The remaining are porines or transporters (20%), metabolic enzymes (16%), activities related to protein translation (5%) and orfs of uncharacterised function (38%). Amongst the later, we have selected the operon PP2810-13 to make an exhaustive analysis of the CbrB binding sequences, together with those of crcZ and crcY. We describe the implication of three independent non-palindromic subsites with a variable spacing in three different targets; CrcZ, CrcY and operon PP2810-13 in the CbrAB activation. CbrB is a quite peculiar σN—dependent activator since it is barely dependent on phosphorylation for transcriptional activation. With the depiction of the precise contacts of CbrB with the DNA, the analysis of the multimerisation status and its dependence on other factors such as RpoN o IHF, we propose a model of transcriptional activation.CbrAB is a high ranked global regulatory system exclusive of the Pseudomonads that responds to carbon limiting conditions. It has become necessary to define the particular regulon of CbrB and discriminate it from the downstream cascades through other regulatory components. We have performed  The ability to recognize and convert external environmental stimuli into appropriate physiological responses is of fundamental importance for all organisms. Several distinct global regulatory networks can participate in co-ordinating gene expression programmes under different situations. In bacteria, signal transduction is predominantly mediated by two-component regulatory systems (TCSs) consisting of a sensor kinase (SK) and a cognate response regulator (RR), which are the principal tools by which bacteria sense and respond to different external signals .Pseudomonas are ubiquitous and very versatile, capable of adapting to very different habitats due to a complex regulatory network that ensures the appropriate responses to each context. The control on cellular carbon-nitrogen balance and determination of the order in which growth substrates are degraded is, in particular, mediated by the TCS CbrAB, which is exclusive of the Pseudomonaceae and found in the genera Pseudomonas . The. ThecrcZcrcZ expression . In contrast, it was subsite F1 (TGTTAC to gactct) the subsite that most affected crcY expression , whilst it was R1 (GTAACA to agcctc) the most important for PP2810 expression . Substitution of subsites R1 or R’ of PcrcY had no effect on crcY expression, and R2 had a milder effect than F1  resulted in an inability of CbrB to bind to the DNA fragment, although they retained partial activity for transcriptional activation with two conserved subsites  did not cause any obvious alteration.Substitution of any of the subsites F1, R1 or R2 for Psubsites . This facrcY and PP2810 had a parallel effect on both expression and binding to the DNA. On the other hand, binding of CbrB to PcrcZ is somehow more sensitive, and alteration of any of the three subsites prevented CbrB binding. However, due to the high efficiency in transcriptional activation from the crcZ promoter, the effects on transcription of the altered F1 and R1 sequences are only evident under conditions of lowest CbrB activity. Altogether, these results suggest that a clear preferential binding subsite for CbrB cannot be defined but rather, there is an accumulative effect of each one, and their relative contribution depend on the target region.Interestingly, alteration of the putative CbrB-binding subsites in the targets PP2810 was examined by DNase I footprinting analysis. The direct interaction of CbrB with the promoter region revealed some protected areas and also regions of hypersensitivity to DNAse I digestion, thus suggesting distortion of the DNA in this area upon CbrB binding  of 3.76 S. The slight asymmetry seen to the right of the peak indicates the existence of certain aggregation of the sample . The molin score  of 146, crcZ, crcY and PP2810) are not palindromic, and CbrB is mainly detected as a monomer in solution, we aimed to determine if in the presence of a DNA containing the CbrB binding sites or the phosphorylation state of CbrB could promote the multimerization of the protein. Addition of a 72 bp DNA fragment containing the subsites F1, R1 and R2 of the promoter region of crcZ to the CbrB sample in the size exclusion chromatography did not change this pattern. Similarly, CbrB treatment with acetyl phosphate in an attempt to phosphorylate it neither changed this pattern  of new target genes coding for proteins. The fact that an important number of regulatory and signal transduction proteins are among the potential targets for CbrB regulation (20% of the targets were involved in regulatory processes), indicates that the CbrB regulon could be very large. On the other hand, 38% of the CbrB targets were annotated as hypothetical proteins that limited the interpretation of its CbrB-mediated control. One of these targets, operon PP2810-PP2013, has structural homology to an RND- type efflux pump that is activated in situations of carbon limitation according to our results. Its promoter arrangement is coherent with the disposition of the CbrB, IHF and σN consensus binding sites found in the crcZ and crcY promoters, and the fact that it is a target of different nature than non-translated regulatory RNAs, led us to use it for its dissection in this work.While the CbrAB two-component system has been widely described in the assimilation of different amino acids as carbon sources , 12, howN-dependent promoters. We are aware that many targets with less recognizable σN sites could be overlooked. In this sense, it has been reported that poor σN sequences are tightly regulated by Enhancer Binding Proteins (EBPs) when IHF contributes to adopt the appropriate topology for the formation of the initiation complex [We have identified 61 targets from the ChIP-Seq analysis that were selected under very restrictive conditions with the purpose to determine a DNA binding sequence when CbrB acts as transcriptional activator of σ complex , 24. On N promoters have been reported to occasionally act as repressors of the transcription of some genes with σ70 promoter sequences [N-dependent promoters were included. Nevertheless, three of the target genes validated by quantitative RT-PCR resulted to be repressed . Although sequences for CbrB binding were identified in the DNA fragment obtained from the ChIP-Seq analysis, its disposition with respect to the putative σN promoter was incoherent, so our interpretation is that their transcription is not dependent on this sigma factor.Also, some EBPs of this family such as NtrC or FleQ, which usually activate transcription of σequences –27. ThisBioinformatics analysis using MEME or FIMO   on the p10-ACA site [Also, two of the targets from the analysis resulted to be not CbrB -regulated, although PP3009 even contained a potential CbrB binding site. This might mean that CbrB, as activators having a short recognition sequence such as NifA that recognises a single TGT-NACA site , could bin silico prediction together with the mutational analysis in the CbrB subsites that reveal the different relevance of each one according to the context, suggests that CbrB may bind to the DNA in a relatively relaxed (variable) manner. The participation of IHF may contribute to the contextualization to favor CbrB activation in sites with different affinities.The lack of a restricted consensus sequence for CbrB binding to make an N-dependent promoters probably to facilitate oligomerization of contiguously bound dimers, an absolute dependency on more than one target site is unusual, and in some cases, such a NifA, a single palindromic sequence is found in its binding sites. Intriguingly, this is the case of NorR where three inactive NorR dimers bind to three adjacent UAS, resulting in an increased local concentration of NorR thus facilitating oligomerisation upon activation of the regulatory GAF domain[in vitro suggest that it does not bind as a dimer. Rather it appears to initially bind as a monomer to each one of the sites. Although there is no evidence of oligomerisation of CbrB in solution, the formation of structures of higher order is the most rational prediction to stabilise the complex in vivo. Each particular binding site has an accumulative effect upon CbrB binding, although neither of the three is essential for transcriptional activation. Actually, one of the subsites is almost dispensable at the crcY and PP2810 promoter regions; only at crcZ, where there is a higher conservation of the sites and higher level of expression, a greater participation of each UAS is observed, although there is still a substantial activation after mutating any of the subsites. As shown for PP2810, even though the subsites at the promoter region are less conserved, the proper spacing of the UAS is preserved and its expression is tightly regulated  as the nitrogen source. Luria–Bertani (LB) was used as a rich medium[Pseudomonas or E. coli strains, respectively. When required, antibiotics and other additives were used at the following concentrations (μg ml−1): ampicillin (Ap) 100; carbenicillin (Cb) 500; kanamycin (Km) 25; rifampicin (Rf) 20 and 5-bromo- 4-chloro-3-indoyl-β-D-galactopyranoside  25. All reagents were purchased from Sigma-Aldrich.The bacterial strains, plasmids and oligonucleotides used in this work are summarized in ch medium. CultureE. coli DH5α was used as a host in all cloning procedures. Plasmid DNA was transferred to E. coli and P. putida strains by transformation[All DNA manipulations were made using standard protocols. Plasmidformation or electformation, respectPP2810 promoter region to lacZ were constructed by PCR amplification of P. putida KT2442 genomic DNA with oligonucleotides PP2810F/PP2810R and PP2810fwmut/PP2810R cloned into the EcoRI- and BamHI-digested vector pMPO234 to give plasmids pMPO420 and pMPO422 respectively. The substitutions of the wild type sequences of F1, R1 and R2 for CbrB binding subsites were generated by PCR extension overlapping fragments as in [P. putida KT2442 genomic DNA as the template and the mutagenic oligonucleotides PP2810mutIF/ PP2810mutIR for F1, PP2810mutIIF/ PP2810mutIIR for R1 and PP2810mutIIIF/ PP2810mutIIIR for R2, with the external nonmutagenic oligonucleotides PP2810R and PP2810fwmut. The final PCR products were cloned as EcoRI/BamHI in pMPO234 to yield pMPO425, pMPO426 and pMPO428, respectively.Plasmid pMPO389 was generated by PCR amplification of genomic DNA using the pair of oligonucleotides crcYSmaI/ crcYBamHI into a BamHI-SmaI digested pUC18Sfi vector. The transcriptional fusions of ts as in  using P.lacZ containing the substituted F1, R1 and R2 CbrB binding subsites for the promoter regions of crcZ were constructed using pMPO1316 as template and the mutagenic oligonucleotides Zmut1F/Zmut1R, Zmut2F/Zmut2R, Zmut3F/Zmut3R, respectively with the external non-mutagenic oligonucleotides ZbisBam/ fuscrcZFEco. The final PCR products were cloned as an EcoRI/BamHI fragment into pMPO234 to yield pMPO436, pMPO437 and pMPO438, respectively. In the case of the promoter region of crcY, plasmid pMPO1314 was used as the template and the mutagenic oligonucleotides Ymut1F/ Ymut1R were used to substitute F1, Ymut2F/ Ymut2R for R1, Ymut3F/ Ymut3R for R’ and Ymut4F/ Ymut4R for R2 site. The external non-mutagenic oligonucleotides to generate the overlapping mutated fragment were FusioncrcYRbisBam and crcYSmaI for the fragment bearing the mutated F1 and FusioncrcYRbisBam and FusioncrcYF for the fragments containing the mutated R1, R' and R2. After EcoRI/BamHI directed