Atoh7 exhibits a dynamic tissue and cellular expression pattern. Although the resulting Atoh7 retinal lineage contains all seven major cell types, only retinal ganglion cells require Atoh7 for proper differentiation. Such specificity necessitates complex regulation of Atoh7 transcription during retina development. The Notch signaling pathway is an evolutionarily conserved suppressor of proneural bHLH factor expression. Previous in vivo mouse genetic studies established the cell autonomous suppression of Atoh7 transcription by Notch1, Rbpj and Hes1. Here we identify four CSL binding sites within the Atoh7 proximal regulatory region and demonstrate Rbpj protein interaction at these sequences by in vitro electromobility shift, calorimetry and luciferase assays and, in vivo via colocalization and chromatin immunoprecipitation. We found that Rbpj simultaneously represses Atoh7 transcription using both Notch-dependent and –independent pathways.In vertebrate retinal progenitor cells, the proneural factor  Among these factors, Atoh7  is dynamically expressed by subsets of embryonic RPCs and required for retinal ganglion cell (RGC) formation6. Without Atoh7 function, essentially all RGCs fail to differentiate and adult animals lack optic nerves6. Although the Atoh7 retinal lineage includes all seven major cell types, gene activity is only required for RGC genesis. To understand how Atoh7 acts as an RGC competence factor requires deeper understanding of its mode of action as a DNA-binding protein, and the mechanisms tightly regulating its mRNA and protein. Here we focus on particular aspects of Atoh7 transcriptional regulation.During vertebrate embryonic development, multipotent retinal progenitor cells (RPCs) undergo a prolonged period of differentiation during which six neuronal  and one glial (Müller) cell type are produced and assembled into a highly laminated tissue. Multiple signaling pathways and transcription factors, including proneural basic helix-loop-helix (bHLH) transcription factors, regulate retinal cell fate, as reviewed inAtoh7 genomic architecture defined two enhancer regions on the 5′ side of the lone Atoh7 coding exon, each containing multiple conserved noncoding elements (CNEs)10. In mice, the distant, shadow enhancer is 9.5 kb and the proximal, primary enhancer is 1.5Kb upstream of the Atoh7 ATG start codon. The primary enhancer is further subdivided into two CNEs, termed distal and proximal. The distal CNE contains validated Pax6 (paired domain) and Neurog2 (E box) binding sites, through which Pax6 activates transcription, and Neurog2 drives the initial wave of retinal neurogenesis12. However, the activities of these two factors cannot account for the rapid downregulation of Atoh7 in subsets of RPCs at differentiation.Previous studies of vertebrate Atoh7 expression at the molecular level, in the context of retinogenesis. Canonical Notch signaling initiates with a Delta-like or Jagged/Serrate ligand on one cell binding to a Notch receptor on an adjacent cell. This triggers proteolytic cleavage of the receptor to ultimately release its intracellular domain (termed NICD). The NICD complexes with a CSL protein  in human/mouse, Su(H) (Suppressor of Hairless) in Drosophila, Lag-1 (lin12 and glp-1 phenotype) in C. elegans) and the Maml (Mastermind-like) co-activating factor. This protein complex activates downstream target gene transcription by binding to a variety of CSL sites within noncoding DNA16. Well-characterized effector genes of the Notch pathway include the Hes (Hairy/Enhancer of Split (E(Spl)) and Hey gene families, which encode transcriptional repressor proteins19. Intriguingly, during Drosophila retinal development, Notch signaling reiteratively regulates Atonal transcription21. In undifferentiated cells anterior to the eye disc morphogenetic furrow (a moving differentiation boundary), Notch signaling activates Atonal in a continuous stripe of cells. But more posteriorly, the Notch complex, via E(Spl) activity, suppresses Atonal