Circadian rhythms are endogenous 24 h cycles that persist in the absence of external time cues. These rhythms provide an internal representation of day length and optimize physiology and behaviour to the varying demands of the solar cycle. These clocks require daily adjustment to local time and the primary time cue (zeitgeber) used by most vertebrates is the daily change in the amount of environmental light (irradiance) at dawn and dusk, a process termed photoentrainment. Attempts to understand the photoreceptor mechanisms mediating non-image-forming responses to light, such as photoentrainment, have resulted in the discovery of a remarkable array of different photoreceptors and photopigment families, all of which appear to use a basic opsin/vitamin A-based photopigment biochemistry. In non-mammalian vertebrates, specialized photoreceptors are located within the pineal complex, deep brain and dermal melanophores. There is also strong evidence in fish and amphibians for the direct photic regulation of circadian clocks in multiple tissues. By contrast, mammals possess only ocular photoreceptors. However, in addition to the image-forming rods and cones of the retina, there exists a third photoreceptor system based on a subset of melanopsin-expressing photosensitive retinal ganglion cells (pRGCs). In this review, we discuss the range of vertebrate photoreceptors and their opsin photopigments, describe the melanopsin/pRGC system in some detail and then finally consider the molecular evolution and sensory ecology of these non-image-forming photoreceptor systems. Instead of passively responding to these changes, organisms have evolved an endogenous representation of the 24 h day—a circadian timing system. These circadian clocks set the time or phase at which physiological and behavioural events occur with respect to the external 24 h environmental cycle. In this way, change can be anticipated, and physiology is optimized to the varying demands of night and day. This allows the organism to exploit the changed conditions as soon as they take place; avoiding the time lost in physiological and behavioural adjustments. However, the circadian system will only provide a selective advantage if biological time remains synchronized (entrained) to environmental time. Thus, the circadian oscillator requires a daily synchronization with the external environment via time cues termed zeitgebers (time givers) . The sysOpn4). In this review, we will consider the range of vertebrate photoreceptors and their opsin photopigments and then provide an overview of the melanopsin/photosensitive retinal ganglion cell (pRGC) system in detail. Finally, we will discuss the molecular evolution of the opsin photopigments and consider the different selective pressures acting upon visual pigments and non-image-forming photopigments in general.The study of the photoreceptors mediating irradiance-detection tasks such as photoentrainment has led to the identification of a range of vertebrate opsins, and perhaps most remarkably, the identification of a novel photoreceptor system within the mammalian retina, a subset of retinal ganglion cells (RGCs) that express the photopigment melanopsin Opn4; The lateral eyes are the most familiar photoreceptive site in vertebrates. The classical photoreceptors of the vertebrate retina consist of the rods and cones. Rods mediate scotopic (dim light) vision, providing low-resolution but high sensitivity, whereas cones are involved in photopic (bright light) vision, and enable high-resolution colour vision/contrast detection. Rod and cone light detection is characterized by rapid and transient electrical responses. The graded potentials from these receptors are processed by inner retinal neurons prior to advanced visual processing in the brain. Light information reaches the visual centres of the brain via topographically mapped axons of the RGCs that form the optic nerve . But in Rutilus rutilus), a subtype of the horizontal cell, termed HC-RSD, expresses both melanopsin and vertebrate ancient (VA) opsin and shows depolarizing responses to light that are maximally sensitive to approximately 477 nm. These cells have longer integration times than rods or cones and maintain their responses when classical photoreceptor inputs are saturated by background light  possesses photosensitive horizontal cells. In the cyprinid retina of the roach ((b)epiphysis cerebri). Here we will use the term pineal complex to refer to the intracranial pineal proper as well as the parapineal and the extracranial ‘third’ eyes found in tuatara , some lizards (Squamata) and frogs (Anura). The intracranial parapineal organ only occurs in some species of fish, and remarkably little is known about the physiological functions of this enigmatic organ (Stirnorgan) of anuran amphibians and the parietal eyes found in lizards. The parietal eye shows remarkably structural similarity to the lateral eyes, with a transparent cornea and lens    as well (c)Phoxinus phoxinus). These fish still demonstrated colour changes in response to light, leading to the suggestion of ‘deep-diencephalic photoreceptors’ (Anguilla anguilla) by λmax at 492 nm. A recent reanalysis of the original data suggests that the λmax may be closer to 483 nm . Although this action spectrum inferred the biochemistry of the photopigment, the precise molecular identity still remains unresolved. Attempts to characterize these photoreceptors have involved the use of immunocytochemical techniques employing antibodies raised against rod and cone photopigment opsins or other elements of the phototransduction cascade. Such approaches either failed to localize opsins within the avian hypothalamus or produced ambiguous results owing to the use of unknown epitopes Xenopus laevis melanopsin has been implicated Xenopus laevis melanophores in vitro  of zebrafish can be entrained to light after being isolated and maintained 3.cis-retinaldehyde (A1), while in many freshwater vertebrates the chromophore is based upon 11-cis-3-dehydroretinal (A2) (trans state (a). The conformational change of the chromophore allows the opsin to interact with a G-protein (transducin) and triggers the phototransduction cascade, ultimately giving rise to a change in receptor membrane potential (λmax) of the pigment may vary widely across the visible spectrum (ultraviolet at 360 nm to deep red at 620 nm), all these pigments have the same characteristic shape, a bell-shaped curve (b)  . The firns state a. The cootential . All vited curve b (Dartnaurve (b) .c). These seven α-helices form a bundle within the membrane, creating a hollow cavity on the extracellular side that serves as a binding site for the chromophore, retinal. There are also several other features usually present in an opsin: the retinal attachment site is a lysine residue (at position 296 numbered as in bovine rod opsin) located in the seventh transmembrane domain which binds the chromophore via a protonated Schiff base linkage; a glutamate counterion at position 113 in the third transmembrane domain; a glutamate, arginine and tyrosine  tripeptide for G-protein interaction; and cysteines (C) at positions 110 and 187 for disulfide bridge (Opsins are members of the superfamily of G-protein-coupled receptors (GPCR) which function through the activation of a guanine nucleotide binding protein (G-protein) and an effector enzyme. All opsins consist of a single polypeptide chain of 340–500 amino acids that form seven α-helical transmembrane regions connected by cytoplasmic and extracellular loops c. These A wide range of opsins have been identified within the vertebrates, with currently 15 distinct gene families . These a(a)Opn1 for cone opsins and Opn2 for rod opsin. Bovine rod opsin was the first opsin to be cloned and sequenced  λmax of 480–520 nm; ultraviolet/violet sensitive (SWS1) λmax 355–435 nm and short-wave sensitive (SWS2) λmax 415–470 nm. The various cone classes show 40–50% amino acid identity to each other cis-retinal transporter or an all-trans retinal photoisomerase , retinal G-protein-coupled receptor (RGR) and peropsin. RGR was originally identified from a bovine RPE library and although it contains the lysine required for chromophore attachment it has a histidine at the