cloning into pMPO234, plasmids pMPO439, pMPO440, pMPO441 and pMPO442 were generated, respectively. Plasmids pMP1342 and pMPO1343 were constructed by PCR amplification of genomic DNA using the pair of oligonucleotides foot2810smaF+/ foot2810XbaR+ and foot2810XbaF-/ foot2810smaR- for the top and bottom strands of the DNA respectively, and the fragments were cloned into a XbaI–SmaI digested pUC18Sfi vector.The transcriptional fusions to crcZ, PcrcY and PPP2810 transcriptional fusions to lacZ and their mutagenized versions in P. putida KT2442 and MPO401 under different growth conditions. Preinocula of bacterial strains harbouring the relevant plasmids were grown to saturation in LB and minimal medium with succinate 20 mM as carbon source. Cells were then diluted to 0.05 of A600 in LB, to 0.1 of A600 in a minimal medium with succinate and to 0.3 of A600 in a minimal medium with oxaloacetate and shaken until they reached the mid-exponential phase (A600 = 0.25–0.5). β-galactosidase activity was determined from SDS- and chloroform-permeabilised cells as previously described[E. coli were transferred by conjugation to P. putida in the presence of the helper vector pRK2013.Steady-state β-galactosidase assays were used to examine the expression of the P-1. CbrB samples were incubated in presence/absence of 4.5 nM 80pb-DNA fragment containing the crcZ promoter region and/or 80 mM acetyl phosphate as in [CbrB was overexpressed and purified as previously described . CbrB Mote as in  where neSedimentation velocity experiments were carried out at 20 °C in an Optima XL-A analytical ultracentrifuge (Beckman Coulter) at Instituto de Química-Física Rocasolano, CSIC, Madrid. Measurements were performed in CbrB binding buffer at 45,000 rpm using a protein concentration of 60 μm. Differential sedimentation coefficients c(s) were calculated by least squares boundary modelling of sedimentation velocity profiles using the program sedfit .crcZ and PcrcY promoter regions  for crcZ and crcY or CbrB binding buffer  for the others reactions. The reactions were performed as in [crcZ and crcY and 1.5 nM for the remaining promoters), 100 ng μl−1 salmon sperm DNA, 250 ng μl−1 BSA and increasing amounts of purified protein CbrB  using an oligonucleotide specific for the labelled strand in each case (sec2810cad- and sec2810cad + for the top and bottom strands of PPP2810) and was run with the partially digested DNA used as a size marker.CbrB DNase I footprinting was performed essentially as described. The DNAP. putida KT2442 or its derivative MPO401 was cultivated in LB or minimal medium with succinate as carbon source. The saturated preinocula were diluted to A600 = 0.05 in LB, and 0.1 in a minimal medium with succinate from saturated cultures in the same medium and grown to mid-exponential phase. Since the strains did not grow with OAA as carbon source, the cultures were inoculated at A600 = 0,3 and were induced in the medium for 4 h, then collected and frozen at -80°C. RNA extraction was prepared as described [crcZ, QflgBF/ QflgBR for PP4391, QPP0952F/ QPP0952R for PP0952, QPP1206F/ QPP1206R for PP1206, QPP3009/ PP3009_for PP3009, QPP3074F/ QPP3074R for PP3074, QPP3420F/ QPP3420R for PP3420, QPP3656F/ QPP3656R for PP3656, QPP4050F/ QPP4050R for PP4050, QPP4486F/ QPP4486R for PP4486 and QPP4643F/ QPP4643R for PP4643) as previously described [escribed . QuantitP. putida KT2442 were independently grown up to mid-exponential phase in a minimal medium containing oxaloacetate as carbon source from pre-culture grown on succinate at 30°C. 1/10 volume of fresh crosslinking solution  was added and incubated at room temperature for 15 min. The reaction was stopped by the addition of 1/20 volume of 2.5 M glycine, and the cells were swirled briefly and incubated at room temperature for 5 min. Cells were rinsed twice with ice cold PBS, and collected at 2000 rcf for 10 min and frozen at -80°C. The pellet was resuspended in 10 ml of Lysis Buffer , and sonicated with a Bioruptor® UCD-200 (Diagenode) in an ice-water bath in 6 cycles of 30 sec on/off for 15 min, until DNA was sheared up to 200–300 bp fragments. For CbrB-DNA bound immunoprecipitation, 80 mg of CbrB antibodies coupled to Protein A Dynabeads (Invitrogen) in washing buffer  following the manufactures’ indications. The complex was incubated overnight at 4°C at a rotatory shaker for specific binding. The antibodies were removed by immobilization of the magnetic beads and after washing twice, the supernatant was removed for antigen elution by denaturation. The extract was resuspended in 40 μL of elution buffer  and incubated at 65°C for 15 min. Then, the supernatant was incubated for 30 min with RNaseA (50ug/mL) at 37°C and de-proteinised with proteinase K (0.4 mg/mL), incubated for 2 hours at 37°C and further inactivated at 55°C for two hours. DNA was purified with the QIAquick PCR Purification Kit , the sample was eluted in 100 μl of mQ H2O and stored at -20°C.Three biological replicates of 250 ml batch cultures of P. putida KT2440 using TMAP 4.4.23–1 with Torrent Suite (version 4.4.3) from PGM. Data were uploaded to the Galaxy Web platform to use the public server at usegalaxy.org [Sequencing libraries were prepared using the Ion ChIP-Seq library preparation kit for PGM (Thermo Fisher Scientific Inc). Ten ng of IP DNA was used as input. Bi-directional 200 bases sequencing was perform using a PGM Ion-Torrent system in Genomic´ s Core Facility at Cabimer. Two technical replicates were sequenced and quality was verified by a FastQC analysis (Babraham Bioinformatics). Reads were aligned on laxy.org  and BAM S1 FigIn cyan the total peaks after peak calling, in green the peaks with a p-value<2, in red the selected DNA fragments after the σN filtration used for the search of a CbrB binding consensus sequence. Bam files representing the reads are shown below.(TIF)Click here for additional data file.S2 FigLinear dsDNA fragments containing the promoter regions of PP4391, PP3074, PP4050 in the presence increasing amounts of CbrB (indicated on top) were used. In parenthesis the coordinates of the DNA fragments used for the EMSA referred to the ATG.(TIF)Click here for additional data file.S3 FigcrcZ (A) and crcY::lacZ (B) transcriptional fusions  of cultures of the KT2442 wild-type and the MPO401 (ΔcbrB) mutant strains, grown in LB (white bars) or in minimal medium containing succinate (black) or oxaloacetate (grey bars) as carbon sources. The values are the average of at least three independent assays. The error bars indicate the standard deviation of the means. Stars designate p-values for the Student's t-test for unpaired samples not assuming equal variance and are referred to the wild type strain. *: p<0.05; **: p<0.01; ***:p<0.005.Expression was measured as β-galactosidase activity of the (TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.


Guanine-quadruplex (G4) motifs, at both the DNA and RNA levels, have assumed an important place in our understanding of the biology of eukaryotes, bacteria and viruses. However, it is generally little known that their very first description, as well as the foundational work on G4s, was performed on protozoans: unicellular life forms that are often parasitic. In this review, we provide a historical perspective on the discovery of G4s, intertwined with their biological significance across the protozoan kingdom. This is a history in three parts: first, a period of discovery including the first characterisation of a G4 motif at the DNA level in ciliates ; second, a period less dense in publications concerning protozoa, during which DNA G4s were discovered in both humans and viruses; and third, a period of renewed interest in protozoa, including more mechanistic work in ciliates but also in pathogenic protozoa. This last period has opened an exciting prospect of finding new anti-parasitic drugs to interfere with parasite biology, thus adding new compounds to the therapeutic arsenal.  Traditionally DNA is viewed by the public, and indeed by the vast majority of scientists, as a double helical structure composed of four nucleotides, pairing adenine-thymine (A-T) and cytosine-guanine (C-G) , as obse(>2)NxG(>2)NxG(>2)NxG(>2) [+, or to a lesser extent Na+, and destabilised in the presence of Li+ [The G4 motif is a family of structurally-diverse four-stranded secondary structures composed of stacks of planar guanine tetrads connected by intervening loop sequences following the general sequence G)NxG(>2)   the multiplicity of their genomic fragments , and  there is no trace of staining of the replication band, indicating that, in ciliates, G4s are resolved during replication, and (2) that G4 motifs were only present in the macronucleus of Stylonychia and not in the micronucleus, suggesting a difference of DNA organisation between the two nuclei [Stylonychia has two nuclei assuring two different biological functions. The macronucleus is the centre of the metabolic activity and undergoes drastic remodelling during the reproductive life cycle, while the micronucleus codes for the germline genetic material [For more than ten years 1995 to 2006), only a few papers were published on G4s in protozoans. However, after the first period of description and characterisation, a series of articles began to appear related to the biological relevance of G4s, and some of these again took advantage of ciliate protozoa. First, some G4 structure-specific antibodies were produced: an important new research tool for the field. Using another o nuclei . This obmaterial . It is n006, onlyS. lemnae. They reported the essentiality of the two Telomere End Binding Proteins, TEBPa and TEBPb, for the formation of G4s in vitro and in vivo, and further demonstrated that TEBPb phosphorylation was necessary for G4 formation [Tetrahymena and Euplotes. In a primer extension assay using Na+-stabilised intermolecular G4s and telomerase from both organisms, they described the intermolecular G4 as being an excellent substrate for the enzyme, suggesting that, at least in vitro, telomere extension is mediated by the presence of the G4 motifs [A few years later, using the Sty49 antibody, Paeschke et al. published the first functional study on the regulatory role of telomeric G4s in ormation . Meanwhi4 motifs .Oxytricha and Tetrahymena sequences, changing the stabilising cations in G4s in crystal form or in solution [Oxytricha nova TEBP and Na+-stabilised G4 telomeric sequence [Secondly during this period, more structural work was performed using NMR and crystallography on solution ,29,30,31sequence . Thus, aThe third period of the history between G4s and protozoa saw the research expand to pathogenic protozoa as well. This period can be split into two different paths. First, the characterisation of the motif and its various biological functions was further pursued in ciliates and then extended to eukaryotic pathogens. Second, G4s began to be pursued as a potential target for anti-parasitic drugs.Oxytrichia T4G4 telomere repeat with the addition of locked nucleic acids, which are analogues of nucleic acids with a locked ribose in C3′-endo, forbidding a syn or anti conformation depending on the pre-existing chemical arrangement [Oxytrichia or elsewhere. In fact, the G4 research community remains preoccupied to this day with the question of whether all motifs observed in vitro can actually exist in vivo as well.This period opened with the discovery of a previously-undescribed form of G4: the V4-motif introduced by Nielsen et al. To achieve this, they used the angement . The aut4G4 repeat was