expression in cells adopting non-R8 photoreceptor fates. This latter activity embodies the classic neurogenic role of Notch signaling. Importantly, these phases of Atonal regulation are separated in both time and space, and utilize distinct Atonal enhancers, as reviewed in22.Given that Notch signaling regulation of bHLH factors occurs widely, including throughout the vertebrate nervous system, we wish to understand how this pathway controls in vivo targets. However, the probability of any gene being a direct target is enhanced when its expression is affected by Notch pathway loss- and gain-of-function mutants. Previous examinations of mouse retinal mutants showed that loss of Notch1, Rbpj or Hes1 derepresses Atoh7 mRNA expression and RGC neurogenesis29. By contrast, overexpression of activated Notch1 (NICD1) stimulates proliferation, thereby blocking Atoh7LacZ expression and RGC formation30. Yet, the mechanism(s) underlying Notch pathway regulation of Atoh7 remain unresolved.Predicted CSL binding sites are interspersed throughout metazoan genomes, but it is challenging to know which are Hes1, which in turn would repress Atoh7 transcription. But, other models are possible, including Rbpj direct regulation of Atoh7, either in a corepressor complex (repression), or in a Notch complex (activation)32. However, all genetic data consistently support Rbpj suppression of Atoh7 transcription. In this study, we characterize four CSL binding sites situated in Atoh7 5′ regulatory DNA. We found that at the peak of Atoh7 retinal expression, Rbpj occupies one CSL site, R3 within the distal primary enhancer, and this binding site mediates transcriptional repression. We also discovered that simultaneous loss of all four binding sites reduced Atoh7 transcription, suggesting a distinct mode of Rbpj regulation, consistent with locus priming for efficient transcriptional regulation by other factors33. Importantly, both Rbpj-mediated repression at site R3, and activation via multiple sites are Notch-independent, although Notch1/3 signaling has a measurable impact on Atoh7 mRNA levels. Overall our data are suggestive of three separate, yet simultaneous modes of Rbpj regulation: locus priming, direct repression and canonical Notch pathway suppression. We propose that integrated regulatory inputs are important for precise control of Atoh7 pulsatile expression in the developing retina.A classical view of Notch regulation of bHLH factors holds that an activated Notch protein complex transcriptionally activates Atoh7 transcriptional activity. Because the Atoh7 primary enhancer recapitulates endogenous mRNA expression in frog, chick, zebrafish and mouse transgenic studies, and contains two CNEs, it was the strongest candidate to contain CSL binding sites34. However, we used the CSL consensus TRANSFAC MATCH algorithm (5′-[C/T]GTG[G/T]GAA-3′) to perform an unbiased search of 6 Kb of noncoding DNA surrounding the Atoh7 coding exon15. Four putative CSL sites (termed R1–R4) were identified; all positioned 5′ to the ATG codon . Interestingly, site R3 is situated in between previously characterized Pax6 and Neurog2 (E4/E3) binding sites  binding sites relative to don Fig. . Three odon Fig. . Of the don Fig. , with ontes Fig. 8,10,11. tes Fig. . In the 35. Previously, a rat anti-Rbpj antibody was used in our exploration of Notch signaling during mouse lens development37. Here we further confirmed the specificity of this monoclonal antibody, during mouse retinogenesis, and evaluated its usefulness in biochemical assays   Fig. . Howeverion Fig. . We alsocts Fig. .Figure 2in vivo embryonic retinal development. The spatiotemporal expression pattern of Atoh7 protein is highly dynamic, restricted to the central optic cup at E11.5 , using bacterially expressed and purified mouse Rbpj protein (residues 53–474), previously shown to bind DNA42. Rbpj protein was incubated with biotin-labeled double stranded oligonucleotides, in which the putative binding site is centrally located  and the