position of the potential counterion b, the pineal complex is photosensitive in non-mammalian vertebrates and multiple opsins have been isolated from this part of the brain.As discussed in §2(i)in vitro expression and reconstitution of pinopsin with 11-cis-retinal. All report the formation of a blue-sensitive pigment, but with slightly different λmax values: approximately 470 nm (Podarcis sicula) suggested that pinopsin was only expressed in the pineal complex Ictalurus punctatus; In situ hybridization showed that lamprey parapinopsin is expressed in the photoreceptor cells located in the dorsal region of the pineal and parapineal organs. The authors also demonstrated that lamprey parapinopsin photopigment has a λmax at 370 nm and that UV light causes cis–trans isomerization of its retinal chromophore, forming a stable photoproduct with λmax at 515 nm Most recently, studies on another photoreceptive structure, the parietal eye of a lizard, have identified expression of two opsins within the same photoreceptor, a blue-sensitive pinopsin and a novel green-sensitive opsin named parietopsin . Parieto(iv)et al. Vigh-Teichmann and colleagues first reported the presence of opsin immunoreactivity in the teleost pineal in the early 1980s (Vigh-Teichmann (d)Salmo salar) (Danio rerio) (Cyprinus carpio)  (Rutilus rutilus) (Petromyzon marinus) pinopsin originally described by Yokoyama and Zhang   and was tivelis)  and roacrutilus) . Phylogerutilus) . Lampreynd Zhang  is now cnd Zhang .λmax between 460 and 480 nm when expressed in vitro and reconstituted with 11-cis-retinal   (Rutilus rutilus)  and short (VAS) form, which vary in the length of their C-terminal tails . Both isoforms were functionally expressed in human embryonic kidney cells 293S but only VAL appeared capable of forming a photopigment when reconstituted with 11-tivelis)  and roacrutilus) . The newXenopus tropicalis), Reptilia (Anolis carolinensis) and the Elasmobranchii , but have failed to find any VA homologues within the mammalian lineage . This surprising finding raises important questions as to the possible function of this opsin within vertebrate taxa.These results in teleost fish prompted the search for orthologues of VA opsin in other vertebrate classes, but until recently attempts have met with failure. This restricted taxonomic distribution of the VA opsins was puzzling as most other opsins classes span multiple vertebrate taxa. Recent and unpublished studies have led to the isolation of the full-length sequence of chicken VA. The gene contains an open reading frame of 972 base pairs and encodes a predicted protein of 323 amino acids. Further studies have also identified VA-like genes in the Amphibia ((e)Opn3 was originally termed encephalopsin, and reported to be an extra-ocular opsin with strong expression in mouse brain and testis, with lower levels in the heart, liver and kidney Fugu and encodes a predicted protein of 402 amino acids, containing all of the essential features of an opsin photopigment including a lysine residue at position 296. Interestingly the Schiff base counterion, usually a glutamate, is substituted by a tyrosine in both Fugu and zebrafish TMT. TMT opsin shows 33–39% identity when compared to other vertebrate opsins Xenopus .Melanopsin was originally isolated from the photosensitive melanophores of ebrafish . Recentl(h)Opn5) was identified in 2003 using a bioinformatic approach rd/rd). These animals lack rods, and show a greatly reduced number of cones. As might be expected, rd/rd mice fail to show any classical visual responses to light. Studies were then undertaken to determine whether the circadian system of rd/rd mice was similarly impaired. When a mouse is housed under a light–dark cycle, its circadian wheel running behaviour is entrained. As would be expected of a nocturnal species, mice are largely active during the dark and inactive in the light. Under conditions of constant darkness, entrainment is lost and circadian behaviour drifts or freeruns with a recurring period approximately 23.5 h, starting its activity cycle approximately 0.5 h earlier every day. If the mouse is then exposed to a pulse of light shortly after activity onset it will delay the onset of activity the following day. The magnitude of this phase delay (Δϕ) is intensity dependent and can be used to determine the sensitivity of circadian responses to light. Both the wavelength and intensity of the light pulse can be systematically varied and the effect of these treatments on delaying wheel running behaviour can be assessed in a dose-dependent manner to produce a series of irradiance response curves.Initial studies to determine which retinal photoreceptors mediate circadian photoentrainment took advantage of a naturally occurring mutation in mice, termed retinal degeneration (rd/rd mice (C3H) and wild-type controls (C57) demonstrated that these mice could still entrain, although the threshold for entrainment in rd/rd animals was approximately 2 log units higher  In mammals, light-induced pupil constriction is regulated by both rods and cones, but still occurs in animals showing profound damage to these photoreceptors. Not unreasonably, it was assumed that the residual pupil light response was owing to the survival of a few rod and/or cone photoreceptors. Pupil measurements were undertaken in rd/rd cl mice and showed that these animals maintained a pupillary light response. Although less sensitive than congenic wild-type animals, rd/rd cl mice retained the ability to fully constrict their pupils . The known visual pigments of the mouse have λmax values of 360, 508 and 498 nm for the ultraviolet-sensitive cone, long-wavelength sensitive cone and rod pigments, respectively. None of these classical photoreceptors could account for the pupillary responses to light rd/rd cl retina −/−Opn4) exhibited attenuated phase-shifting and pupillary responses to light, as well as reduced period lengthening in constant light (LL) cis-retinal, an approach which has been particularly successful with visual pigments. This study produced a functional photopigment that was capable of activating transducin with a λmax between 420 and 440 nm, an absorption maxima considerably shifted away from OP480   are needed for these responses to light, that light will ultimately trigger the release of intracellular calcium, and that this may involve a Gq-type G-protein rather than transducin. Furthermore, melanopsin acts as a bistable pigment able to regenerate (recycle) its chromophore  using all-trans-retinal and long-wavelength light in a manner reminiscent of the invertebrate photopigments i/G0 class of G-proteins), phosphodiesterase and closure of cyclic nucleotide gated channels and a hyperpolarizing membrane potential. By contrast, invertebrate phototransduction, most extensively characterized in Drosophila, involves activation of a Gq/G11-type G-protein, activation of phospholipase C (PLC), gating of transient receptor potential (TRP) channels and the depolarization of membrane potential   shows that TRPC3 channels can generate a light-activated photocurrent in the presence of melanopsin (q/G11 that in turn activates a PLC-β. PLC-β generates inositol triphosphate (IP3) and diacylglycerol (DAG), which may ultimately modulate a TRPC channel, possibly via a protein kinase C (PKC). Most recently combined pharmacolgical and anatomical approaches have suggested TRPC7 as the channel . Remarkably the genetic ablation of Prkcz mimics precisely the melanopsin knock-out phenotype in a battery of behavioural and pupillometric tests , changes in the spectral composition of light also occur and are known as the Chappuis effect . As the The spectral tuning of vertebrate opsins will also be influenced by their evolutionary history . For exa6.Considerable progress has been made in the last decade in characterizing the photoreceptors and photopigments mediating non-image-forming responses to light, such as photoentrainment. While over a dozen different opsin photopigments have been identified in recent years , we are