clearly an ideal model. First, Abu-Ghazalah et al. in 2009 looked at the composition of the G-track and the T-loop of Oxytricha G4s. Using oligonucleotide sequences of Oxytricha telomere repeats with the addition or deletion of a G in the G-track or a T in the T-loop, they showed that stability correlates with the number of guanine residues. They then demonstrated an effect of the T-loop length on the global conformation. Lastly, they ran stability experiments with all residues of the T-loop replaced by adenosine, showing different behaviours of G4s with T-loops versus A-loops [Tetrahymena by changing the first guanine of the G-track to an adenine (T2G4 -> T2AG3). They showed that the substitution does not impair G4 formation but may change the topology of the G4 and also reduce its stability [Secondly, the importance of sequence diversity within the G4 motif itself, which had thus far been largely neglected, was now studied, with ciliate telomeres again being the model sequences of choice. For this work, the highly homogenous Ttability . This woOxytricha and Tetrahymena restructure their somatic genome during sexual differentiation and during this process up to a third of the genetic material is excised and lost. The lost fragments, also called internal eliminated sequences (IES), have the particularity to be outside of the telomeric regions and flanked by cis-acting sequences, one of the most studied being the polypurine tract (A5G5) [5G5 is a G4 forming sequence, Carle et al. demonstrated that the protein Lia3 was binding to parallel G4s, as well as ensuring DNA cleavage. When the LIA3 gene was knocked out, excision of the IES was prevented and the presence of the G4 was also essential for the binding of Lia3 [Indeed, the second conclusion of the work done by Abu-Ghazalah et al. opened the perspective to study diverse, non-telomeric G4 motifs, which also occur in ciliate genomes. Ciliates like t (A5G5) ,35,36. W of Lia3 . Thus, GLeishmania parasites are G-C-biased.Using bioinformatic tools, it was reported in 2007 that 40% of human gene promoters are carrying a Putative G-Quadruplex Sequence (PQS), and that PQSs appear on average approximately every kilobase in this genome . This, tPlasmodium falciparum, which is extremely A-T-biased. They identified only 63 PQSs in non-telomeric sequences and, interestingly, these were concentrated in the upstream regions of a subset of the var virulence genes (the ‘group B’ var genes). This gene family encodes the major variant antigen of this parasite, P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1), which is expressed at the surface of the infected erythrocytes. Each parasite genome has ~60 var variants; their expression is mutually exclusive and is regulated by epigenetic silencing and switching [var B upstream sequence, leading to a new hypothesis that var gene expression might be regulated via structure-specific helicases that can target G4s [P. falciparum, called PfBLM [var gene expression, but curiously Claessens et al. found that var gene expression increased in their knockout, whereas Li et al. found that expression of the gene family was completely shut down. The reason for this difference remains, at present, unclear.In 2009, Smargiasso et al. published the first bioinformatic PQS screen of the genome of the malaria parasite witching . The autrget G4s . Two reced PfBLM ,42. BothP. falciparum parasites to continually evade the immune system, antigenic switching amongst a ~60-gene family is not enough: var genes must also recombine to create new variants [var gene expression. Stanton et al. found 80 PQSs in an updated version of the P. falciparum reference genome and reported a strong spatial association between PQSs and var gene recombination events. Furthermore, it was again suggested that G4-targetting DNA helicases could regulate this process in the parasite [P. falciparum RecQ helicase in var gene recombination. This time, the PfWRN helicase was knocked down, and recombination events in the var gene family dramatically increased  [P. falciparum parasite has evidently evolved to retain G4-forming sequences in a highly A-T-rich genome for a very specific purpose: regulating both the expression and the evolution of a key virulence gene family.In order for variants . In 2016parasite . Indeed, effect) . OverallPlasmodium. They included 2-quartet as well as 3-quartet motifs and therefore described a higher number of PQSs ). Concentrating on these potential rG4s, they then re-analysed various RNA-seq datasets and ribosome footprinting studies and concluded that translation efficiency was reduced at certain lifecycle stages in PQS-harbouring genes [P. falciparum, and the first to suggest a G4-based expression-regulating system in a protozoan.Finally, in 2016, Bhartiya et al. performed another bioinformatic G4 screening on 6 different species of ng genes . A mechaP. falciparum has the most A-T-rich genome yet sequenced, but other protozoa do exist with similarly biased genomes. One such organism is the environmental Mycetozoa Distyostelium discoideum. Bedrat et al. recently developed a bioinformatic program, G4Hunter, which uses a new and more sophisticated algorithm to detect the presence of G4 motifs based on genome sequences: they included two A-T-rich protozoa in their analysis, D. discoideum and P. falciparum. The number of predicted G4s from this new algorithm remained low, depending on the parameters: between 249 and 1055 for D. discoideum , but the authors hypothesised that the few PQSs retained in the D. discoideum genome could be informative to study [5G5) located in a putative promoter of two divergent D. discoideum genes [to study . Therefoum genes . A bioloP. serpens kDNA [Trypanosomatid family. However, interest was raised again in Trypanosomatidae with the study of human pathogens such as Trypanosoma brucei, two subspecies of which cause human sleeping sickness; Trypanosoma cruzi, causing Chagas disease; and Leishmania spp, responsible for the complex of diseases called leishmaniasis [L. tarentolae, revealing DNA sequence together with epigenetic base modifications. They demonstrated the presence of PQSs surrounding regions of β-d-glucosyl-hydroxymethyluracil, or ‘base J’, a Trypanosomatid-specific epigenetic modification of thymine. The Trypanosomatidae group of protozoa is characterised by unusual poly-cistronic transcription: base J is a marker of transcriptional strand-switching between poly-cistrons and it is also present in abundance in the telomeres (99%). They verified their discovery using a plasmid carrying the detected PQS, (GGGTTA)10, and found that even plasmids carrying only this sequence without any coding sequence were modified to harbour base J. This result suggests that the presence of a G4 is a hallmark for base J positioning [After the demonstration of G4 formation in ens kDNA , the fiemaniasis . It was itioning . Again, T. cruzi and L. tarentolae, they made the same conclusion as previously, and finally hypothesised that kDNA replication versus kDNA transcription is directly linked to the presence of intra-molecular G4 structures in the pre-kRNA. When the rG4 is formed on the neo-synthetised pre-kRNA, it cannot recognise PQSs on the maxicircle, leading to active transcription and inactive maxicircle replication. On the contrary, when the rG4 in the pre-kRNA molecule is unwound, it allows the formation of a kDNA/kRNA hybrid, preventing transcription and activating kDNA replication [Trypanosomatidae biology.Regarding the same family of parasites, and after the discovery of G4 formation at the RNA level in humans , Leeder lication . Yet agaP. falciparum with var gene recombination, or they regulate gene expression, as in Trypanosomatids with the placement of base J. Furthermore, the first demonstration of G4 presence at the RNA level—now reported in T. brucei—gives a role to the rG4 in regulating transcript editing. Regarding the pathogenic species, most of these mechanisms are essential for parasite survival and this makes G4s potential targets for anti-parasitic drugs.Overall, G4 motifs have been shown to play several different roles in the biology of protozoa. They either organise the genome, as in ciliates with germline formation or in The second path of G4-related research that remains very active today is represented by the discovery of new anti-protozoan drugs to fight pathogenic species. In the era of drug-resistant pathogens, new pharmaceutical compounds are necessary to complete the pharmacopeia. In this regard, non-pathogenic protozoa can be useful as well – before directly working on pathogenic organisms, which require a specialised biosafety level of confinement, ciliates have sometimes been used as models to test anti-G4 drugs . In pathP. falciparum telomeres was made in 2008 by De Cian et al., at the same time as the description of the structure and characteristics of P. falciparum G4s. They showed that known G4-binding compounds, developed primarily for human G4s, also bound to P. falciparum G4s more effectively than to duplex DNA. However, none of the 12 compounds tested showed a particular discrimination between Plasmodium and human G4 structures in vitro [P. falciparum when exposed to two drugs previously tested by De Cian et al., TMPyP4 and telomestatin. While the first showed the highest reduction in telomerase activity, the second showed the highest parasite growth inhibition. It is important to note that G4-targetting was not proven and telomestatin activity could interfere with other pathways inside the parasite [P. falciparum telomere erosion, suggesting a common mode of interference with telomerase activity [The very first report of compounds binding to G4s in in vitro . Neverthparasite . Howeveractivity .T. b. brucei, L. major and P. falciparum. Amongst the seven, two derivatives showed a particular action against T. b. brucei and a limited toxicity in human cells, making them potential drug candidates against the T. brucei subspecies responsible for sleeping sickness [P. falciparum, Harris et al. demonstrated G4-related anti-parasitic activity of a candidate anticancer drug, a fluoroquinolone called quarfloxin. Quarfloxin showed rapid killing activity against the erythrocytic forms of the malaria parasite in culture and could represent a good drug for repurposing against malaria. Unlike telomestatin, quarfloxin did not apparently cause telomere erosion: it may instead exert its toxicity via genes encoding non-telomeric G4s [While the first compound screens focused entirely on molecules capable of inhibiting telomerase activity, others focused on molecules capable of interfering with the G4s scattered across the genome and regulating different biological functions, as described earlier in this review. Belmonte-Reche et al. recently published a screen of seven carboxy-NDI derivatives against sickness . Focusineric G4s .As can be observed throughout this review, the history of G4 discovery is intertwined with protozoan biology, from the understanding of genome organisation to drug development. Looking at the large diversity of biological systems that represent the protozoan phylum, it is safe to assume that we still have a lot to learn from these small but far-from-simple organisms. Furthermore, the presence of G4s within the telomeres, regulatory elements and coding regions of many pathogenic protozoa makes them a structure of choice to develop new compounds or repurpose or modify existing ones, thus opening new perspectives for the treatment of major parasitic diseases.