non-specific sequence (GCTACTCATACCTAGAACG), respectively, and detected binding from the Hes1 site (~1 μM Kd), but did not detect binding from the non-specific site (data not shown). The results showed Rbpj bound to three of the four putative CSL sites with comparable affinity to the well-characterized Hes1 site  to quantitate binding, using purified Rbpj with the oligomeric DNA duplexes that correspond to the four putative CSL binding sites Fig. . As posiite Fig. 43. Howevity Fig. . While win vivo retinal development. Previously we demonstrated that Pax6 occupies consensus site J in the human ATOH7 gene, using chromatin from Ad12Her10 retinal cell line10. As a positive control, we performed Pax6 ChIP in parallel here, using mouse E14.5 retinal chromatin. This age was selected because it is the peak of Atoh7 expression44. Dissected retinas from individual embryonic litters were pooled and frozen en masse (n = 3 litters). After lysis, cross-linking, quantification and sheering, chromatin was immunoprecipitated with rat anti-Rbpj, rabbit anti-Pax6 or relevant IgG controls (see Methods). The antibody-bound chromatin complexes were purified, crosslinks reversed, and the isolated genomic DNA used as a template for real-time PCR. Each primer set amplified an amplicon specific for each putative binding site , or a corepressor complex (repression). To address these possibilities formally we used luciferase reporter assays to measure the activity and requirement for each site individually, versus simultaneous mutation of all 4 sites, within a previously identified primary gene Atoh7 enhancer10. However, first we compared Atoh7 primary gene enhancer expression to that of the endogenous protein, which had not been previously reported46. For this we created a transgenic construct with 2.4Kb of mouse Atoh7 5′ DNA joined to a minimal human β-globin promoter (BG) and red fluorescent monomeric Cherry reporter (mCherry)47. We then used antibody colabeling to evaluate mCherry expression in retinal cryosections from E13.5 transient transgenic embryos. We found that endogenous Atoh7 significantly overlaps with Cherry+ cells  were simultaneously mutated, there was a significant decrease in Atoh7 transcriptional levels  or Notch3 (NICD3) in our luciferase assays. Each receptor alone, as well as in combination, was previously shown to be required in vivo for particular aspects of Atoh7 expression26. Plasmids containing the NICD1, NICD3, or a mixture of the two, were cotransfected with either the Atoh7 wild type or ΔR1–R4 mutant luciferase constructs53. Increasing NICD1 levels stimulated Atoh7 transcription but had no impact on the ΔR1–R4 mutant  in the E13.5 mouse retina -paired site), consists of two CSL binding sites in a head to head configuration separated by ~16 nucleotides64. These SPS elements are occupied by dimeric NICD/Rbpj complexes, to regulate transcription68. Although the Atoh7 upstream CSL binding sites lack a canonical SPS arrangement, there remains some possibility for such a mechanism, since a cryptic CSL element in the Hes5 promoter acts in a dimeric SPS complex65.Bioinformatic analysis of noncoding sequences surrounding the mouse in vitro, but was the only site that ChIPped CSL in vivo with statistical significance. Similar phenomenon has been observed for Su(H), the fly CSL ortholog, binding of the sparkled (spa) enhancer in Drosophila, whereby the low affinity Su(H) sites are critical for proper gene expression and patterning driven by spa69. The location of R3 within the distal CNE is flanked by a Neurog2-dependent Ebox (30 bp upstream) and a Pax6 binding site (20 bp downstream). Such spacing allows one or two helical turns to separate each of these sites. In the developing pancreas, this same distance is permissive for Rbpj physical interaction with the bHLH transcription factor Ptf1a70. Interestingly, Ptf1a is also expressed in the developing retina and influences neurogenesis of several retinal cell types73. In Drosophila and Xenopus there are other