Formation of site specific genomic double strand breaks (DSBs), induced by the expression of a pair of engineered zinc-finger nucleases (ZFNs), dramatically increases the rates of homologous recombination (HR) between a specific genomic target and a donor plasmid. However, for the safe use of ZFN induced HR in practical applications, possible adverse effects of the technology such as cytotoxicity and genotoxicity need to be well understood. In this work, off-target activity of a pair of ZFNs has been examined by measuring the ratio between HR and illegitimate genomic integration in cells that are growing exponentially, and in cells that have been arrested in the G2/M phase.enhanced green fluorescent protein (EGFP) reporter gene was used to measure ratios between HR and non-homologous integration of a plasmid template. Both in human cells (HEK 293) containing the consensus ZFN binding sites and in cells lacking the ZFN binding sites, a 3.5 fold increase in the level of illegitimate integration was observed upon ZFN expression. Since the reporter gene containing the consensus ZFN target sites was found to be intact in cells where illegitimate integration had occurred, increased rates of illegitimate integration most likely resulted from the formation of off-target genomic DSBs. Additionally, in a fraction of the ZFN treated cells the co-occurrence of both specific HR and illegitimate integration was observed. As a mean to minimize unspecific effects, cell cycle manipulation of the target cells by induction of a transient G2/M cell cycle arrest was shown to stimulate the activity of HR while having little effect on the levels of illegitimate integration, thus resulting in a nearly eight fold increase in the ratio between the two processes.A reporter cell line that contained consensus ZFN binding sites in an The demonstration that ZFN expression, in addition to stimulating specific gene targeting by HR, leads to increased rates of illegitimate integration emphasizes the importance of careful characterization of ZFN treated cells. In order to reduce off-target events, reversible cell cycle arrest of the target cells in the G2/M phase is an efficient way for increasing the ratio between specific HR and illegitimate integration. Currently, gene targeting by homologous recombination (HR) is the standard method utilized for precise genome modification of mammalian cells. In this strategy the cellular DNA repair pathway, HR, mediates exchange of sequences between a given donor DNA sequence and a homologous genomic target sequence . AlthougCaenorhabditis elegans [Drosophila [With the aim of developing methods for more efficient and specific gene targeting, various alternative strategies have been exploited including the use of modified donor DNA, recombinant enzymes and modified viral vectors -6. Never elegans , Drosophosophila , zebra fosophila ,17, planosophila ,19, ratsosophila  and humaosophila ,21,22.enhanced green fluorescent protein (mEGFP) gene in human embryonic kidney 293 cells. The donor plasmid, given as a template for HR repair of the DSB formed, also included an expression cassette for the puromycin resistance (PuroR) gene. Accordingly, by quantifying the number of puromycin resistant colonies formed with and without expression of the ZFNs, the level of illegitimate integration could be measured and used as an indication of the specificity of the ZFNs. In 293-Flp-mEGFP cells that contained the canonical ZFN binding sites and in wild type (wt) 293 cells devoid of the ZFN binding sites, the levels of illegitimate integration were increased 3.5 fold upon ZFN exposure. Analysis of the integrity of the mEGFP gene in puromycin resistant colonies showed that the PuroR expression cassette had not integrated at the position where the ZFNs were intended to create a DSB. Interestingly, co-occurrence of both specific HR and illegitimate integration could be observed in a fraction of ZFN treated cells. Importantly, transient cell cycle arrest in the G2/M phase during ZFNs exposure did not only boost rates of HR, but also increased the ratio of specific HR versus illegitimate integration by nearly eight fold.For practical applications of ZFN induced HR it is essential that all possible adverse effects that ZFNs and donor plasmids might have on the target genome are well characterized. Formation of off-target genomic DSBs could cause unwanted effects on the targeted genome including, in the worst case, transformation due to genetic rearrangements -25. In tmEGFP gene were created and named 293-Flp-mEGFP. The mEGFP gene in the 293-Flp-mEGFP cell line does not encode for a functional EGFP protein due to the introduction of a premature stop codon together with a two bp deletion in the EGFP coding sequence (CDS)  used contained a truncated wt EGFP gene (ΔEGFP) lacking the first seven bases of the EGFP CDS  Fig. . The twoDS) Fig. . BindingCDS Fig. . In addicin Fig. .mEGFP gene [In non-transfected and in 293-Flp-mEGFP cells transfected with the ZFNs alone, no green cells could be detected (detection limit ~1 in 100 000) . When either ZFN-L or ZFN-R was individually transfected with the pDonor, an increase in the frequencies of puromycin resistant colonies to 55 × 10-5 was observed. Compared to the level of illegitimate integration obtained with the pDonor alone this increase was not statistically significant (P = 0.084). However, when 293-Flp-mEGFP cells were transfected with both ZFN-L and ZFN-R simultaneously together with pDonor, a significant increase (up to 99 × 10-5) of puromycin resistant colonies could be observed (P = 0.020). As a further control of the specificity of the ZFNs, their influence on illegitimate integration was also quantified in wt 293 cells that did not contain the mEGFP target gene incorporating the canonical ZFN binding sites. When wt 293 cells were transfected with either ZFN-L or ZFN-R individually along with the pDonor construct, no increase in illegitimate integration could be observed  in all the 25 colonies (not shown). Taken together these results established that the increased rates of illegitimate integration following ZFN expression was not due to genomic integration of the PuroR cassette in the genomic mEGFP site where the ZFNs were designed to form a DSB. Given that that the average occurrence of the canonical 18 bp ZFN recognition sequence statistically is only one in the mammalian genome, the observed increased rates of illegitimate integration following ZFN expression was most likely due to formation off-target genomic DSBs by the ZFNs.To investigate whether the observed increase rates of illegitimate integration after ZFN exposure was due to an integration of the Generally, the repair of genomic DSBs is carried out by either the HR or the non-homologous end joining (NHEJ) repair pathway . HoweverΔEGFP and the PuroR regions from the pDonor construct must have been stably integrated into the genome of the target cells. Theoretically, the ΔEGFP and PuroR segments from the pDonor could either have been integrated jointly as a single fragment or they could have been integrated as multiple fragments. Given that the analyzed cells displayed green fluorescence due to HR between the genomic mEGFP gene and the ΔEGFP region in the pDonor, integration of the ΔEGFP and PuroR regions as a single fragment would require that the PuroR region to be positioned downstream (3') of the mEGFP gene  was obtained in 24 of the 26 colonies analyzed (not shown).In the green fluorescent 293-Flp-mEGFP cells that also exhibited puromycin resistance, both the ene Fig. . To stud P1 Fig. , specifiΔEGFP region and the PuroR region from the pDonor construct had been incorporated in the genome via multiple integration events. Only in 8% of the clones the ΔEGFP and PuroR regions had integrated as a single fragment "in frame" with the genomic mEGFP. Our observed genomic incorporation of both homologous and non-homologous sequences at a DSB in 8% of the clones is in accordance with Richardson et al. who demonstrated that a DSB repair event could be initiated by homologous invasion at one side and completed by NHEJ at the other end [Taken together, the analysis of the quality of the HR reaction established that in the majority (92%) of the clones that had undergone concurrent HR and illegitimate recombination, the ther end .EGFP expression plasmid (pEGFP-c1) displayed similar frequencies both in untreated and nocodazole treated cells (not