Retinal neurogenesis occurs through partially overlapping temporal windows, driven by concerted actions of transcription factors which, in turn, may contribute to the establishment of divergent genetic programs in the developing retina by coordinating variations in chromatin landscapes. Here we comprehensively profile murine retinal progenitors by integrating next generation sequencing methods and interrogate changes in chromatin accessibility at embryonic and post-natal stages. An unbiased search for motifs in open chromatin regions identifies putative factors involved in the developmental progression of the epigenome in retinal progenitor cells. Among these factors, the transcription factor LHX2 exhibits a developmentally regulated cis-regulatory repertoire and stage-dependent motif instances. Using loss-of-function assays, we determine LHX2 coordinates variations in chromatin accessibility, by competition for nucleosome occupancy and secondary regulation of candidate pioneer factors. Cristina Zibetti et al. present ATAC-seq, RNA-seq and ChIP-seq analyses of murine retinal progenitors at early and late developmental stages. They find a central role for the LIM homeodomain transcription factor LHX2 in the global control of chromatin accessibility and identify Lhx2-dependent candidate pioneer factors that may contribute to the establishment and maintenance of open chromatin in the retina. A subset of transcription factors, commonly referred to as pioneer factors, occupy compacted chromatin regions to initiate or mediate chromatin opening, enabling nucleosomal accessibility to the transcriptional apparatus and instructing recruitment of accessory factors to reprogram cellular identity.Retinal neurogenesis occurs through partially overlapping temporal windows giving rise to individual cell types, as result of temporal patterningEven though the expression patterns have been extensively characterized for most of the retinal transcription factors, the mechanisms by which these coordinate epigenetic changes across retinal development remain elusive, as they are limited to select cases and often perturbed by experimental biases intrinsic to cell cultures systems and specific genetic background.7. While Lhx2 mutants are anophthalmic, display hypoplasia of the neocortex and severe anemia, neuroretina specific loss-of-function of Lhx2 causes severe microphthalmia, loss of expression of a subset of retinal progenitor cells (RPC)-specific genes and ectopic expression of hypothalamic genes8.The LIM homeodomain transcription factor LHX2 has been characterized in a broad range of developmental contexts and it is required for the eye field specification and the morphogenesis of the optic cup through cell autonomous regulation of gene expressionLhx2 has been observed as early as embryonic day e8.5, it can be detected in the retinal neuro-epithelium and retinal pigmented epithelium at e10 and ciliary margin by e14, becoming progressively restricted to the inner nuclear layer. In the post natal retina, LHX2 is expressed in mitotic retinal progenitor cells that co-express VSX2 and mKi67. By post natal day P7, LHX2 expression becomes restricted to differentiating Müller glia and is preserved in adult Müller glia, a subset of bipolar and starburst amacrine cells9. Early embryonic deletion of Lhx2 results in proliferative defects of glial committed precursors while early post natal ablation, as well as overexpression9 results in the loss of glial markers and dysmorphic apical structures.Ocular expression of Hes5-mediated Notch signaling effectors10, its function does not seem to be confined to the differentiation of glial precursors: early embryonic ablation of Lhx2 at e12.5 results in the depletion of multipotent retinal progenitors and overproduction of retinal ganglion cells (RGCs), while overproduction of rods is observed three days later. This suggests LHX2 may contribute to the maintenance of embryonic progenitors in a proliferative and multipotent state, by restricting their exit from the cell cycle and allowing for the generation of later cell types within discrete temporal windows11.While temporally controlled conditional knockouts indicate that LHX2 induces and stabilizes the retinal glial fate during first post natal week of development, by sustaining the expression of the Chx10-Cre:eGFP transgenic line12 that allows preferential retention of proliferating retinal progenitors at early and late stages of retinal development. We query the deriving epigenomic and transcriptional profiles obtained from natively extracted retinal progenitor cells for LHX2 representation and function over the course of retinal neurogenesis and we show LHX2 controls chromatin accessibility by modulating expression and co-opting action of developmentally regulated transcription factors with high pioneer potential.To investigate the mechanistic basis by which LHX2 orchestrates diverse developmental programs, we perform flow cytometry sorted analysis of dissociated retinal cells obtained from the neuro-retinal reporter Chx10-Cre:eGFP transgene12 from embryonic day (E)14 and post natal day (P)2 retina, representing early and late stages of retinal progenitors competence, respectively1. The brightest fraction of GFP-positive cells expresses the retinal progenitor-specific markers VSX2 (Chx10), mKi67 and CCND1 and was separated from the dim and GFP-negative -positive cell fractions at E14 and P2 and assessed for reproducibility    Lhx2 cKO conditions  and P2 . Furthermore, LHX2-binding sites were coupled with bimodally distributed H3K27ac profiles at P2, a characteristic of active enhancers. Exons and ncRNA were also found among the enriched categories , where co-localization of transcripts with H3K27ac meta-profiles may potentially underlie the biogenesis of enhancer-templated eRNAs. The LHX2 targets identified in the E14 RPCs open chromatin regions were involved in regulatory RNA pathways, promoter opening and cell cycle checkpoints 17, SoxE (SOX8/9)18, and ASCL119 were found at P2 16. Sox2 is expressed in early and late-stage RPCs and is also a transcriptional target of LHX2 . We then estimated transcription factor-specific chromatin opening indexes from ATAC-Seq developmental time pointsnts Fig. , chromatnts Fig.  in RPCs Chx10Cre;Lhx2lox/lox retinas, the average cut frequency at LHX2 motif center was reduced and in P2 Lhx2lox/lox retinas a minor reduction in the average cut frequency  transcription factors SOX2, SOX8, and SOX9 in the post natal retina has been shown to promote amacrine cells formation in Lhx2 loss-of-function affects gene expression at post natal stages may be, in part, result of LHX2 residual expression in neonatal electroporated cells, where the perdurance of the GFP reporter in progenitors exiting the cell cycle after transfection may contribute to the profiles analyzed. However, differential motif enrichment analysis indicates the post natal retinal transcriptome relies on a broader set of transcriptional networks, which may compensate for Lhx2 ablation, limiting the magnitude of the transcriptional variations. This would be particularly relevant in the post natal retina as opposed to the embryonic knock-out, as the endogenous expression of LHX2 that precedes its experimental ablation at P0, hence a phasic drop in protein expression, may enable a feed-forward mechanism.While LHX2 has a prominent role in the regulation of the transcriptome of early progenitors, a more moderate effect can be seen at post natal stages. The lower extent to which Kip1 and GLUL in Müller Glia is reduced both by overexpression23 and downregulation of Lhx2 in the neonatal retina9, suggesting a balanced amount of LHX2 protein may be necessary to mediate the gliogenic process and a graded transcriptional response of target genes. Transcriptional de-regulation of P27Kip1 and Glul may ultimately result from threshold responses that override the feed-forward regulatory loop controlling Lhx2 expression.Notably, immunostaining for P27 Lhx2 loss-of-function affects genes involved in the cell cycle checkpoints, DNA replication, retinal disease phenotypes and the Notch signaling pathway, consistent with previous findings10. LHX2 has been previously shown to act as a regulator of genes that control cortical neuronal subtype identity by interaction with members of the nucleosome and histone deacetylase remodeling complex, although the mechanistic basis behind its regulatory role had not been investigated24.LHX2 regulatory role likely relies on remodeling of nucleosomes units, variably coupled with active enhancers marks, flanked by active promoter and non-coding regions within modules of coordinately accessible chromatin. As a result, Lhx2 loss-of-function.LHX2 affects both local and global chromatin accessibility by competition for nucleosome occupancy, whereby secondary regulation of chromatin remodeling factors and pioneer factors is likely to concur to the widespread decrease in accessibility, observed upon Lhx2-coordinated variations in local accessibility, paired with epistatic variations in gene expression, as is the case of Sox2, and/or by steric interaction of predicted co-factors, both of which would presumably result in altered transcription factors recruitment and nucleosome remodeling.Secondary regulation of candidate pioneer factors may be achieved transcriptionally, by Lhx2 loss-of-function and/or across development. These genes include Hes5, Neurod4, Meis1, Rax, Dlx2, and Neurod1. Additional transcription factors, whose encoding genes are transcriptionally targeted by LHX2, exhibit high predicted pioneer potential such as ATF1, ATOH7, CREB1, DEAF1, EGR1, ETS2, ETV5, HEY1, HEYL, MYCN, NR2F2, PROX1,REST, SP4, TEAD1, VAX2, VSX2, ZBTB33, and ZFX.Transcriptional dependence of candidate pioneer factors on LHX2 can be exemplified with the following cases, wherein variations in local accessibility at their closest cis-regulatory sites bound by LHX2 co-occur with variations in expression levels upon Lhx2-dependence than would be expected based on peaks association by proximity to the closest promoter region. This may partially reflect the preponderant distribution of LHX2-binding sites at intergenic regions, exerting distal regulation of target genes. On the other hand, the extent to which Lhx2 loss-of-function impacts retinal gene expression in early RPCs, may depend on collateral transcriptional networks evoked by LHX2, whereby the observed variations in gene expression following E14 Lhx2 loss-of-function would be amplified by Lhx2-dependent transcription factors.The E14 retinal transcriptome shows greater Lhx2 loss-of-function at E14. Affinity might be affected at both stages for many of the predicted interactors, as shown in their meta-profiles distribution and mean scores from motif centers. Moreover, nucleosomes depletion was observed at motif center of NF-I, KLF4/9, ASCL1, and HES5, while variations in nucleosomes coverage were detected upon Lhx2 loss-of-function, suggesting LHX2 may be necessary to stabilize candidate pioneer co-factors prior to or during recruitment to target sites.Among the many transcription factors predicted to co-occur with LHX2 ChIP-Seq peaks, some had predicted pioneer potential for which we observed variations in footprints counts upon 20, a process that is dependent on LHX210, Ascl1 plays an essential role in conferring neurogenic competence to late-stage RPCs22. Among additional LHX2 targets, Pax6 and Sox2 instead show broad temporal expression profiles26 and exert distinct functions across development31. Some of the known and candidate pioneer factors that exhibit developmentally regulated nucleosome-binding signatures, like NF-I, SOX2, KLF4, may preserve chromatin in a highly compacted state at embryonic states, while mediating chromatin unfolding later in development.While several of the predicted pioneer transcription factors affected by LHX2, such as HES5, SOX8/9, and NFIA, play important roles in the control of retinal gliogenesisChx10-Cre:eGFP transgene12 from embryonic day (E)14 and post natal day (P)2 retina, representing early and late stages of RPC competence, respectively. Flow-sorted cells were profiled by RNA-Seq and ATAC-Seq, relying on direct in vitro transposition of sequencing adapters into native chromatin. ChIP-Seq was then performed on a select transcription factor candidate, identified in the preliminary screening of the ATAC-Seq-derived open chromatin regions. ChIP-Seq-binding sites for the candidate factor were ultimately integrated and compared with the