examples of CSL/Rbpj protein interactions with bHLH factors that activate transcription75. Hence, we cannot discount the possibility that Neurog2 (or another bHLH factor) may physically interact with Rbpj. Alternatively, Rbpj occupancy of site R3 might affect local chromatin architecture, thereby displacing activating factors. Because we detected both Rbpj and Pax6 occupancy in E14.5 retinal chromatin (within the same preparation), a mutually exclusive binding mechanism would seem implausible. One caveat was the use of whole retina chromatin, which could obscure distinct configurations of transcription factor binding at the Atoh7 primary enhancer, among a heterogenic population of retinal cells. For example, in mitotically-active RPCs that do not express Atoh7, Rbpj could act as a repressor at site R3. When these cells enter their terminal mitosis, they may activate Atoh7 expression, via Pax6 or Neurog2 binding, which would also displace nearby Rbpj-corepressor complexes. Conversely, during terminal differentiation, this relationship could be reversed, with Rbpj-corepressor binding at site R3 dislodging either Pax6 or Neurog2. Only the generation of single-cell genomic datasets can map the occupancy of particular enhancer binding site to the developmental status of cells, at distinct stages of retinogenesis.Among the CSL sites analyzed here for the embryonic retina, R3 stands out as unique. Counterintuitively, CSL has the weakest affinity for R3 in vivo context for our biochemical data, via direct comparison of Rbpj and Atoh7 protein expression patterns. Rbpj is well established as ubiquitously expressed, so co-localization with Atoh7 protein was already predicted. But, transcription factors that are coexpressed with their target genes typically activate transcription, not repress it. Indeed, Hes1, a known repressor of neurogenesis, displays mutually exclusive expression with βgal in Atoh7LacZ/+ eyes26. Yet, our transcriptional activity data clearly indicate that Rbpj repression of Atoh7 is the major mode of regulation, which correlates with all previous genetic findings29. However, we also note that Rbpj does not appear uniformly expressed, with brighter anti-Rbpj labeling coinciding with Atoh7 expression. The significance of this observation remains unclear. Better understanding will require the determination of which Rbpj regulation activities at work during distinct stages of retinal cell development. Moreover, we must clarify whether Notch1/3 signaling invokes canonical pathway regulation, namely Notch-Rbpj-Maml binding to Hes gene promoters, which may in turn directly repress Atoh7 transcription.Here we also provided some insight into Atoh7 locus open and primed 33, until its rapid, pulsatile expression is needed. Higher levels of Rbpj protein may subsequently be required to also engage in the activities of Notch and co-repressor complexes, which act at different regulatory sequences, and possibly at different rates to shut down Atoh7 transcription. Clearly additional transcription factors must simultaneously regulate Atoh7 (positively or negatively) since brighter-labeled Rbpj cells are capable of Atoh7 co-expression. The integration of these multiple modes of regulation allows for more precise modulation of target gene mRNA levels, particularly during highly dynamic developmental processes.While highly speculative, we propose that lower levels of Rbpj protein expression are sufficient for binding to multiple CSL sites, which contributes to keeping the All mice were housed and cared for in accordance with the guidelines provided by the National Institutes of Health, Bethesda, Maryland, and the Association for Research in Vision and Ophthalmology, and conducted with approval and oversight from the Cincinnati Children’s Hospital Research Foundation and UC Davis Institutional Animal Care and Use Committees.Rbpjtm1Hon conditional allele (termed RbpjCKO) were maintained on a 129/SvJ background and genotyped as described41. α-Cre transgenic