shown). Increased rates of HR following nocodazole induced G2/M cell cycle arrest are in agreement with Urnov et al. who observed increased levels (4.8 fold) of ZFN mediated HR in cells reversely arrested in the G2/M phase using vinblastine [Cell cycle status is an important factor in the decision whether a genomic DSB is repaired by HR or NHEJ. While NHEJ is active throughout the cell cycle, HR is mainly active in the late S and G2/M phases . To examlevels 4. fold of When the rate of illegitimate integration was compared between exponentially growing and G2/M arrested cells, only minor variations was observed Fig. . ConsequStimulation of targeted genome modification by ZFNs has proven to be very successful . NeverthEGFP specific ZFNs has been investigated by measuring their influence on the levels of illegitimate integration of the given donor plasmid. We have shown that upon expression of the ZFNs in human 293 cells, the levels of illegitimate integration of the donor plasmid was increased by about 3.5 fold in cells containing the canonical ZFN binding sites and in cells devoid of the ZFN target sequences. In cells that contained the canonical ZFN binding sites, genomic analysis of the mEGFP target gene revealed that the donor DNA had not integrated at the position where the ZFNs where intended to form a DSB . In addition, the levels of illegitimate integration obtained with the ZFNs, although present, were only moderately increased compared to the benchmark I-SceI endonuclease. Interestingly, Moehle egration .The main impediment in gene targeting approaches is the unfavorable ratio between high frequency of illegitimate integration and low frequency of specific HR . By enabBy enabling the formation of site specific genomic DSBs over a range of genomic targets, the development of ZFNs has brought the field of targeted genome modification a substantial step forward. Next generation ZFNs with further improved properties  as exempThe demonstration that ZFNs in addition to stimulate specific gene targeting by HR also can produce increased rates of illegitimate integration emphasizes the importance of cautious characterization of cells that have been treated with ZFNs. Reversible cell cycle arrest of the ZFN targeted cells in the G2/M phase is a simple and efficient way of positively altering the ratio between specific HR and illegitimate integration.mutEGFP (mEGFP) gene codes for a non-functional EGFP protein due to the presence of a premature stop codon (GAC to TAA) at position 229-231 and a two bp deletion (AT to --) at position 235-236 in the EGFP CDS. The pFRT/mEGFP construct used for making the 293-Flp-mEGFP cell line was made by cloning the mutEGFP sequence into pEF5/FRT/V5-D-TOPO . pFRT/EGFP (expressing wt EGFP) was made by cloning the wt EGFP CDS from pEGFP-c1  into the same construct. The pDonor construct incorporated a truncated EGFP sequence (promoterless) lacking the first seven bases (ΔEGFP) together with a PuroR expression cassette that had been cloned into pBluescript-SK   were generously provided by Samgamo BioSciences, Inc.  and have been described elsewhere . The mutas) Fig. . The pEGABE-puro  construcABE-puro EGFP (mEGFP) gene, was generated by using Invitrogen's Flp-In system according to their instructions. Briefly, the stable 293-Flp-In cell line  that contains a single copy genomic integration of the Flp recombination target (FRT) site was transfected with the expression construct for the Flp recombinase (pOG44) together with the plasmid containing the mEGFP gene and a FRT site (pFRT/mEGFP). As a result of Flp recombinase mediated recombination, the mEGFP gene will be integrated in the genomic FRT site of the 293-Flp-In cells where its expression is driven by the human Elongation Factor 1α (EF1α) promoter , was purchased from ATCC . The 293-Flp-mEGFP cell line, containing a single copy genomic integration of the mutated ter Fig. . All celCells were transfected in 12 well plates with pDonor (0.5 μg), pZFN-L and pZFN-R (0.05 μg each) as indicated in the figures. The total amount of DNA in each well was adjusted to be 0.6 μg by addition of the pBlueskript vector . Transfection was done using Lipofectamine (LF) 2000 (Invitrogen) according to the manufactures instructions . Two days after transfection, cells were plated out both in new 12 well plates for analysis of HR and in 60 mm dishes (600 000 cells/dish) for analysis of illegitimate integration. For analysis of HR, the amount of green cells was quantified (three days after transfection) on a Partec PAS flow cytometer . Cell sorting was performed with the FACSaria cell-sorting system . For analysis of illegitimate integration of the pDonor plasmid, cells were grown in the presence of 1.0 μg/ml puromycin . Following 10 days of growth in selective medium, puromycin resistant colonies were visualized by methylene blue staining  and quantified using the GeneTools image analysis software .One day after transfection of 293-Flp-mEGFP cells with pDonor, cells were incubated overnight with growth medium containing 0.1 μM etoposide (Sigma-Aldrich). Three days after transfection the cells were plated in 60 mm dishes for puromycin selection and for measurement of the PE as described below. Etoposide treatment of the cells did not influence the PE (not shown).2 in complete growth medium in 60 mm dishes. Following 14 days of expansion, the number of colonies formed was counted and the percentage of colony forming cells was calculated.PE was determined by plating 20 cells/cmmEGFP gene in non-green puromycin resistant 293-Flp-mEGFP cells was done by PCR analysis on their genomic DNA . Nocodazole (0.1 μg/ml) was applied to the cells together with the transfection mixture and was kept on the cells for 24 hours before the cells were allowed to re-enter cell cycling. Cell cycle analysis was done on fixed cells (70% EtOH) stained with propidium iodide (PI) . Cells were replated for quantification of illegitimate integration and PE two days after transfection as described. The PE for exponentially growing cells was 33% while for G2/M arrested cells the PE was 5%.PuroR: puromycin resistance gene; wt: wild type; ZFNs: zinc-finger nucleases.bp: base pairs; CDS: coding sequence; CMV: cytomegalovirus; DSBs: double-strand breaks; EF1α: elongation factor 1α; EGFP: enhanced green fluorescent protein; FACS: fluorescent activated cell sorting; FRT: Flp recombination target; HR: homologous recombination; LF: Lipofectamine; NHEJ: non-homologous end joining; OPEN: Oligomerized Pool Engineering; PE: plating efficiency; PI: propidium iodide; PAO and SK designed the study and experiments. PAO carried out the majority of the experiments with the assistance of MR and MG. PAO was the primary author of the manuscript. SK conceived of the study and participated in its design, coordination and contributed to the writing of the manuscript. All authors approved the final manuscript.


Alkylating agents can cause latent and permanent damage to the bone marrow. We compared the long term effects of melphalan on a number of immune and haemopoietic functions of plasmacytoma bearing BALB/c mice with that of normal mice treated with a similar dose of melphalan. The drug administered orally at a dose of 250 micrograms and 400 micrograms on day 14 and 24 following i.m. inoculation of MOPC-315 plasmacytoma cells resulted in cure of the mice. Their spleen cells showed a permanent impairment of MLR activity, T-cell number and IL-2 production as well as a mild suppression of NK activity for one year after cessation of melphalan therapy. The number of B cells was elevated. In contrast, plasmacytoma-free mice treated with melphalan retained long term normal immune functions, although shortly after melphalan therapy a temporary suppression was noted. On the other hand, melphalan was responsible for bone marrow myeloid stem cell damage since the number of myeloid progenitor cell (CFU-GM) colonies was reduced in both melphalan-treated groups compared to untreated normal controls. Plasmacytoma bearing mice had a shorter survival. These results demonstrate that some late sequelae of alkylating agents are not due to the drug alone; shorter survival and T-cell deficiency are related to the previous presence of the tumour.