age-matched RNA-Seq and ATAC-Seq profiles from control and knock-out retinas. For each experimental condition involving a point-source factor and broad regions  a minimum of 20 million uniquely mappable reads or ≥10 million uniquely mappable reads for each biological replicate were collected, according to the ENCODE’s guidelines. For point-source datasets, non-redundant mapped reads were retained for downstream analysis.The experimental pipeline involves the generation and integration of high-throughput sequencing libraries from purified fractions of murine retinal progenitor cells (RPCs). Fluorescence-activated cell sorting (FACS) was adopted to isolate RPCs from mice expressing the RPC-specific Chx10-Cre:GFP mice12 were purchased from the Jackson Laboratories. Retinas were freshly dissected, incubated in a suspension of papain and DNAse for 30 min at 37 °C, inactivated with bovine serum albumin, resuspended in equilibrated Earle’s balanced salt solution and subject to fluorescence-activated cell sorting (98–99% purity) where viability was assessed by propidium iodide exclusion. Cell fractions were collected on poly-d-lysine coated slides, fixed in 4% paraformaldehyde for 10 min, permeabilized in TritonX-100 and stained for CHX10 , GFP , mKi67 , or CCND1 . The brightest fraction, differing of fourfold mean intensity for GFP relative to the dim fraction, was always retained for subsequent processing and hereafter referred to as GFP-positive, RPC-enriched fraction. Lhx2 conditional embryonic knockouts were obtained by crossing Chx10-Cre:GFP with Lhx2lox/lox mice, and harvesting at E1432. Post natal Lhx2 knockouts were generated by electroporation of pCAG-Cre-GFP construct into P0.5 wild-type CD-1 animals or Lhx2lox/lox animals. Retinas were harvested at P2, dissociated, and GFP-positive electroporated cells were isolated by FACS. Overall electroporation efficiency was 2–3%.CD-1 mice of either sex were euthanized at embryonic day 14 (E14) and post natal day 2 (P2) according to Johns Hopkins IACUC-approved protocols. Timed pregnant CD-1 mice were obtained from Charles River Laboratories. Chx10-Cre-GFP + ve and GFP-ve retinal fractions was processed as previously described13. Briefly, chromatin was extracted and processed for Tn5-mediated tagmentation and adapter incorporation, according to the Manufacturer’s protocol  at 37 °C for 30 min. Reduced-cycle amplification was carried out in presence of compatibly indexed sequencing adapters. The quality of the libraries was assessed by fluorometric DNA incorporation-based assay (Thermo Fisher ScientificTM) and automated capillary electrophoresis  and up to four samples per lane were pooled and run as 50 bp paired ends on a HiSeq2500 Illumina sequencer.Chromatin derived from flow-sorted TM) and automated capillary electrophoresis .RNA was processed with Qiagen RNAeasy Mini kit, subject to DNAse digestion, and samples with a minimum RNA integrity number (RIN) of seven were further processed for sequencing. Libraries were prepared using Illumina TruSeq RNA Sample kit  following the manufacturer’s recommended procedure. Briefly, total RNA was denatured at 65 °C for 5 min, cooled on ice, purified and incubated at 80 °C for 2 min. The eluted mRNA was fragmented at 94 °C for 8 min and converted to double stranded cDNA, end repaired, A-tailed, and ligated with indexed adaptors and run on a MiSeq Illumina sequencer. The quality of the libraries was assessed by fluorimetric RNA incorporation-based assay , rabbit anti-H3K27Ac , or the corresponding isotype controls (Abcam); retained on agarose beads (Invitrogen), washed and purified by organic extraction. Amplicons corresponding to the targeted transcription factors and other candidate cis-regulatory regions that exhibit LHX2-binding sites and syntenic unbound regions had been verified by SYBR qRT-PCR (Agilent Technologies) as we previously reported10. Amplicons exhibiting enrichment of anti-LHX2 fractions over isotype control include Six6, Fgf15, Rax, Vsx2, Dct, Car2, NeuroD1, Neurod4, and Neurog2. Libraries were processed according to the manufacturer’s protocol (TruSeq Nano DNA Library Prep Kit). The quality of the libraries was assessed by fluorometric DNA incorporation-based assay (Thermo Fisher) and automated capillary electrophoresis (Agilent Technologies) and libraries  were run on a HiSeq2500 Illumina sequencer.ChIP was performed as described previously34, and differentially expressed genes (DEGs) were identified by Cuffdiffs35, imposing a cutoff q-val = 0.05 for pairwise comparison.RNA-Seq reads were aligned to the mouse transcriptome (mm9 UCSC build) using Tophat236. Uniquely mappable reads from ChIP-Seq were retained for peak calling by MACSs37  q-val cutoff = 1E-02, λ = 1000–10,000 bp unless indicated otherwise).Bowtie2 was used for ChIP-Seq and ATAC-Seq read alignment on the mouse genome (mm9)37. Correlations between open chromatin states identified by pairwise comparison of normalized read counts in P2 and E14 GFP-positive flow-sorted retinal progenitor cells was computed with Jaccard38. The Jaccard index was estimated as the number of peaks that overlap between two peak files, divided by the union of the two files.Open chromatin regions were identified in ATAC-Seq data using MACS240. Annotation was performed by proximity to the closest transcription start site.Footprints and nucleosomes were identified as described previouslyp-val threshold = 0.0001, min FE = 4, FDR = 0.001, max tags per position = 1, normalization to input, or isotype control) and subject to comparison with ATAC-Seq peaks  where co-occurrence was defined by physical overlap, allowing a max distance of 20 bp from peak summits over 3000 bp for confocality. For direct comparison of ChIP-Seq and ATAC-Seq data, high-confidence peaks with the highest differential in accessibility between nucleosomal units and flanking nucleosomes-free, transposon-accessible regions, and a minimum distance of 300 bp were identified from two ATAC-Seq replicates.High-confidence ChIP-Seq peaks were identified from at least two experimental replicates .41 was done with the following parameters  by relative alignment length (rl) divided by the overall alignment length. 43.Hierarchical clustering of probabilistically assigned motifs (Jaspar 2016 non-redundant core vertebrates)44.For ChIP-Seq analysis, binomial probability analysis of regulatory transcription factor motifs was calculated in the overall set of high-confidence peaks . The regions size was empirically determined and motifs were found by cumulative binomial distribution of known position weight matrices assuming a random representation of decamers. Motif finding was performed on the repeats masked mm9 murine genome, optimized for the top enriched 20 putative motifs, randomized and repeated twice to estimate FDR45 was used to scan the genome for candidate binding sites of different transcription factors. We then used BPAC to identify the actual binding sites among these candidate sites40. The number of active binding sites was analyzed at E14 and P2. Genome-wide changes in footprint counts and nucleosome occupancy for individual transcription factor motifs were estimated for all candidate binding sites at E14 and P2. Footprints scores were calculated for LHX2, KLF9/13, NF-I (NFIA/B/X), and SOX2. T-test on mean footprints scores distributed 200 bp from motif centers were calculated for the paired control and Lhx2 cKO conditions.FIMOLhx2 cKO) with q-val (FDR) < 0.05 were retained for downstream analysis, unless otherwise specified. Two-tailed t-test was adopted to compare the average footprints counts for candidate pioneer factors in control and Lhx2 knock-out conditions.Co-occurrence statistics for point-source and broad regions of interest was computed by hypergeometric test with a default minimum overlap of 1 bp, unless otherwise specified. Coverage was adopted as reproducibility metrics for ChIP-Seq and ATAC-Seq experimental replicates (fraction of reads/10 Million non-redundant uniquely mappable reads) and FPKM for RNA-Seq, where correlation was reported by Pearson’s or Spearman’s coefficient. Binomial probability analysis of regulatory transcription factor motifs was applied genome-wide to identify enriched position weight matrices (PWMs) and clustered by average linkage. Fisher’s exact test was adopted to compute gene ontology enrichment and corrected for multiple hypothesis (Bonferroni–Hochberg) and RNA-Seq-derived gene sets from flow-sorted retinal cell fractions and control vs. experimental conditions (Further information on experimental design is available in the Reporting SummarySupplementary InformationDescription of additional supplementary itemsSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4


Numerous genetic risk loci are found to associate with major neuropsychiatric disorders represented by schizophrenia. The pathogenic roles of genetic risk loci in psychiatric diseases are further complicated by the association with cell lineage- and/or developmental stage-specific epigenetic alterations. Besides aberrant assembly and malfunction of neuronal circuitry, an increasing volume of discoveries clearly demonstrate impairment of oligodendroglia and disruption of white matter integrity in psychiatric diseases. Nonetheless, whether and how genetic risk factors and epigenetic dysregulations for neuronal susceptibility may affect oligodendroglia is largely unknown. In this mini-review, we will discuss emerging evidence regarding the functional interplay between genetic risk loci and epigenetic factors, which may underlie compromised oligodendroglia and myelin development in neuropsychiatric disorders. Transcriptional and epigenetic factors are the major aspects affected in oligodendroglia. Moreover, multiple disease susceptibility genes are connected by epigenetically modulated transcriptional and post-transcriptional mechanisms. Oligodendroglia specific complex molecular orchestra may explain how distinct risk factors lead to the common clinical expression of white matter pathology of neuropsychiatric disorders. The highly complex etiology of neuropsychiatric disorders, which is influenced by both genetic predisposition and environmental factors, has been a major challenge in understanding these devastating diseases, like schizophrenia. In recent years, genomic studies have uncovered the complex genetic architecture of psychiatric disorders including thousands of genetic loci . MultiplApart from the genetic component, environmental risk factors, including biological and psychosocial ones, are also involved in mental diseases onset . EnvironDecades of extensive investigations revealed aberrant neuronal circuit assembly and synaptic malfunction of neuropsychiatric diseases, which forms the mechanisms for most current antipsychotics and new therapeutic development . Recent Moreover, in recent years, neuropathological, neuroimaging, and genetic studies clearly revealed developmental defects in oligodendroglia/myelin formation and disrupted white matter integrity in major psychiatric disorders, including schizophrenia, depression, and bipolar disorders . PostmorHowever, several questions need further discussion. How do genetic alterations and epigenetic dysregulations influence oligodendroglia besides neurons? Are there any cell-type-specific or developmental-stage-specific mechanisms in oligodendroglia? Whether and how do multiple risk factors coordinate to play roles in oligodendroglia? In this mini-review, we will try to address these questions based on emerging concepts of genetic and epigenetic findings in oligodendroglia during the pathogenesis of psychiatric disorders.de novo single nucleotide and insertion/deletion variants in schizophrenia , which are crucial for oligodendroglia development and myelination, are robustly dysregulated in major mental illnesses . Recent Furthermore, several studies found a dysregulation of epigenetic regulators in the brains of psychiatric cohorts, including histone modification factors , DNA methylation factors  and microRNAs . TherefoIn our previous study, we found that histone acetylation could affect transcription of FEZ1, a well-defined schizophrenia risk gene , in oligStrikingly, in a recent study, researchers analyzed genetic variants in glial type-specific schizophrenia risk genes by cross-referring GWAS data to previously published microarray database, and identified 1650 schizophrenia associated genes highly enriched in oligodendroglia . Further1, these genes were involved in the histone modifications, transcription