, which may have limited the chance to accumulate homozygous mutants in the pool.VSG silencing. An N-terminal half of fission yeast MCM-BP is sufficient for MCM4 interaction los1 clones. Therefore, it is possible that a C-terminally truncated Tbmcm-BP protein is expressed and acts as a dominant negative allele by competing with wild-type MCM-BP for the interaction with MCM subunits. In addition, in six clones, transposons were inserted at intergenic regions, which could disrupt functions of untranslated regions (UTRs) and affect expression levels of neighboring genes. In fact, in one los clone (clone 10), Tb927.4.4520 expression was increased 5 fold proving that a transposon insertion can interfere with gene expression near its target site. Spontaneous loss of heterozygosity in Leishmania major has been reported to be 10−4∼10−6 and increased 20–1,000-fold after mutagen treatments T. brucei, by sequentially treating transposon-targeted trypanosomes with increasing amount of puromycin together with mutagens. We conclude that our transposon-mediated random mutagenesis can be a useful tool for a large-scale genetic screen once properly tuned and it could isolate not only ‘loss-of-function’ mutants, but also ‘gain-of-function’ and overexpression mutants.Despite the shortfall, it is noteworthy that our screen isolated a true positive clone that affects VSGs, and transcriptional VSG switching (in situ switching) in T. brucei. Depletion of TbORC1, a protein involved in replication initiation, derepressed BES-associated silent VSGs and metacyclic VSGs in BF trypanosomes and increased the in situ switching rate in situ VSG switching VSGs was also observed in BF cells depleted of chromatin assembly factors, TbASF1A and TbCAF-1b, and the linker histone H1 VSGs VSG silencing are linked. However, because TbMCM-BP-depleted cells showed stronger derepression near telomeres than silent BES promoters, TbMCM-BP should have roles in VSG silencing independently of chromatin remodeling proteins.A number of recent studies demonstrated that chromosome maintenance factors are required for silencing of BES promoters and VSG regions XenopusTbORC1, MCM subunit genes, and TbMCM-BP affected G2 progression in BF cells, only TbMCM-BP depletion caused significant accumulation of anucleated cells (zoids). Therefore, the role of TbMCM-BP seems to be specific at least during cell cycle progression. It appears that TbMCM-BP is mostly chromatin-unbound because we were not able to immunoprecipitate much DNA with TbMCM-BP (data not shown) suggesting that TbMCM-BP plays its role in chromosome maintenance via transient interaction with chromatin or via indirect interaction with chromatin.Recently, replication origins have been mapped along all 11 megabase chromosomes by TbORC1 chromatin IP taz1Δ  can be relieved by a top2 mutant Xenopus, MCM-BP is required at the late S phase to remove MCM, probably to block re-replication Topoisomerase 2α (Top2 in yeasts) is required for centromere maintenance and for replication termination VSGs than at silent BES promoters. Therefore, TbMCM-BP may not have a direct effect on the activity of silent BES promoters. Transcription initiates at silent BESs but the transcripts are attenuated VSGs in TbMCM-BP-depleted cells seem to be polyadenylated, because they were readily identified in oligo-dT-reverse transcribed cDNA, and VSGs expressed from silent BESs were detected by immunoblot and IFs. We do not know whether the role of TbMCM-BP in VSG silencing is dependent on TbRAP1, but it is possible that TbMCM-BP functions with some chromatin binding proteins with greater affinities to telomeres or subtelomeric regions. Chromatin changes along silent BESs may facilitate transcription elongation, leading to a disproportional increase of transcripts from the most distally positioned VSG gene.Derepression was much stronger at subtelomeric PAG RNAs, which are transcribed from an RNA pol I promoter that is repressed in BF. This transcription unit is located between two convergent RNA Pol II polycistrons. Chromatin remodellers, TbISWI and TbNLP, are also required for repression of procyclin