Accurate determination of orthology is central to comparative genomics. For vertebrates in particular, very large gene families, high rates of gene duplication and loss, multiple mechanisms of gene duplication, and high rates of retrotransposition all combine to make inference of orthology between genes difficult. Many methods have been developed to identify orthologous genes, mostly based upon analysis of the inferred protein sequence of the genes. More recently, methods have been proposed that use genomic context in addition to protein sequence to improve orthology assignment in vertebrates. Such methods have been most successfully implemented in fungal genomes and have long been used in prokaryotic genomes, where gene order is far less variable than in vertebrates. However, to our knowledge, no explicit comparison of synteny and sequence based definitions of orthology has been reported in vertebrates, or, more specifically, in mammals.We test a simple method for the measurement and utilization of gene order  in the identification of mammalian orthologs by investigating the agreement between coding sequence based orthology (Inparanoid) and local synteny based orthology. In the 5 mammalian genomes studied, 93% of the sampled inter-species pairs were found to be concordant between the two orthology methods, illustrating that local synteny is a robust substitute to coding sequence for identifying orthologs. However, 7% of pairs were found to be discordant between local synteny and Inparanoid. These cases of discordance result from evolutionary events including retrotransposition and genome rearrangements.By analyzing cases of discordance between local synteny and Inparanoid we show that local synteny can distinguish between true orthologs and recent retrogenes, can resolve ambiguous many-to-many orthology relationships into one-to-one ortholog pairs, and might be used to identify cases of non-orthologous gene displacement by retroduplicated paralogs. The accurate determination of orthology is central to comparative genomics. Pinpointing the origin of new genes, understanding the evolution of new gene families, and assessing the impact of gene and genome duplication events all require the accurate assignment of orthology between genes in distinct genomes. In complex genomes with large gene families this task requires differentiating between genes that have diverged through a speciation event (orthologs) and those derived through duplication events within a species . Determination of orthology and paralogy is especially challenging in vertebrates species such as the mammals. Very large gene families, high rates of gene duplication and loss, multiple mechanisms of gene duplication, and high rates of retrotransposition all combine to make the determination of orthology between vertebrate genes difficult.Given the importance of accurate orthology assignment, many methods have been developed to identify orthologous genes. Most of these methods rely upon analysis of the inferred protein sequence of the genes in question by clustering the results of protein sequence comparisons results in the classification of putative orthologs. Examples of this approach include reciprocal best BLAST hits and more inclusive BLAST based clustering methods. Splitting of these clusters based on relative similarity can distinguish between older and newer duplication events and is implemented in the widely used Inparanoid algorithm  and relaMore recently, methods have been proposed that use genomic context in addition to protein sequence to improve orthology assignment. These methods have been most successfully implemented in fungal genomes ,4, and hIn this study, we evaluate a simple method for using gene order  in the identification of mammalian orthologs. We explicitly compare the relative performance of local synteny and Inparanoid for inferring orthology within this clade and show that local synteny alone is sufficient to identify orthologs with an accuracy comparable to that of Inparanoid. By analyzing cases of discordance between local synteny and Inparanoid we show that local synteny can distinguish between true orthologs and recent retrogenes, can resolve ambiguous many-to-many orthology relationships into one-to-one ortholog pairs, and can highlight possible cases of parental gene displacement by retrogenes.Several orthology inference methods, such as Inparanoid  and OrthHomo sapiens (human), Pan troglodytes (chimp), Mus musculus (mouse), Rattus norvegicus (rat), and Canis familiaris (dog). To avoid protein family-specific bias in this analysis, we chose one homologous pair from each Pfam family. For each pair we computed Protdist and the degree of local synteny between the two genes. Figure To validate the use of local synteny for inferring orthology, we evaluated the correlation between Protdist and local synteny using a dataset derived from the Pfam protein family database. Pfam families are highly accurate protein families based on protein domains . We randTheoretically two non-orthologous genes should not share homologous neighboring genes. However, there is a small probability of homology matches occurring by chance. Moreover rearrangements, insertions, and deletions will lead to loss of local synteny between orthologous genes. In order to account for these events, we wanted to determine an optimal window size and match percentage that could reliably identify orthologs based on local synteny. In Figure Since many orthology detection methods use more complicated algorithms than just coding sequence similarity, a high correlation between Protdist and local synteny Figure  is not sFigure Because Inparanoid is one of the most widely used ortholog definition methods -19 and iIn order to identify the source of this discordance we employed intron-based evidence. Specifically, for each gene pair used in the test we computed the intron conservation ratio (ICR) between the two genes defined as follows (for more details see Methods):Figure All the non-syntenic Inparanoid ortholog pairs with zero ICR contain one intronless copy and one intron-bearing member. Based on local synteny information and intron conservation ratio, these intronless copies are probably retrotransposed (RT) copies of the original orthologs. Of all the samples, 1.2% are non-syntenic 0-ICR Inparanoid orthologs. Inparanoid likely included RT copies in these orthologous groups because the RT copies have not diverged sufficiently to be distinguished from their parent gene. In 0.2% of samples Inparanoid chose RT copies even though there were other syntenic high-ICR copies. Figure The rest of non-syntenic 0-ICR Inparanoid orthologs (1.0%) of all samples are classified as one-to-many orthologs by Inparanoid. In many of these one-to-many orthologs, syntenic high-ICR pairs are chosen as "core orthologs" (based on the Inparanoid scores) with additional RT copies added to the orthology group because their protein sequences are still close to those of the original copies. By using local synteny, the RT copies in one-to-many orthologs would be distinguished from the other members. Thus, local synteny based orthology separates RT copies from those generated by speciation or other duplication mechanisms and can be more informative in recovering the evolutionary history of a gene family.All non-syntenic Inparanoid orthologs with an ICR > 0 1.3%) are likely to result from the loss of local synteny. They each have one homologous match between neighboring genes (lower than the threshold of being syntenic) and are selected from distant species pairs (i.e. not human-chimp or mouse-rat). Each of these distant pairs is part of larger orthology groups (5-species), in which the counterparts in closer species pairs have higher local synteny (more than 1 match). This loss of local synteny likely results from rearrangements and gene insertions or gene losses in more distant species. See Additional file % are likThere are 2.3% syntenic non-Inparanoid orthologs with an ICR ≥ 0.5 Figure . The majIn the non-syntenic non-Inparanoid case Figure , where tSince local synteny is able to differentiate some speciation and duplication events, a phylogenetic tree (or duplication-speciation history) of ambiguous many-to-many orthologs may be determined using local synteny information. In mouse-to-rat ortholog definitions from Inparanoid, there are 131 many-to-many ortholog groups. The majority (~75%) of many-to-many groups are comprised of DNA-mediated duplicated copies  while ~20% have true orthologs  as well as non-syntenic intronless copies (probably from RT events).We present two many-to-many Inparanoid ortholog groups where the local synteny determines the order of evolutionary events in the gene family. One of them is an example of orthologs from distant DNA-mediated duplication event(s) followed by possible rearrangements or gene gains/losses, and then speciation event Figure . In thisAnother case where local synteny clarifies the history of closely related groups of duplicates can be seen in a mouse-rat many-to-many Inparanoid ortholog group including RT copies Figure . This exThe accurate determination of orthology is critically important to comparative genomics. However it has been a challenge to compare the various orthology determination methods without a reliable gold-standard orthology dataset . The staIn Figure The FN rate of local synteny based orthology is higher than those of Blastp based orthologies . This may be due to distant paralogs retaining flanking genes, but diverging in their coding sequences enough to be distinguishable by Blastp. RT miscalls by Inparanoid  the estimated FN