mice were maintained on a CD-1 background and genotyped as described38. Le-Cre mice were maintained on an FVB/N background and genotyped as described40. Z/EG lineage tracing mice (Tg(CAG-Bgeo/GFP)21Lb3/J) use the CMV enhancer/chicken actin promoter to constitutively express lacZ, which is replaced with eGFP expression upon Cre activation39. These mice were acquired from Jackson Labs (Stock Number 003920), maintained on a CD-1 background and genotyped as in39. Embryonic gestational age was determined by timed matings, with the date of the vaginal plug as E0.5.A Atoh7 genomic DNA (nucleotides −3032 to −503 containing the primary enhancer but lacking the TATAA box) was PCR amplified using primers with engineered XbaI and BglII restriction sites, cloned into the Xba I-Bam HI sites of the pBGnCherry vector47 in the normal transcriptional orientation, and verified by Sanger sequencing. The Atoh7 fragment was PCR amplified (EXPAND Hi-Fidelity polyermase) from a previously subcloned 6.5 Kb mouse Atoh7 genomic DNA template3, digested with BglII and XbaI and then purified. The pBGnCherry vector47 contains a minimal human β-globin promoter and monomeric Cherry red fluorescent protein reporter cassette (mCherry), as well as a synthetic amino terminal nuclear localization signal (MAPKKKRKVEDV) downstream of the BamHI site. There is no intrinsic activity of this vector in transgenic mice47. Linearized DNA was microinjected into CD-1 mouse pronuclei by the CHRF Transgenic Core Facility. F0 embryos were collected at E13.5 and screened for live mCherry fluorescence with a Leica MZ12 dissecting scope equipped with a Texas-Red filter. We harvested 4 Cherry +/44 embryos and each Cherry-positive embryonic head was cryoembedded, sectioned and analyzed using immunohistochemistry and confocal imaging (see below).The upstream, noncoding 2.4 Kb of mouse Atoh7 genes (Gene IDs 53404 and 220202) were aligned using the MacVector Clustal W algorithm (v. 12). CNEs were identified in multiple vertebrate genomes using the UCSC genome browser MultiZ alignment and conservation features and mm10 genome assembly (http://genome.ucsc.edu). Putative CSL/Rbpj binding sites were identified using the TRANSFAC MATCH program with matrices M01111 (V$RBPJK_Q4) and M01112 (V$RBPJK_01). Previously defined Pax6 paired domain and E-box binding sites within the mouse Atoh7 primary distal CNE are included for reference11.Three kilobases of 5′ and three kilobases of 3′ noncoding genomic DNA from the mouse and human 2 to label with chick anti-GFP , rat anti-Rbpj , rabbit anti-Atoh7 , or goat anti-mCherry polyclonal  primary antibodies. Secondary antibodies were directly conjugated to Alexa Fluor 488 , Alexa Fluor 594 , Alexa 594 , Alexa Fluor 647 , or Dylight 649 . Microscopic imaging used either a Zeiss Axioplan fluorescent microscope with a black and white camera, Apotome deconvolution device, and Axiovision (v. 7.0) software, or a Leica DM5500 microscope equipped with a SPEII solid state confocal and Leica LASAF software. All digital micrographs were electronically adjusted equivalently for brightness, contrast and pseudocoloring using Adobe Photoshop CS5 software.Embryonic heads were fixed in 4% paraformaldehyde/PBS for 1 hour at 4 °C, processed through a sucrose/PBS series, cryoembedded and sectioned at 10 μm. Immunohistochemistry using our lab protocolRbpjCKO/CKO, Le-Cre;RbpjCKO/+ and Le-Cre;RbpjCKO/CKO embryos and flash frozen. Twenty lenses (10 pairs) of the same genotype were pooled and lysed in RIPA buffer  containing Complete protease inhibitors (Sigma 11697498001). Total protein concentrations were determined by Bradford assay . NuPAGE 4–12% Bis-Tris gels  were loaded with 20ug of total lens protein per gel lane, electrophoresed and transferred onto nitrocellulose membranes . Standard western blotting was performed, using rat anti-Rbpj  or mouse anti-β-actin  primary antibodies, followed by HRP-conjugated anti-rat IgG or mouse IgG secondary antibodies . Blots were developed using a Supersignal