and PAGs VSG loci, but might work together with these chromatin factors in silencing of a chromosome-internally located RNA Pol I-transcription unit. Depletion of TbCAF-1b led to derepression of silent VSGs specifically in late-S phase and G2 VSGs was observed during all cell cycle in TbASF1A-depleted cells VSG derepression does not seem to depend on the phase of the cell cycle, or on abnormalities in cell cycle. However, we were able to detect silent VSG expression only in a subset of TbMCM-BP-depleted cells by IF, and a more quantitative method would be necessary to firmly determine if there is a correlation between TbMCM-BP-mediated VSG silencing and cell cycle. Collectively, data from various genes suggest that networks of multiple complex pathways are involved in silencing VSG and procyclin transcription loci in BF trypanosomes. It has been difficult to perform complicated genetic analysis in T. brucei, partly because generation of mutations has relied solely on sequential transfections using limited number of selection markers, due to inability to reproduce meiotic stages in culture. With our loxP-Cre and conditional KO systems, we can now analyze, relatively easily, genetic interactions between multiple key factors by creating double or triple mutants and this will be helpful to get more detailed molecular understandings on complex mechanisms, such as antigenic variation.Depletion of TbMCM-BP also increased the levels of procyclin and VSG silencing factor has been selected by a phenotype-based large-scale screening approach, and T. brucei is the first organism in which MCM-BP was identified in such way. TbMCM-BP is strongly associated with four T. brucei MCM-subunits, MCM4-MCM7, and MCM8, a subunit that is uniquely co-purified only with MCM-BP in T. brucei. Whether or not this TbMCM-BP's unique composition has special roles in antigenic variation in T. brucei remains to be determined.This is the first time that a Trypanosoma brucei bloodstream forms (strain Lister 427 antigenic type MITat1.2 clone 221a) were cultured in HMI-9 at 37°C PUR), luciferase (LUC), and emerald-GFP (emGFP) genes, and targeting sequences immediate downstream of a BES promoter. The targeted BES was identified by PCR analyses using a primer set specific to the insert and ESAG7 located downstream of a BES promoter and using a polymorphism of ESAG6 Triple-reporter cell line (HSTB-10): PF427 was transfected with a linearized plasmid (pHJ1) containing a puromycin-resistance (mariner transposase (HSTB-39): The HSTB-10 was transfected with a pHJ2 to stably express mariner transposase.Reporter cell line expressing the TbMCM-BP knock out (KO) cell lines: one allele of TbMCM-BP was deleted (HSTB-317) using PUR conjugated with Herpes simplex virus thymidine kinase gene (HSVTK or TK) flanked by loxP sites so that the markers can be removed by expressing Cre-recombinase and be reused HYG-TK, and 1042 bp downstream homology sequences were transfected either into wild type or HSTB-317.TbMCM-BP RNAi cell lines (HSTB-464 and -465): the wild-type MCM-BP allele in the sKO (HSTB-317) was epitope-tagged in situ with 3xmyc using one-step PCR epitope tagging plasmid pMOTag53M TbMCM-BP to make HSTB-464 and -465.TbMCM-BP conditional knock out (cKO) cell line (HSTB-660): HSTB-317  was transfected with pLEW100-Cre-EP1 PUR-TK marker was removed by expressing Cre recombinase to obtain HSTB-647. The wild type allele of TbMCM-BP in the HSTB-647 was then replaced with a TbMCM-BP cKO cassette containing MCM-BP-myc followed by HYG-TK and flanked by loxP sites (pDS24), to make HSTB-660.TbMCM-BP cKO cell line with a silent BES marked with a triple reporter (HSTB-683 and -684): HSTB-660 was transfected with pHJ1 to randomly target one of silent BESs with markers, PUR-LUC-emGFP. Targeting was confirmed by PCR analyses and puromycin sensitivity at 10 µg/ml.mcm-BPΔHYG-TK/MCM-BP) was transfected with pDS15 (pMCM-BP-PTP-NEO). The tag sequence was fused to the C-terminus of an endogenous TbMCM-BP allele by targeted insertion of the plasmid pDS15. Cells expressing MCM-BP-3xmyc, MCM2-flag, and MCM5-flag were constructed using one-step PCR