coactivation/corepression, transcription initiation and chromatin remodeling. Moreover, 59% of these genes were well-defined transcription factors. As the transcription factors are crucial for oligodendroglia differentiation and myelination (We further analyzed aforementioned gene transcription set, which included 123 genes in total. As described in Entrez Gene databaselination , both thHowever, whether and how the oligodendroglia specific risk factors, including the above-mentioned transcription factors, coordinate to affect oligodendroglia function and myelination, except for their individual roles, is vastly unclear. For example, HDAC activity is necessary for oligodendroglia differentiation . When ovBesides epigenetic factors involved in gene transcription, some post-transcriptional regulators are also well-recognized as risk factors in oligodendroglia. For example, substantial evidence supports that quaking is a glia-expressed schizophrenia risk factor ,b. The qGenetic alterations, as a large component of mental disease etiology, are not only affecting neuronal function, but also myelinating oligodendroglia. Moreover, these alterations need to induce functional effects through interaction with epigenetic regulators. Genetic variants located in gene coding regions could lead to aberrant protein conformation and posttranslational modifications. Genetic variants within non-coding regions are mostly included in cis regulatory elements including promoters and enhancers, and show deleterious effects by affecting chromatin accessibility and transcription factor binding motif Figure .Transcription and epigenetic regulation are important for oligodendroglia development, and are also affected during oligodendroglia pathogenesis. Ordered chromatin remodeling and transcription factor expression pattern during oligodendroglia development and myelin formation raise the possibility that genetic alterations and epigenetic dysregulations may exhibit functional effects at specific developmental stage based on the expression pattern and functional needs of the epigenetic factors. Moreover, multiple risk factors in oligodendroglia could functionally interplay at transcriptional and post-transcriptional level to affect oligodendroglia function during the pathogenesis, suggesting that malfunction of a molecular orchestra involving distinct risk factors may lead to the common pathophysiology of psychiatric diseases.XC and JG had designed this study and drafted the manuscript. All authors contributed to reviewing and editing the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sox2 deletion caused widespread reduction of Pol II-mediated long-range interactions and decreased gene expression. Genes showing reduced expression in Sox2-deleted cells were significantly enriched in interactions between promoters and SOX2-bound distal enhancers. Expression of one such gene, Suppressor of Cytokine Signaling 3 (Socs3), rescued the self-renewal defect of Sox2-ablated NSCs. Our work identifies SOX2 as a major regulator of gene expression through connections to the enhancer network in NSCs. Through the definition of such a connectivity network, our study shows the way to the identification of genes and enhancers involved in NSC maintenance and neurodevelopmental disorders.The SOX2 transcription factor is critical for neural stem cell (NSC) maintenance and brain development. Through chromatin immunoprecipitation (ChIP) and chromatin interaction analysis (ChIA-PET), we determined genome-wide SOX2-bound regions and Pol II-mediated long-range chromatin interactions in brain-derived NSCs. SOX2-bound DNA was highly enriched in distal chromatin regions interacting with promoters and carrying epigenetic enhancer marks.   •Sox2-bound enhancers are enriched within long-range interactions in neural stem cells•SOX2 loss decreases chromatin interactivity genome-wide•Sox2-bound enhancers from interactions activate reporter genes in zebrafish forebrain•Socs3, a gene downregulated in Sox2 mutant NSCs, rescues their self-renewal Bertolini et al. report that long-range chromatin interactions in neural stem cells (NSCs) are enriched in Sox2-bound enhancers; in Sox2-deleted NSCs, interactions are reduced. Genes downregulated in Sox2-deleted cells are enriched in interactions with enhancers normally Sox2-bound. Overexpression of Socs3, a gene downregulated in mutant NSCs, rescues long-term NSC self-renewal. SOX2 mutations cause genetically dominant nervous system disease involving hippocampus and eye defects, epilepsy, and learning disabilities (OMIM 206900). In mice, Sox2 ablation causes similar defects, such as hippocampal hypoplasia, microcephaly, ventral forebrain depletion, and anophthalmia, some of which may result from a defect in NSC self-renewal  are critical for brain development and for postnatal maintenance of neurogenesis in specific brain areas. SOX2, a transcription factor (TF) essential for pluripotency , is also-renewal . These icultures . SOX2 fucultures .Transcriptional regulation is mediated via DNA looping between gene promoters and their corresponding distal enhancers, often located at large distances and skipping intervening genes . Genome-Sox2-dependent gene regulation in NSCs, as well as genes involved in Sox2-dependent maintenance of long-term NSC self-renewal. We thus deleted Sox2 in NSCs in mouse embryonic brain and studied the effects of embryonic loss of Sox2 on RNA expression in neonatal NSCs grown in vitro  to identify RNA polymerase II (Pol II)-mediated long-range interactions in embryonic stem cells (ESCs) and in brain-derived NSC or progenitor cell cultures . In the itro see  and its Sox2-ablated mice and their control non-deleted littermates  and control  NSCs efficiently expand in culture at early passages  termates . Freshlypassages , howeverpocampus . Sox2-deSox2 loss on the genome-wide pattern of Pol II-mediated long-range chromatin interactions, we first performed ChIA-PET analysis with anti-polII antibodies, specific for the preinitiation complex  brain cells to determine Pol II-mediated long-range connectivity (distant enhancer-promoter and promoter-promoter connectivity)  versus WT NSCs  (in situ ChIA-PET) , proximity ligation was performed in permeabilized intact nuclei, followed by ChIP and a transposase-mediated “tagmentation” to generate PETs for sequencing analysis  . We begactivity)  that perctivity)  neonatesctivity)  are refein wTR1) . Specifiin wTR1)  and S2. hIA-PET) . In the analysis . As suchractions ; Table 1ractions , line 3 in situ proximity ligation adopted in the improved ChIA-PET method effectively captures specific Pol II-mediated long-range interactions between regulatory elements through the Pol II ChIP enrichment (both DNA regions connected by the interactions) (The richment . From a richment . Among tactions) . We defiactions) B. We furin situ ChIA-PET, providing a higher depth in the intra-molecular ligated PETs, showed a reduction in frequency instead of a complete loss of the interactions  B. The nuractions ; the aboractions .Sox2, chromatin connectivity was substantially altered genome-wide, with an overall decreased interaction frequency, in particular at selected loci.In conclusion, the data indicate that in the absence of Sox2 ablation point to a role for SOX2 DNA binding within chromatin in the generation and/or maintenance of long-range interactions. We thus identified SOX2-bound sites through genome-wide ChIP-seq of WT brain-derived NSCs in culture (+ and H3K4me1+), as well as “poised” (H3K4me1+ only) enhancers, with the potential to be activated   . For a s+. On the other hand, within H3K27ac+ regions, a much smaller proportion of promoters than of distal regions were SOX2-bound  and more frequently in intronic and intergenic distal regions  C. Approx+, H3K4me1+) and the remainder with either one or the other mark; among non-SOX2-bound distal anchors, ∼70% were associated with both marks and ∼15% with either (not shown). From now on, we refer to these interactions as “promoter-enhancer” (P-E) interactions. Importantly, SOX2-positive epigenetically marked (EM) distal regions (whether H3K27Ac+ and/or H3K4Me1+) were significantly more involved in interactions than SOX2-negative EM regions: this was observed in the original ChIA-PET (TR1), as well as in in situ ChIA-PET data (TR2 and TR3)  carried a SOX2-bound site within at least one of the two interacting anchors  and S5. and TR3) B and theand TR3) E.In conclusion, the high enrichment of SOX2-positive EM regions within distal anchors in P-E interactions points to a functional role of SOX2 binding at the level of these interactions.Interestingly, the loss of SOX2 in MUT NSCs does not lead to important changes (loss or gain) in the patterns of histone modifications: enrichment in H3K27ac, H3K4me1, both, or none .https://enhancer.lbl.gov)   and S5.  reduced . These dSp8, Cxcr4, Sox3, Nr2f1, Irx1, Socs3 and c-fos . Interestingly, higher expression levels were also associated with an increase in number of interactions per gene, particularly with P-E interactions, which show a clear trend toward higher expression values for every added “enhancer” .To determine how the presence of Pol II-mediated interactions is reflected into gene expression levels, we subdivided genes according to the Pol II ChIA-PET connectivity of their promoters  non-conD. Then, −16 for every pair considered), for TR1, TR2, and TR3  (Sox2 loss) was able to rescue long-term self-renewal in MUT NSC, we expressed in MUT NSCs the Socs3 gene, an inhibitor of Jak/Stat signaling, which antagonizes precocious differentiation of NSCs into astroglia  in MUT NSCs , most or all neurospheres (from transduced MUT NSCs) contained GFP+ cells, and over 70% of the cells were positive by fluorescence-activated cell sorting (FACS), indicating a strong enrichment of the transduced cells; eventually, all cells became GFP+ . This result identifies a SOX2-regulated gene, involved in SOX2-dependent interactions, whose abnormal regulation in MUT NSCs may be responsible for their defective long-term maintenance.30 days) . To evalstroglia . Socs3 iMUT NSCs  and S5 aMUT NSCs E, includMUT NSCs . We tranges 8–12 B and 6C.ame GFP+ D. Note tSox2 decreases the expression of ∼1,000 genes. The identification of thousands of epigenetically defined enhancers involved in long-range interactions allowed us to demonstrate that SOX2-bound long-range interactions represent the most relevant functional category associated with the observed gene downregulation in MUT NSCs  at either promoters or enhancers. The loss of MUT NSCs D. mRNAs Sox2, there is a reduction in interactions frequency, which is detected by ChIA-PET  and the ensuing global chromatin conformation changes; an additional contribution to interaction loss might be represented by the transcriptional deregulation of many SOX2-controlled transcription factors  and S5 pSox2 MUT cells could in principle be ascribed to either loss of an effect of SOX2 on the gene promoter, or to loss of an effect on a connected enhancer. We demonstrate a predominant role of SOX2-bound enhancers versus non-SOX2-bound enhancers on the regulation of their connected genes (Sox2 ablation), or both.The decreased transcription of ∼1,000 genes  and S5 ied genes D; in coned genes ; our dated genes B and 5D.ed genes  that P-EThe role of interactions in controlling gene activity has been addressed by the knockout of genes encoding CTCF or Cohesin components in regulating chromatin interactions in cell lines. Only moderate transcriptional deregulation was observed in connection with widespread and deep changes in long-range interactions ; these pSox2 MUT NSCs (Sox2 was ablated at a stage (E11.5) when specific EM might already have been established within NSCs; SOX2 might be initially important in determining the transition from an ectodermic cell to a NSC, and thus the establishment of proper chromatin EM, but might not be required afterward for their maintenance. These observations dissociate the presence of an epigenetic EM from the actual presence of a critical transcription factor on interacting promoter-enhancer complexes.While SOX2 is bound to a very large proportion of epigenetic enhancers in NSC and plays an important role in the regulation of a subset of genes, epigenetic marks on enhancers are not lost in MUT NSCs . This miSox2 is expressed in NSCs throughout life , encoding well-known regulators of cell proliferation, are expressed at high levels in WT NSCs and are substantially downregulated in Sox2 MUT cells  TF gene  abnormalities, some degree of mental retardation, and eye defects .