West Pico Chemiluminescent substrate kit , Kodak standard x-ray film and film developer.Pairs of lenses were harvested from E14.5 et al., 2008) and 1 nM of labeled oligonucleotide complexes, in the presence of 1.9 ng/μl poly[d(I-C)] , were resolved on a 6% DNA retardation gels  in 0.5 × TBE buffer and then transferred to nylon membranes. The LightShift Chemiluminescent EMSA kit assay  was performed on the blots, which were developed using the Chemiluminescent Nucleic Acid Detection kit , Kodak x-ray film and film developer.Single-stranded complementary oligonucleotides containing predicted CSL binding sites were labeled using a Biotin 3′ end DNA labeling kit . Double-stranded DNA probes were made by annealing biotin-labeled complementary oligonucleotide pairs at room temperature for one hour. 0.5 μM purified mouse Rbpj protein   in the syringe and Rbpj (~10 μM) in the cell and consisted of 40 injections of 7 μl each. Data analysis used the ORIGIN software and was fitted to a one-site binding model, with binding data representing the average of n = 3 experiments.The production and purification of bacterially expressed Rbpj protein, residues 53–474, has been describedDT) Fig.  were hyd76 with several modifications. 30 E14.5 CD-1 pooled embryonic retinas were crosslinked with 1% formaldehyde and the reaction stopped by addition of 125 mM final concentration of glycine. Chromatin was sheered to 300–1000 bp size range with a Bioruptor UCD-200 sonicator + chiller (Diagenode), for 20 minutes at high power with 15 sec ON/30 sec OFF cycles. Either 3 μg rat anti-Rbpj antibody  or rat IgG  were incubated with 40 μg sonicated chromatin overnight at 4 °C. Immune complexes were collected with Protein G agarose beads , washed several times and eluted using 0.5 M NaHCO3, 1% SDS elution buffer. Pax6 ChIP was run in parallel from each retinal chromatin prep, by incubating 20 μg of sheared chromatin with 1 μg anti-Pax6 , or rabbit IgG , coupled to Protein A sepharose beads . Input and immunoprecipitated chromatin samples were initially analyzed by performing 30 cycles of PCR amplification and agarose gel electrophoresis, then quantified by real time PCR, using Table ChIP was performed as describedAtoh7 locus was previously cloned into the pGL2 luciferase vector10. Rbpj binding site mutations were generated using PCR-based site directed mutagenesis77 and verified by Sanger Sequencing. Either 3.5 × 105 HEK293T or 5 × 105 AD12HER10 cells were plated per well of a 6-well tissue culture plate. After 48 hours (~60% confluency) cultures were transfected according to the Fugene6  protocol with a 5:1 Fugene6 to DNA ratio, with the DNA constituting 500 ng of luciferase plasmid and 50 ng of Renilla control plasmid (pRL). In Notch ICD overexpression experiments, 100 ng of NICD1/pBK-CMV, 100 ng of NICD3/p3XFLAG-CMV-7TM, or a mixture of 50 ng of each plasmid were cotransfected with the luciferase and Renilla plasmids. Cells were washed in PBS, harvested 48 hours after transfection in 500 μL 1 × PLB (Promega) and cell pellets stored at −80 °C. Cell extracts were assayed in technical triplicate using the Dual Luciferase Assay System  on a Perkin Elmer Victor X5 workstation. Luciferase activity levels were normalized to the control Renilla activity, and p-values determined with GraphPad Prism (v6) software, using a two-tailed, unpaired t-test with equal standard deviation and assuming a Gaussian distribution.The upstream 2.6 Kb noncoding DNA from the mouse Total RNA was extracted using the RNeasy micro kit  from 1 pair of dissected E13.5 retinas, or using Trizol (Invitrogen Cat No 15596026) for HER-10 cells. For embryonic retinal RNA 100 ng was reverse transcribed into cDNA using the iScript Synthesis kit and product protocol . For HER-10 cells, 6.5 μg of total RNA was first treated with 1U of 10 U/μL of DNase (Roche Cat No. 04716728001) by incubating at 37 °C × 35 min, 80 °C × 5 min, 90 °C × 3 min. Then 2 μg of treated RNA was used for cDNA synthesis, with Superscript III and manufacturer protocol . Both experiments included a mock synthesis  performed in parallel. For embryonic retinas, 1 μL of cDNA was combined with individual primer sets (Supplemental Table All data generated or analyzed during this study are included within this published article and its Supplementary Information files.Supplemental Files