rates from OrthoMCL are the highest in this experiment (approximately 0.3). This is opposite from the result of Chen et al.  where OrSynteny information has been used to cluster synteny blocks between two related genomes in order to detect orthologous gene pairs ,21 and tAnother similar approach to local synteny was used to reconstruct phylogenetic gene trees from coding sequence similarity and local gene order . In the Finally, MSOAR  uses comMost importantly, when the orthology is defined by codon sequence similarity, testing any hypothesis of selective pressures on orthologous gene presents tautological challenges. Since orthology detection by local synteny is not based on the comparison of coding sequence information between candidate orthologs, testing the selective pressure between orthologs becomes a valid comparison of largely independent variables. However, gene order also degrades over evolutionary time. How well synteny will be able to effectively identify ancient orthologs remains to be seen.Gene duplications are a major force in genome evolution . Genes aSometimes the difference between coding sequences of parental genes and RT copies is not large enough to distinguish the RT copy from the parent. RT copies, however, do not share introns or flanking genes with the parental paralog. Therefore, local synteny and gene structure can often separate the RT copy from the original gene. However, when neither has an intron, only local synteny will distinguish parental and RT copies. Based on our random sampling, approximately 8~10% of Pfam orthologs are intronless gene pairs.Reconstructing phylogenetic trees informs our understanding of the evolutionary history of gene families. Using tree reconciliation between a species tree and gene tree we can identify duplication and lost events on the tree. However by distinguishing two duplication mechanisms, DNA-mediated and RNA-mediated, not just identifying duplication events, we can sometimes place duplication events in an appropriate phylogenetic context. For example, as Figure In this paper, we investigate the concordance between coding sequence based orthology (Inparanoid) and local synteny based orthology. In the 5 mammalian genomes studied, 93% of the sampled inter-species pairs were found to be concordant between the two orthology methods, illustrating that local synteny can largely substitute for coding sequence in identifying orthologs. However, 7% of pairs were found to be discordant. Discordance is often associated with evolutionary events like retrotransposition, iterative DNA-mediated duplication, and genome rearrangement. Analysis of discordant cases between local synteny and Inparanoid shows that local synteny can differentiate between true orthologs and recent retrogenes, can split ambiguous many-to-many orthology groups into more precise one-to-one ortholog pairs, and, when employed in a genome-wide screen, might help in highlighting possible cases of non-orthologous gene displacement by retrocopied paralogs in mammalian genomes.Five species analyzed  were obtained from Ensembl release 48 . We onlyLocal synteny is measured by homology between the neighboring genes of two genes of interest. In this study the maximum unique homology matches between two sets of six neighboring genes (three upstream and three downstream neighbors) was used. The matches do not need to be co-linear or between genes on the same strand/orientation either  BLASTP: homology detection using E-value cutoff (= 1e-5)2) SBH: Single-way or One-way Best Hit. 'Best-hit' is defined as the hit (or multiple hits tied) with the highest E-value 3) RBH: Reciprocal Best Hit. 'Best-hit' is defined as same as SBH 4) Inparanoid (v2.0): bit score cutoff = 50 bits and sequence overlap cutoff = 0.55) OrthoMCL (v1.4): E-value cutoff = 1e-5 and MCL inflation index = 1.5; MCL package (v02-063) was used.The frequency table of agreements and disagreements between orthology detection methods was calculated and used for LCA. LCA was performed using the LEM package  with defGene structure similarity is measured by the intron conservation ratio (ICR) between two intron-bearing genes . For genFor the discordant cases between Inparanoid, local synteny, and/or ICR based orthology  any significant Blastp hits other than the sampled pairs  the other Inparanoid orthologs in all 5 species in order to confirm rearrangement history in those families  Inparanoid orthologous counterparts of non-Inparanoid sampled pairs in Figure Mouse-rat many-to-many Inparanoid ortholog groups were collected and analyzed to reconstruct their evolutionary history by considering the genomic location and intron content of their member genes. Two example ortholog groups Figure  were choJJ performed the programming and the data analysis. JJ, IIM and CEN participated in the design of the study and drafted the manuscript. All authors read and approved the final manuscript.Various numbers of neighbors. False positive (FP) and false negative rates (FN) of local synteny measures to the Inparanoid orthologs and Ensembl orthologs, with using different number of neighbors.Click here for fileTabular format of Figure Click here for fileExample of loss of local synteny case. One example of loss of local synteny along the evolutionary distance.Click here for fileTabular format of Figure Click here for fileTabular format of Figure Click here for fileLCA with a CFactor model. Another LCA model to account for the dependency between orthology detection methods and a corresponding FP/FN graph.Click here for file


Current outcome predictors based on "molecular profiling" rely on gene lists selected without consideration for their molecular mechanisms. This study was designed to demonstrate that we could learn about genes related to a specific mechanism and further use this knowledge to predict outcome in patients – a paradigm shift towards accurate "mechanism-anchored profiling". We propose a novel algorithm, PGnet, which predicts a tripartite mechanism-anchored network associated to epigenetic regulation consisting of phenotypes, genes and mechanisms. Genes termed as GEMs in this network meet all of the following criteria: (i) they are co-expressed with genes known to be involved in the biological mechanism of interest, (ii) they are also differentially expressed between distinct phenotypes relevant to the study, and (iii) as a biomodule, genes correlate with both the mechanism and the phenotype.This proof-of-concept study, which focuses on epigenetic mechanisms, was conducted in a well-studied set of 132 acute lymphoblastic leukemia (ALL) microarrays annotated with nine distinct phenotypes and three measures of response to therapy. We used established parametric and non parametric statistics to derive the PGnet tripartite network that consisted of 10 phenotypes and 33 significant clusters of GEMs comprising 535 distinct genes. The significance of PGnet was estimated from empirical p-values, and a robust subnetwork derived from ALL outcome data was produced by repeated random sampling. The evaluation of derived robust network to predict outcome (relapse of ALL) was significant (p = 3%), using one hundred three-fold cross-validations and the shrunken centroids classifier..To our knowledge, this is the first method predicting co-expression networks of genes associated with epigenetic mechanisms and to demonstrate its inherent capability to predict therapeutic outcome. This PGnet approach can be applied to any regulatory mechanisms including transcriptional or microRNA regulation in order to derive predictive molecular profiles that are mechanistically anchored. The implementation of PGnet in R is freely available at  Though phenotype-genotype-network" method (PGnet), a set of known biological mechanism-anchored genes are required as the "seed" (input). Although this method can be generalized to other molecular mechanisms and other diseases, we focused this study on epigenetic alterations in acute lymphoblastic leukemia (ALL).We hypothesized that those co-expression modules, which are predictive of outcome, can be computationally derived from genes known to regulate or to be regulated by epigenetic mechanisms in previous studies and from novel microarray expression specifically designed for a new phenotype for which the epigenetic mechanisms may not be well understood ,5. NearlARACNE has been shown to be effective in practice by using a mutual information theoretic approach which focuses on direct co-expression of genes [FunNet, addresses multiplicity by combining gene expression data with Gene Ontology [StAM, identifies expression signature by focusing on biological processes which can characterize subgroup of patients [i) they are associated ab initio to the biological mechanism of interest (here epigenetics), and ii) they are derived from the a non-parametric statistic taking into account comprehensive expression patterns of the every gene in the microarray rather than from a subset of the differential-expressed ones.To compute co-expression modules of genes in disease and to infer their interplay generally require the integration of data from a wide variety of sources ,17. For of genes ,25. HoweOntology ,27 or KEOntology  annotatiOntology . AnotherWe hypothesize that using supervised pair-wise measurements from microarray data together with robust feature selection technology , we are We selected a large array dataset published by the Downing research group (ALL arrays)  that comGene Ontology terms and PubMed were used to identify genes with epigenetic effects. Genes were subsequently mapped to Affymetrix probe-sets and curated into eight categories.compdiagTools [The 132 ALL arrays were normalized with the variance stabilization and calibration normalization (vsn) method  using BiiagTools . We theniagTools  to elimiThe construction of a network comprising "Leukemia Phenotypes" (LPs), "epigenetic seed Genes" (ESGs) and co-expressed genes required six steps . Results were denoted as stats was used to calculate the PCC and the package Twilight [At the same time, all genes were also sorted based on their adjusted Student t-test conducted between the phenotype of interest against the remaining pooled phenotypes. Results were denoted as onductor ,33 packaTwilight  was usedOrderedList [Ms = , where each score si, j assessed the pair-wise similarities between two vectors. The fist vector is the ordered co-expression coefficients VESGi and the second one is the ordered differential expression statistics VLPj. The similarity score gives higher weights to ranking extremes: the top and bottom ranks in both lists. In our method, we compared the ranking of genes in the co-expression set with those gene ranks from the phenotypic set. This resulted in a total of two comparisons for each phenotype/"seed gene" combination (correlation and anti-correlation).To compare two ordered lists of gene expressions, we used eredList -39 from eredList ,33, a noTo evaluate the statistical significance of the similarity score, we generated 2,000 controls through the permutation of each list of gene ranks and calculated empirical p-values based on random scores. Two networks were generated. In this proof-of-concept study, the arbitrary but uniform significance threshold , circle (predicted GEMs) and box (phenotypes); red (up-regulated), blue (down-regulated) and grey for vertex of a gene had more than one linkage and was up-regulated in one condition but down-regulated in a different condition  in the epigenetic context of this network meet all of the following criteria: (i) they are co-expressed with genes known to be involved in the biological mechanism of interest, (ii) they are also differentially expressed between distinct phenotypes relevant to the study, and (iii) as a biomodule, genes correlate with both the mechanism and the phenotype.Using Gene ontology, we conducted an enrichment of the molecular function and biological processes among the genes identified in the PGnet biomodules in order to characterize biologically the network and we also reviewed the literature for the genes involved in the biomodules associated to BCR-ABL, T-ALL and hyperdiploidy. Thus the resulting set of genes termed as The predictive capabilities of the derived network were evaluated with two approaches: (a) quantitative computational studies of the accuracy of the predictor of outcome, and (b) qualitative comparison of the PGnet method to that of another reverse engineering one.verification [(a) To demonstrate the accuracy of the derived network to predict relapse, we performed a conservative evaluation consisting of one hundred three-fold cross-validation (CV) studies of the PGnet method. In other words, as shown in Figure fication . A robusfication . We furtfication . The rob(b) Finally we compared our results to those obtained by a straightforward reverse engineering method (ARACNE).ab initio among genes differentially expressed in ALL samples suggesting a biological relevance in ALL .Seventy-one distinct epigenetic seed genes were identified in the literature review and denoted as "seed gene" candidates for input in PGnet . A summary of predictions is provided in Suppl. Table 2 .We built a gene-phenotype network specific for epigenetic genes clusters in ALL as shown in Figures  associated with leukemia relapse : SMARCA4 is a drug target candidates in hyperdiploid multiple myeloma [BAZ2A is a novel nucleolar chromatin remodeling machine [SMARCC2 was among the top discriminating genes in the good prognosis subgroup of MLL [-16, Suppl. Table 6  PGnet and FunNet combine supervised technology and non-parametric methodology while ARACNE uses information theory; (ii) inputs to PGnet are expression levels and phenotypic associations of interest such as seed genes whereas FunNet requires full expression together with a reference list of all transcripts to be analyzed and ARACNE uses expression data exclusively; (iii) FunNet abstracts transcriptional functions from co-expression layer; and consequently (iv) PGnet's output is a tripartite network consisting of co-regulated genes and clinical/genetic characteristics of interest while ARACNE's or FunNet's outputs are uni-partite graphs. (v) The significant threshold of PGnet relates to the complete ordering of all genes to be analyzed whereas the significant threshold of FunNet is related to the co-expression of single gene. (vi) PGnet not only provides a degree of association between phenotypes but also sheds light on whether there was concordance in the directionality of the changes in expression level.PGnet parallelizes two input vectors and finds sets of GEMs via vectorial enrichment optimization. Using measurements of differential expression and co-expression together, PGnet is more reliable in discovering phenotype-specific biomodules that are consistent across every patient than a simplified method that analyzes the expressed pattern of the epigenetic seed genes (ESGs) alone. First, a simplified method identifies none of GEMs from PGnet. However, we have shown some evidence indicating that the GEMs are more likely to be involved in specific epigenetic events than those directly calculated to be correlated to a phenotype of interest. Second, simpler alternate methods relying on co-expression or differential expression separately would identify only a the subset of ESGs from PGnet (data not shown), because these methods use an arbitrary threshold for significance of each gene and neglect the joint analysis of co-expression patterns with those of differential expression. In contrast, the "ESG-phenotype" linkage, which we proposed in PGnet, would be significant even if the epigenetic seed gene itself is not "significantly" differentially expressed in the linked phenotype , which could also be utilize to infer regulation (activation or suppression) .The authors declare that they have no competing interests.Lussier was responsible of the overall experimental design and contributed to the R&D of the methods. Yang contributed significantly to the design, methods development and implementation, as well as the majority of the computations. Xie and Sun contributed to the design, methods and epigenetic gene lists. Yang, Huang, Chen, and Lussier contributed to the evaluation, discussion and conclusion.Supplementary Methods – Supplementary Methods.Click here for fileSupplementary Table 1 – The eight categories of epigenetic key terms and their corresponding 71 epigenetic seed genes.Click here for fileSupplementary Table 2 – Thirty-three significant "LP-ESG" linkages contributed by corresponding GEMs (parameter T = 200)Click here for fileSupplementary Table 3 – The predicted GEMs of 117 significant "LP-ESG" linkages based on optimal parameters (Ts)Click here for fileSupplementary Figure 2 – The Design of the two Evaluations (A and B).Click here for fileSupplementary Figure 3 – The ROC curve of the computational evaluation method B of PGnet-predicted GEMs and ESGs signature associated with ALL relapse.Click here for fileSupplementary Figure 4 – The precision_recall results of the computational evaluation A of PGnet-predicted GEMs and ESGs signature associated with ALL relapse.Click here for fileSupplementary Table 5 – Robust GEMs and ESGs Predictive of Leukemia Relapse.Click here for fileSupplementary Table 4 – The significantly enriched GO items among the predicted GEMs that linking to phenotypes of interested.Click here for fileSupplementary file Figure 5 – Expression pattern of ALL phenotype "Hyperdiploid>50" specific GEMs by PGnet.Click here for fileSupplementary Table 6 – Previously reported facts about predicted GEMs.Click here for fileSupplementary Table 7 – Comparison GEMs in PGnet with genes identified by ARACNE.Click here for fileSupplementary Table 8 – Comparison of the results from ARACNE and PGnet for leukemia phenotypes.Click here for fileSupplementary Figure 1 – PGnet: significant "phenotypes in ALL – GEMs – epigenetic seed genes"Click here for file