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact Silvia K. Nicolis . ce as in . The expAB and tupl wild-type zebrafish strains were maintained and bred according to standard procedures  B27 in DMEM F12 with Glutamax), supplemented with 10 ng/ml EGF, 10 ng/ml of basic fibroblast growth factor (FGF) with 0.2% (vol/vol) heparin. We cultured wild-type and mutant cells in parallel for two initial passages  , followeAfter obtaining appropriate numbers of wild-type and mutant cells, we started collecting part of the neurospheres at each passage, continuing the culture of the remaining cells as long as the growth of mutant cells was comparable to that of wild-type cells. In order to proceed to ChIA-PET and ChIP-seq experiments, we mechanically dissociated and crosslinked the neurospheres as described below.ChIA-PET experiments for TR1were performed as described , using pin situ Pol II ChIA-PET 10 million formaldehyde crosslinked cells for each analysis ,  were suspended with 100 μL 0.55% SDS and incubated at RT for 10 min, 62°C for 10 min and 37°C for 10 min to permeabilize nuclei, which was followed by addition of 25 μL 20% Triton X-100, incubation of 30 min at 37°C to quench SDS, followed by addition of 50 μL of blunt-end four-cutter AluI , 50 μL 10 × CutSmart buffer and 275 μL H2O and incubation at 37°C overnight for in situ digestion. After pelleting and washing once with 1 mL 1 × CutSmart buffer, the nuclei were suspended in 500 μL A tailing solution composed of 50 μL 10 × CutSmart buffer, 10 μL BSA , 10 μL 10 mM dATP , 10 μL Klenow (3′-5′ exo-) , and 420 μL H2O and incubated at RT for 1 hr. The in situ proximity ligation with biotinylated bridge linker was performed by adding 200 μL 5 × ligation buffer , 6 μL Bridge linker (200 ng/ul), 10 μL T4 DNA ligase  and 284 μL H2O and incubating at 16°C overnight. The nuclei with in situ proximity ligation were then subjected to sonication and chromatin immunoprecipitation with anti-Pol II antibody , Tn5 tagmentation, and biotin selection; PETs were amplified by PCR and sequenced.For the in situ ChIA-PET protocol, ChIA-PET data were processed with ChIA-PET Utilities, a scalable re-implementation of ChIA-PET tool. Briefly, sequencing adaptors incorporated during the tagmentation reaction in the library construction process were removed from the paired reads. Tags identified (> = 18bp) were mapped to mouse genome (mm9) using BWA alignment , intra-chromosomal (L tags and R tags mapped onto the same chromosome with genomic distance > 8Kb) and self-ligation PETs (L tags and R tags mapped onto the genome ≤ 8Kb). Multiple intra-chromosomal PETs whose respective ends were found within 1 Kb were then clustered as iPET-2, 3 … We further performed statistical assessment of the PET clusters interaction significance using ChiaSigScaled, a scalable re-implementation of ChiaSig , with maximum distance 1 Mb. Because the methods used to fragment chromatins and generate tags for sequencing were different in TR1, TR1 was not included in the SCC analysis. As TR1 used sonication shearing and MmeI digestion while TR2 and TR3 used AluI digestion and Tn5 transposon-based tagmentation, the exact anchor locations defined by these two methods cannot be directly compared.NSC from 6 wild-type forebrains, at P0 , were inCells were fixed sequentially with di(N-succimidyl) glutarate and 1% formaldehyde in phosphate-buffered saline and then lysed, sonicated and immunoprecipitated as described previously . The raw reads were mapped to the mouse genome (mm9 including random chromosomes) with Bowtie  version By using default significance thresholds, this resulted in 18,359 SOX2-bound peak regions for the first (“spheres”) ChIPseq. Since fixation of whole neurospheres might not, in theory, be equally efficient for internal relative to external cells, we also performed another ChIP experiment (“singles” ChIPseq). Read mapping and peak calling were performed with the same parameters used in the first experiment, producing 43,070 bound regions at the same significance thresholds. Of the first dataset (“spheres” ChIPseq), the vast majority  were contained in the latter (“singles” ChIPseq) peak list. Since also the 13% non-overlapping peaks for the first experiment showed enrichment in the corresponding loci in the second ChIPseq, even if below the “peak detection threshold,” we kept for all the subsequent analyses presented in the paper all the peaks returned by the first experiment (“spheres”).The difference between “spheres” and “singles” ChIPseq appears to be due mainly to the presence in “singles” of numerous additional small peaks that have no (significant) corresponding peaks in the “spheres” sample, and may represent more marginal binding sites. Indeed, comparison of profiles between “spheres” and “singles” showed little differences between them, indicating that the main binding sites are very similar (data not shown).Finally, over 50% of the ca. 18400 peaks observed in our forebrain NSC “spheres” are found also within the ca. 24800 peaks detected in a ChIPseq analysis of the NS-5 cell line , an ES-dSox2-deleted mice, at P0 (https://openwetware.org/wiki/BioMicroCenter). Obtained sequences were aligned onto the mm9 mouse genome assembly using Bowtie 1.1.0 (http://bowtie-bio.sourceforge.net) excluding reads that had more than 1 mismatch or that could map to multiple genomic locations. MACS2 was used for peak calling  and narrowpeaks were extended to a minimum of 2000 basepairs (bps) to match peak resolution. Overlapping enriched regions were merged and were considered promoters when located within 1000 bps from annotated mm9 transcriptional start sites (TSSs) and considered putative distal enhancers when located more than 1000 bps away from TSSs.NSC were derived from six forebrains of wild-type and 6 forebrains of e, at P0 . NSC were, at P0 . Neurospe, at P0  using 50Co-localization of histone modifications was performed with ChromHMM version 1.4. . BrieflyThe program was run setting a different number of states, and by processing either wild-type (WT) samples alone and mutant (MUT) samples alone, and on both WT and MUT samples combined. In every setting, the model recovered consistently four main states, corresponding to the joint presence of H3K27ac and H3K4me1, either modification alone, and neither modification. More importantly, all the analyses run on the combined WT and MUT samples failed to identify “differential” states in which one of the two modifications was present only in WT or MUT samples. That is, the model built, regardless of the number of states given as input, consistently contained four more states corresponding to 1) the presence of both H3K27ac and H3K4me1 in both WT and MUT samples; 2) to H3K27ac in both WT and MUT samples; 3) to H3K4me1 in both WT and MUT samples; 4) to neither modification in WT or MUT samples.n reads in WT and m reads for MUT, then we compute the probability of finding n and m reads by chance, given N mapped reads for WT and M for MUT, with a Chi-Square test. We considered “differentially enriched” all regions with the resulting p value lower than 10−4.Differentially enriched 200bp samples were identified with an approach similar to the one of ChromHMM, by comparing for each modification the number of mapped reads in the segments in the two WT samples to the number of reads of the two MUT samples. Given a region with 22955616) and unmappable regions. To define unmappable regions, bam-files from all H3K27ac and H3K4me1 ChIP-seq datasets (n = 8) were merged and mapped onto the mm9 genome that was binned in sliding windows of 3000 bp with an overlap of 500 bp. Bins with zero reads were defined as unmappable and thus excluded.Significance of overlap between H3K27ac-enriched regions and SOX2 binding sites was analyzed in comparison to 1,000 sets of random genomic DNA (random sampling). These sets comprised the same number of elements of similar size selected randomly from the mm9 genome excluding GAP regions (UCSC genome browser), blacklisted regions (cite PMID: ® TA Cloning® Kit (Life Technologies). Individual fragments were then transferred, using recombination-mediated, Clonase™-assisted, Gateway® technology (Invitrogen), to the ZED (Zebrafish Enhancer Detection) vector . EGFP-positive (in forebrain) embryos were collected and propagated to generate three independent F1 transgenic lines each by crossing with wild-type animals. In the case of the en1 and en2 c-fos enhancer embryos were analyzed only at F0. To confirm a link between Sox2 and the identified elements, embryos derived from the F1 lines were microinjected in one blastomere at 1-2 cell stage with a sox2 specific morpholino , previously reported to efficiently interfere with sox2 expression in zebrafish, without however causing major morphological defects (sox2 coding sequence was linearized and in vitro transcribed using the SP6 MessagemMachine kit (Ambion). The synthesized mRNA  were amplified from the mouse genome using specific primers  and clon) vector . ZED con defects . To compzed mRNA  was puriRNA extraction was performed on three independent NSC populations for both wild-type and mutant cells, using Trizol and RNeasy Kit (QIAGEN). 1/5 volume of chloroform was added to one volume of Trizol. Aqueuos phase was transferred into a new tube. 1.5x volume of ethanol was added and mixed well. Mixture was was filtered through Rneasy (QIAGEN) column. Column was washed with Buffer RW1. On-column DNase treatment was performed as described by the RNase-Free DNase Kit (QIAGEN). Post treatment column was clean up with Buffer RW1 and two washes of RPE buffer. Column was then dried and total RNA was eluted with RNase-free water. PolyA Stranded Truseq Libraries were generated using the Truseq Stranded mRNA Sample Preparation Kit (Illumina). First, mRNA was purified from 1μg of total RNA using magnetic beads containing poly-T oligos. mRNA was then fragmented and reversed transcribed using Superscript II (Invitrogen), followed by second strand synthesis. Double stranded cDNA was treated with end-pair, A-tailing, adaptor ligation and 8 cycles of PCR amplification.RNA-Seq was performed on triplicates for the two genotypes studied, yielding 51 bp single-end reads. The number of sequences obtained in each sample ranged from 7.5 to 12.5 millions.Read counts and transcript levels for each sample were computed with the RSEM software package version 1.17 , on the Expression boxplots and subsequent tests were generated using the R functions “boxplot” and “Wilcox.test.” Differential expression analysis was performed on the TPM values with the Noiseq package , n (size of the sample), m (number of successes on the population), N (size of the population). The test was applied by computing the number k of genes involved in any type of interaction  in each of the three expression variation groups. Co-association scores were computed starting from the p value p resulting from the test, defined as –log10 p if k was greater than the expected value (hence showing a co-association between the two gene classes greater than what expected by chance), log10 p otherwise (hence showing a negative co-association). Thus, the higher positive co-association scores are, the more significant is the overlap between the two categories considered, with co-association scores greater than 2 showing a statistically significant overlap. Vice versa for negative scores, showing a significantly low overlap between two sets if lower than −2. The coassociation scores have been calculated using genes expressed at levels of at least 5 TPM in wt NSC; we also did the same analysis using all expressed genes: the results are qualitatively similar, and are not shown.Sox2 mutant neurospheres (obtained as in Socs3-expressing vector at a multiplicity of infection (MOI) of 3.5-5.5 and incubated overnight at 37°C. Then 1ml per well of fresh medium was added both to transduced and non-transduced (control) cells. After 4 days, cells were dissociated to single cells, counted, and seeded at a density of 20,000 cell/well in the same EGF medium described above. In addition, 500,000 cells for each sample (from pooled wells) were fixed using PFA 4% and washed in PBS in order to analyze the GFP fluorescence by flow cytometry : 10,000 events were analyzed for each sample. The samples were excited at 488 nm (blue laser) and the resulting fluorescence measured at wavelengths > 530 nm. The results were analyzed using CellQuest Pro software (BD Biosciences).The TWEEN lentiviral vector expressing SOCS3 from the CMV promoter and GFP driven by the human PGK promoter  was tranGSE90561The data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus (GEO). The accession number for the ChIA-PET, ChIPseq and RNAseq data reported in this paper is GEO: The genomic data can be visualized through the WashU browser:http://epigenomegateway.wustl.edu/legacy/?genome=mm9&datahub=https://wangftp.wustl.edu/∼dli/7131149234337a58201ae3da174ecc51/hub&coordinate=chr8:87120161-87587163