Arabidopsis. Furthermore, ten NAC proteins from different NAC subfamilies in Arabidopsis thaliana were selected and confirmed to bind to the MybSt1, NRS1, and NRS2 motifs, indicating that they are recognized commonly by NACs. These findings will help us to further reveal the functions of NAC proteins.NAC  transcription factors play important roles in many biological processes, and mainly bind to the NACRS with core sequences “CACG” or “CATGTG” to regulate gene expression. However, whether NAC proteins can bind to other motifs without these core sequences remains unknown. In this study, we employed a Transcription Factor-Centered Yeast one Hybrid (TF-Centered Y1H) screen to study the motifs recognized by ANAC074. In addition to the NACRS core cis-element, we identified that ANAC074 could bind to MybSt1, NRS1, and NRS2. Y1H and GUS assays showed that ANAC074 could bind the promoters of ethylene responsive genes and stress responsive genes via the NRS1, NRS2, or MybSt1 element. ChIP study further confirmed that the bindings of ANAC074 to MybSt1, NRS1, and NRS2 actually occurred in  Transcription factors (TFs) are a group of regulatory proteins which can regulate the expression of target genes through binding to specific cis-regulatory elements that are often located in the promoters of target genes . AlthougArabidopsis  sequence  sequence [Arabidopsis ANAC069 protein was found to bind to C[A/G]CG[T/G] sequence, in which the sequence C[A/G]CG is similar to the core sequence “CACG” [Arabidopsis NAC1 binds to a 21-bp DNA fragment containing an as-1 element (TGACG), in which the conserved sequence “GACG” is also similar to the “CACG” [CTTCACGCGGG” containing the core sequence “CACG” like other NAC binding elements. This result further suggested that some NAC proteins can bind core sequences “CACG”.The sequence “CCCet genes ,19,20. Isequence , the cone “CACG” . ArabidoArabidopsis transcriptional repressor CBNAC can bind to CBNACBS with a “GCTT” sequence but not the “CACG” sequence [TTAGTTTCGGG” and “CCCTGAATCTCGGG” both contain unknown DNA motifs recognized by ANAC074, and were further studied.However, other NAC proteins appear to bind different sequences. Kim et al. (2007) found that the sequence . Therefosequence , “CCCTGATTAGTTTCGGG” and “CCCTGAATCTCGGG” (insertion is underlined) from both 5’ and 3’ terminals, and cloned the sequences into pHIS2 to drive the HIS3 gene. The interaction between these deleted insertion sequences and ANAC074 was studied using Y1H. The sequences of “AGTT” and “TAGT” abolished the binding to ANAC074 while the sequence of “TAGTT” restored the binding activity , NRS1 mutants , and NRS2 mutants  all lost binding activity with ANAC074, indicating that the bindings of ANAC074 to MybSt1 element, NRS1 and NRS2 are specific  in tobacco also confirmed that the ANAC074 bound to MybSt1, NRS1 and NRS2 elements specifically (The bindings of ANAC074 to NRS1 (TAGTT), NRS2 (GAATC), MybSt1 (GGATA) and their mutants were investigated by Y1H to investigate the specification of binding. The results showed that the ANAC074 can bind to MybSt1 element, NRS1 and NRS2, while the MybSt1 mutants , GTCGA (M2) and TTCGC (M3)  A, indicaArabidopsis using the Patmatch program on Tair [AT4G17500, AT1G74930 and AT4G11280) happen to be the target genes of ANAC069 [The MybSt1, NRS1, and NRS2 were identified using the TF-centered Y1H system ,24. Howe on Tair . The cri ANAC069 . We specATERF-1 (AT4G17500), NRS2 was present in the promoter regions of ORA47 (AT1G74930), and MybSt1 element was included in ATACS6 (AT4G11280)  containing “TAGTT” (NRS1p+), without “TAGTT” (NRS1p-) or with “GGGGG” (NRS1pm: completely mutated NRS1); truncated