epitope tagging method To make a PF cell line expressing TbMCM-BP-PTP, HSTB-586 (Plasmids used for this study are listed in PUR), luciferase (LUC), and emerald-GFP (emGFP) genes, and targeting sequences immediate downstream of a BES promoter. The HSTB-10 was transfected with a pHJ2 to stably express mariner transposase to make HSTB-39. We titrated puromycin concentration for the screen and confirmed that the 100 µg/ml effectively kills the reporter cells with the rate of spontaneous mutation ∼3×10−7. Random insertional mutagenesis was induced by transfecting cells with the donor-plasmid pSGL35 HYG) flanked by mariner inverted repeats (IRs) and a neomycin-resistance gene (NEO) to select cells transformed by the plasmid. The plasmid-transfected cells were expanded under G418 selection until the population reached 5×108 and were distributed in the 96-well plates containing 100 µg/ml puromycin and 40 µg/ml hygromycin. 19 R HYGRPUR clones were isolated and examined for luciferase activity (Promega). The transposon target sites were mapped by inverse-PCR followed by sequencing To make a loss-of-silencing reporter cell line (HSTB-10), PF trypanosomes were transfected with a pHJ1, a triple-reporter plasmid containing a puromycin-resistance (20 and reverse-transcriptase (Stratagene). RNA was amplified using primers specific to individual genes by quantitative PCR using the LightCycler 480 (Roche). Amplified double-stranded DNA product during 40 cycles was detected by SYBR Green I. All measurements were in triplicate and compared with a 1000-fold range of serially diluted standard genomic DNA prepared from the wild-type strain. The sequences of primers are available upon request.Total mRNA was prepared using RNA STAT60 (TEL-Test) as described in manufacturer's protocol and cDNAs were generated using oligo dT8 cells were lysed in lysis buffer (25 mM Tris–HCl (pH 8.0), 1 mM EDTA, 0.5% NP-40, 10% glycerol, 1 mM phenylmethylsulfonylfloride (PMSF), 1 mM dithiothreitol (DTT), protease inhibitor cocktails (Sigma)). Cell lysates were immunoprecipitated and analyzed by immunoblot using with anti-myc  or anti-flag antibodies (Sigma). TAP immunoblot monitoring of TbMCM-BP-PTP was carried out with the monoclonal anti-protein C antibody HPC4 (Roche).About 10Tandem affinity purification of TbMCM-BP-PTP was performed exactly as described in the standard protocol Immunofluorescence was carried out as described previously Cells were fixed with 70% ice-cold ethanol and stained with 0.5 µg/ml propidium iodide to examine cell cycle progression by flow cytometry.Figure S1Depletion of TbMCM-BP by RNAi caused cell growth defect. The −/MYCTbMCM-BP strain was transfected with a construct containing an RNAi cassette specific to TbMCM-BP, which can be induced by adding tetracycline. Depletion of TbMCM-BP by RNAi showed a growth defect, which correlated with TbMCM-BP-myc disappearance. Two independent cell lines were examined and tubulin was used as a loading control. * indicates breakdown product of tubulin.(TIF)Click here for additional data file.Figure S2TbMCM-BP deficiency accumulated anucleated (zoid) cells and abnormalities in cell cycle and division. (A) Profiling of abnormal cells by DAPI staining. Anucleated cells greatly accumulated upon TbMCM-BP removal. Other abnormalities observed less frequently are categorized. (B) Examples of DAPI stained cells with the DNA content of 1N1K, 1N2K, and 2N2K, and of abnormal NK contents.(TIF)Click here for additional data file.Table S1Mapping of transposon-targeted regions in LOS clones.(DOC)Click here for additional data file.Table S2Mass spectrometric identification of non-MCM proteins that co-purified with TbMCM-BP.(DOC)Click here for additional data file.Table S3Unique peptide sequences identified in each protein. Note: Some cases, same unique peptide sequence contained redundant peptide which show(s) different methionine oxidation site (underlined).(DOC)Click here for additional data file.Table S4Trypanosoma brucei strains used in this study.(DOC)Click here for additional data file.Table S5Plasmids used in this study.(DOC)Click here for additional data file.