Current models for the folding of the human genome see a hierarchy stretching down from chromosome territories, through A/B compartments and topologically-associating domains (TADs), to contact domains stabilized by cohesin and CTCF. However, molecular mechanisms underlying this folding, and the way folding affects transcriptional activity, remain obscure. Here we review physical principles driving proteins bound to long polymers into clusters surrounded by loops, and present a parsimonious yet comprehensive model for the way the organization determines function. We argue that clusters of active RNA polymerases and their transcription factors are major architectural features; then, contact domains, TADs and compartments just reflect one or more loops and clusters. We suggest tethering a gene close to a cluster containing appropriate factors—a transcription factory—increases the firing frequency, and offer solutions to many current puzzles concerning the actions of enhancers, super-enhancers, boundaries and eQTLs (expression quantitative trait loci). As a result, the activity of any gene is directly influenced by the activity of other transcription units around it in 3D space, and this is supported by Brownian-dynamics simulations of transcription factors binding to cognate sites on long polymers. Current reviews of DNA folding in interphase human nuclei focus on levels in the hierarchy between looped nucleosomal fibers and chromosome territories ,2. Hi-C—https://doi.org/10.1101/118737). and many other organisms get by without the protein, . TherefThe relationship between structure and function is also obscure . For exaTime-lapse imaging of a GFP-tagged gene in a living mammalian cell is consistent with it diffusing for ∼1 min through a ‘corral’ in chromatin, ‘jumping’ to a nearby corral the next and bouncing back to the original one . ConsequMonte Carlo simulations of polymers confined in a sphere uncovered several entropic effects depending solely on excluded volume ,20. Flexcis contacts (i.e. involving contacts with the same chromosome), or visits others to make trans ones depends significantly on territory shape. Children who buy Mcis contacts plus some trans ones . ModeliA third mechanism involves cohesin—a ring-like complex that clips on to a fiber like a carabiner on a climber’s rope. In Hi-C maps, many human domains are contained in loops apparently delimited by CTCF bound to cognate sites in convergent orientations . Such ‘cWe now review results of simulations involving what we will call the ‘transcription-factor model’. This incorporates the few assumptions implicit in the classical model illustrated in Figure First, and extraordinarily, bound factors cluster spontaneously in the absence of any specified DNA–DNA or protein–protein interactions Figure ). This c2 in Figure The basic mechanism yielding clustering is a simple positive feedback loop which works as sketched in Figure This process drives local phase separation of polymerases and factors, and so naturally explains how super-enhancer (SE) clusters form ). This gin vivo), so they are inevitably in different places in 3D space . This he Figure .A third result is that clusters and loops self-assemble into ‘TADs’ and ‘A/B compartments’ (26–28). Thus, if chromosome 19 in human GM12878 cells is modeled as a string of beads colored according to whether corresponding regions are active or inactive, binding of just red and black spheres (‘activators’ and ‘repressors’) yields contact maps much like Hi-C ones Figure . As neitThe simple transcription-factor model has been extended to explain how pre-existing red clusters can evolve into green clusters, or persist for hours as individual factors exchange with the soluble pool in seconds—as in photo-bleaching experiments ,36). AddThese physical principles lead naturally to a model in which a central architectural feature is a cluster of active polymerases/factors surrounded by loops—a ‘transcription factory’. A factory was defined as a site containing ≥2 polymerases active on ≥2 templates, just to distinguish it from cases where two enzymes are active on one Figure ,40). MucIn all models, a gene only becomes active if appropriate polymerases  and factors are present; in this one, there are three more requirements. First, active polymerases are transiently immobile when active; they reel in their templates as they extrude their transcripts Figure . This cop often visits the nearby green volume, whereas q mainly roams ‘outer space’; simulations and experiments confirm this  re-initiate to reform a loop. Alternatively, e may spend some time diffusing through outer space before rebinding to the same or a different factory. Consequently, as factors and polymerase bind and dissociate, factories morph, loops appear and disappear—and the looping pattern of every chromosomal segment changes from moment to moment. Then, it is unlikely the 3D structure of any chromosome is like that of its homolog, either in the same cell or any other cell in a clonal population.These three principles combine to ensure the structure is probabilistic and dynamic, with current shape depending on past and present environments. For example, as q in Figure These physical principles also lead naturally to an explanation of how genes become inactive. Thus, We now describe results easily explained by this model, but difficult or impossible to explain by others without additional complicated assumptions see also .Contacts seen by 3C-based approaches often involve active promoters and enhancers; for example, FIRES (frequently-interacting regions) in 14 different human tissues and 7 human cell lines are usually active enhancers . SimilarWhile 3C focuses on contacts between two DNA sequences, the ligation involved can join >2 together (24 is the current record), and these again generally encode active sequences ,51. Why Early studies also point to a correlation between transcription and structure. For example, switching on/off many mammalian genes correlates with their attachment/detachment . What unCis Hi-C contacts fall off rapidly with increasing genetic distance, whereas trans ones are so rare they are often treated as background. However, ChIA-PET yields more trans than cis contacts when active sequences are selected by pulling down ERα or polymerase II ; it spends most time roaming ‘outer space’ making a few chance contacts with nearby segments of its own loop, and—if it visits a factory—it cannot initiate in the absence of phospho-NFκB. But once phospho-NFκB appears (10 min after adding TNFα), it initiates. Then, NFκB binding sites in SAMD4A become tethered to the factory, these bind phospho-NFκB, exchange of the factor increases the local concentration, and this increases the chances that other inflammatory genes initiate when they pass by. And once they do, this creates a virtuous cycle; as more inflammatory genes initiate, more NFκB binding sites become tethered to the factory, the local NFκB concentration rises, this further increases the chances that passing responsive genes initiate, and the factory evolves into one specializing in transcribing inflammatory genes. As a result, the rapid concentration of inflammatory genes around the resulting ‘NFκB’ factory yields the rapid increase in cis and trans contacts between them seen by 3C-based methods and RNA-FISH .Studies on mouse eggs and embryos also provide conflicting data. Thus, activity is lost as oocytes mature, and TADs plus A/B compartments disappear ,63,64; tData from zebrafish make unified interpretation even more difficult. In contrast to some cases cited earlier, TADs and compartments exist before zygotic gene activation, and many of each are lost when transcription begins . ClearlySfmbt2 enhancer in mouse ES cells indicates it is the former . The model also explains how enhancers can act over such great distances  Promoters of protein-coding genes are often enhancers of other protein-coding genes (e is an enhancer irrespective of whether it encodes an mRNA or eRNA. (iv) Enhancers act both promiscuously and selectively. They interact with many other enhancers and targets . Then, once e terminates, close tethering is lost—and p remains silent for as long as it remains far from an appropriate factory. RNA FISH experiments on human SAMD4A support this explanation; the promoter is usually silent, but adding TNFα induces successive attachments/detachments to/from a factory  Enhancer activity depends on contact with its target promoter ,72. We sng genes ,75,76. I targets –79, with targets . At the  targets . In our  targets . Thus, wy Figure Aii,iii. y Figure , and thi embryos ,74. Such embryos . Periods factory .e, e’, e’’) in the SE increases the time p spends near a factory (to increase its firing probability).A related conundrum concerns how SEs work. SEs are groups of enhancers that are closely-spaced on the genetic map and often target genes determining cell identity ,84. In FCaulobacter crescentus—which lacks CTCF but possesses TADs—shows it does ; a C to G substitution at position -87 reduces binding, and this reduces HBB expression and causes β-thalassaemia. Therefore, we might expect binding of factors to promoters of coding genes drives phenotypic variation. However, results obtained using GWAS (genome-wide association studies)—an unbiased way of finding which genetic loci affect a phenotype—lead to a different view for many diseases; they are so unexpected that only general explanations are proffered for them (86–88).Classical studies on bacterial repressors  inform our thinking on how regulators work: they act locally as binary switches. We assume eukaryotes are more complicated, with more local switches, plus a few global ones . We are encouraged to think this by studies on some diseases . For exacis and trans (Quantitative trait loci (QTLs) are sequence variants  occurring naturally in populations that influence phenotypes. Most QTLs affecting disease do not encode transcription factors or global regulators; instead, they map to non-coding regions, especially enhancers ,88. eQTLnd trans –92. Addind trans , and onend trans ,92,93. Ind trans . This cob (perhaps an eQTL) might reduce binding of a ‘yellow’ factor and b’s firing frequency, and this has consequential effects on how often d and a are tethered close to the yellow factory—and so can initiate. But this change influences the whole network. By altering positions relative to appropriate factories, an eQTL ‘communicates’ directly with functionally-related targets, and indirectly (but still at the level of transcription) with all other genes around it in nuclear space. This neatly reconciles how eQTLs target functionally-related genes whilst having omnigenic effects .In Figure The idea that altering one loop in a network has global effects was tested using simulations of five factors binding to cognate sites in a 5000-bead string . Additionally, the motor activity behind loop extrusion, if present, may be provided by transcription itself —which is naturally explained by the ‘loop-extrusion’ model ,25,94,95n itself  (Supplemn itself .Second, the structures of mitotic and sperm chromatin pose a challenge to all models . For ourSeeing is believing. While clusters of RNA polymerase II tagged with GFP are seen in images of living cells –102, decIn biology, structure and function are inter-related. Here, we suggest that many individual acts of transcription determine global genome conformation, and this—in turn—feeds back to directly influence the firing of each individual transcription unit. Consequently, ‘omnigenic’ effects work both ways. [Note the term ‘omnigenic’ is used here to include both genic and non-genic transcription units.] In other words, transcription is the most ancient and basic driver of the organization in all kingdoms, with recently-evolved factors like CTCF modulating this basic structure. It also seems likely that transcription factories nucleate related ones involved in replication, repair, and recombination , as wellSupplementary DataClick here for additional data file.