Recent years have seen an explosion of new knowledge defining the molecular events that are critical for development and activation of immune cells. Much of this new information has come from a careful molecular dissection of key signal transduction pathways that are initiated when immune cell receptors are engaged. In addition to the receptors themselves and critical effector molecules, these signaling pathways depend on adapters, proteins that have no intrinsic effector function but serve instead as scaffolds to nucleate multimolecular complexes. This review summarizes some of what has been learned about one such adapter protein, SH2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76), and how it regulates and integrates signals after engagement of immunoreceptors and integrins on various immune cell lineages. In the last twenty-five years, since the initial description of the molecular components of the T cell antigen receptor (TCR), much attention has been focused on understanding the biochemical events that occur when that receptor is engaged by antigen . ElegantSLP-76 was identified originally in a screen designed to discover substrates of the PTKs stimulated by ligation of the TCR . The genWork from several laboratories has defined the binding partners for these three SLP-76 domains. Tyrosines 112 and 128 are in the same motif (DYESP) and when phosphorylated create an excellent docking site for the SH2 domains of the guanine nucleotide exchange factor Vav and the adapter protein Nck ,14. The SLP-76 is found in the cytosol of resting cells. It constitutively binds to its partners that interact via its central proline-rich region, and it inducibly associates with its binders that require tyrosine phosphorylation after receptor engagement (and activation of the receptor-associated PTKs). The ability of SLP-76 to support signaling downstream of both immunoreceptors and integrins appears to require its relocalization from the cytosol to signaling clusters that form near the activated receptors ,23. Our in vivo consequence of SLP-76 loss in the T cell compartment is a complete block in thymocyte development at the double negative 3 (DN3) stage, the point during which the pre-TCR must signal its successful expression following TCR gene recombination. Failed signaling at this point leads to thymocyte death and the absence of double positive (DP) and single positive (SP) cells. Mice lacking SLP-76 therefore have no T cells in their peripheral lymphoid organs. More recent studies making use of "floxed" SLP-76 alleles demonstrate that if the gene for SLP-76 is not deleted until after DP thymocytes appear, SLP-76 loss in this compartment abrogates selection, leading to thymocyte "death by neglect"  and integrin signaling is platelets. Lack of SLP-76 results in failed platelet aggregation or degranulation in response to collagen (which makes use of both integrins and the GPVI receptor that pairs with the common γ chain of Fc receptors) . SLP-76-+/- matings gave rise to the observation that it was possible to genotype the SLP-76 deficiency by eye, as fetuses lacking two SLP-76 alleles appeared grossly hemorrhagic. Later, it was shown that what originally appeared as bleeding was in fact the presence of blood-filled lymphatic vessels, as it was learned that loss of SLP-76 prevented normal separation of blood from lymphatic vessels  present within the CD3 molecules of the TCR on T cells and Fc receptors on other immune cells. The ITAM phosphotyrosines then recruit members of the Syk family PTKs, thus converting the immunoreceptor from an enzymatically inert structure to a receptor with associated PTK activity.Among the substrates that are rapidly phosphorylated by the activated immunoreceptor complex are adapters including the transmembrane protein linker of activated T cells (LAT) . LAT actBecause SLP-76 is also a substrate of Syk family PTKs, activation of immunoreceptors results in SLP-76 tyrosine phosphorylation. This occurs on three residues  within the N-terminus of SLP-76. Y112 and Y128 are in the appropriate motif to bind to SH2 domains of Vav, a guanine nucleotide exchange factor important for cytoskeletal reorganization, and Nck, another adapter essential for full immune cell activation. Y145 has a binding site for the SH2 domain of Tec family PTKs (ITK in T cells and BTK in other immune cells). These PTKs are critical for initiating calcium and ras signaling in immune cells by activating PLC (γ1 in T cells and γ2 in other immune cells). Initiation of this pathway is facilitated by the interaction of SLP-76 with PLCγ1 and PLCγ2 via the proline-rich central region of SLP-76. PLC localization is further facilitated by the interaction of these enzymes with phospho-LAT. The C-terminal SH2 domain of SLP-76 also recruits key signaling molecules, including the serine/threonine kinase HPK-1. Recent data suggest that HPK-1 can phosphorylate SLP-76 on serine residues . The SLPin vivo model systems. Early studies defined the binding partners of LAT and SLP-76, and then mapped residues key for these interactions using cell lines lacking either molecule followed by transfection of wild type or mutant versions of LAT  by transfection of cDNAs. For both J14 and J.CaM2, transfection of cDNA encoding a wild type version of the missing molecule completely restored signaling mediated via the TCR. Subsequent studies demonstrated that mutants of SLP-76 or LAT that could not bind to Gads failed to support TCR-mediated activation of the PLC signaling cascade ,41-43. Cin vivo importance of the SLP-76/Gads/LAT axis was then demonstrated through the use of transgenic animals expressing SLP-76 mutants on the SLP-76-deficient background and through retroviral transduction of SLP-76 null bone marrow with wild type or mutant variants of SLP-76 followed by transplant into Rag-/- recipients , thus making immune cells better able to respond to integrin activation . SeveralADAP becomes tyrosine phosphorylated following immunoreceptor engagement by Fyn, one of the Src family PTKs that is inducibly activated upon TCR engagement . In factAfter the discovery of ADAP and SKAP55, a series of studies were initiated to probe their importance in immune cell signaling. Several groups found that cells lacking these adapter proteins failed to adhere tightly to integrin coated surfaces -55. Thisin vivo structure/function studies of SLP-76 making use of mice transgenic for mutant forms of SLP-76 crossed to SLP-76-deficient animals demonstrated that among the three SLP-76 protein interacting domains, the N-terminal tyrosines were most critical for function  are severely compromised in these mice, sufficient signaling is supported by the SLP-76 mutants to prevent the vascular abnormalities from occurring. This observation provides an important clue that is being pursued to understand what cell type and which receptor on that cell requires SLP-76 to support normal vascular development.Given the poor signaling via the TCR in the Jurkat system, we were surprised to discover that gross thymic development in the Y145F and Y112/128F mice was remarkably intact in terms of overall cellularity and examination of subsets by CD4/CD8 staining. It was evident, however, through staining for CD5 and surface TCR levels that positive selection was likely aberrant. This finding was extended by evaluating TCR Vβ family usage with the observation that in strains that should have eliminated particular TCR families (due to endogenous superantigen expression), deletion was defective (worse in the Y145F compared to the Y112/128F mice). Thus, it appeared that both positive and negative selection may be impacted by altering the SLP-76 tyrosines. This was confirmed by breeding the knockin mice to animals expressing TCR transgenes that should either signal deletion or positive selection. In both circumstances we found substantial defects in selection, again with the Y145F mice demonstrating a more pronounced phenotype than that observed in the Y112/128F animals. Aberrant selection in the thymi of these mice correlated with failed TCR signaling as expected from the studies of the reconstituted J14 mice.trans in a single cell. To approach this question, we bred the Y145F mice to mice expressing Y112/128F SLP-76 resulting in animals with no wild type SLP-76 alleles but instead one Y145F allele and one Y112/128F encoding chromosome. We were surprised that in every assay of TCR function or thymic development, we found a rescue to nearly wild type levels. Thus, it appears that the two SLP-76 mutants can cooperate with each other when expressed in the same cell.Generating knockin mice with complementary tyrosine mutations gave us the opportunity to test the hypothesis that two SLP-76 mutants, each conferring a significant phenotype, could complement each other in in vivo platelet function. For these studies, we applied an irritant (ferric chloride) to the external carotid artery of mice and then measured blood flow in the artery by Doppler. In wild type mice, the irritated artery stimulated a rapid platelet response resulting in occlusion of the vessel by a stable thrombus. The vast majority of SLP-76 Y145F mice failed entirely to occlude the vessel, and in all but one mouse in which a thrombus formed, the clot that did occur was unstable. The phenotype of the Y112/128F mice tended towards defective thrombus formation but was not nearly as pronounced as seen in the Y145F animals. Based on our studies in T cells, we predicted that the two tyrosine mutant alleles would complement each other in trans in the platelet compartment. We were surprised to discover that this was not the case, an observation that we are continuing to pursue.The knockin mice gave us the opportunity also to investigate the importance of the SLP-76 tyrosines in other lineages. Similar to what we discovered in the T cell compartment, platelets expressing either the Y145F or Y112/128F SLP-76 mutants revealed marked signaling abnormalities downstream of the GPVI receptor . Interesin vitro and in vivo. Signals initiated by the binding of immunoreceptors and integrins results in the assembly of large multimolecular complexes that are critical for integration of the myriad of second messenger cascades. These complexes are nucleated by key adapter proteins. Interestingly, while some of the rules for the generation and function of these complexes are the same, the molecular details of how they act appear to differ between cell types and receptor systems. Additional information about how these complexes regulate key biologic outcomes will provide useful clues as attempts are made to manipulate signaling events for therapeutic benefit.Recent years have seen an explosion of new knowledge informing the scientific community regarding the process by which signals are transmitted from engaged receptors on the surface of cells. Immune cells have proven to be an outstanding model system, as they are easily accessible and can be manipulated both