promoter of AT1G74930  containing “GAATC” (NRS2p+), without “GAATC” (NRS2p-) or with “TCCGA” (NRS2pm: completely mutated NRS2); truncated promoter of AT4G11280  containing “GGATA” (MYBp+), without “GGATA” (MYBp-) or with “TTCGC” (MYBpm: completely mutated MybSt1), were respectively inserted into pHIS2 and hybridized with ANAC074 using Y1H assay. The results showed that ANAC074 binds to the truncated promoters with NRS1, NRS2, or MybSt1, but failed to bind with the truncated promoters without or with completely mutated NRS1, NRS2, or MybSt1  A. To stur MybSt1 B. Coexprr MybSt1 C,D. TakeAT4G17500, AT1G74930, and AT4G11280 were target genes regulated by ANAC069 under salt stress condition using Afymetrix Arabidopsis Gene Chips (GSE82274). In this study, ANAC074 binds to the NRS1, NRS2, or MybSt1 motifs in the promoters of AT4G17500, AT1G74930, and AT4G11280. ANAC069 and ANAC074 share down-stream target genes, indicating that there are similar regulatory mechanisms between ANAC069 and ANAC074 and that their transactivation functions are somewhat redundant. The function of ANAC069 in salt and osmotic stress has been confirmed [In the previous study, we found that hips GSE874. In thArabidopsis thaliana plants overexpressing a ANAC074-GFP fusion gene. The three truncated promoter regions of each putative target gene  with NRS1, NRS2 or MybSt1 element were examined. The results showed that the truncated promoters of AT2G21320, AT2G31880 and AT3G13470 all can be amplified from chromatin immunoprecipitated with anti-GFP antibody, but not from chromatin immunoprecipitated with HA antibody     failed to amplify the corresponding PCR products (data not shown).To determine whether the binding of ANAC074 to NRS1, NRS2 and MybSt1 actually occurs in plants, ChIP analysis was performed using antibody . These rd NACRS) A, the prd NACRS) B, and thd NACRS) C. UnfortMdCBF2 promoter in three regions [Interestingly, there are regions where the binding site is present but no PCR product amplification is detected A–C. This regions . It is lMdERF3 (ETHYLENE RESPONSE FACTOR) promoter and activates its transcription. MdNAC047 regulates ethylene production at least partially in an MdERF3-dependent pathway [LeACS2 and LeACS4 [ATERF-1 (AT4G17500), ORA47 (AT1G74930), ACS6 (AT4G11280), BBX18 (AT2G21320), SOBIR1 (AT2G31880), and CPNB2 (AT3G13470) as well as ANAC074 all can respond to ethylene , which suggests they may be involved in the ethylene signaling pathway.Accumulating evidence indicates that NAC gene family members are involved in regulating ethylene signals ,36. MdNA pathway . SNAC4 ad LeACS4 . It is wATERF-1, ORA47, and ACS6, demonstrating that ANAC074 may be as a regulator functioning upstream of ethylene synthesis genes. However, we did not detect the interaction between ANAC074 and the promoters of these three genes using the ChIP experiment in 3-week-old seedlings under normal growth condition. On the one hand, there may be functional redundancy between ANAC074 and other NACs under normal growth conditions in Arabidopsis. It is possible that the promoters of ATERF-1, ORA47, and ACS6 are preferentially identified by ANAC069 or other NACs rather than ANAC074 when Arabidopsis grows normally. On the other hand, the interaction between ANAC074 and the promoter of ATERF-1, ORA47, or ACS6 in Arabidopsis may be determined by the growth period and the environmental conditions of the plants. Perhaps this interaction occurs only during specific growth stage or under certain adverse conditions. Further study was required to reveal when and under what conditions ANAC074 binding to the promoters of ATERF-1, ORA47, and ACS6 occurs in Arabidopsis thaliana.ATERF-1 (AT4G17500) and ORA47 (AT1G74930) both belong to the ERF/AP2 transcription factor family, which plays a crucial role in the ethylene signaling pathway ,40. ATERArabidopsis transcription factor NAC2 is involved in the salt stress response through the ethylene signaling pathway [MdERF3-ethylene-salt tolerance” regulatory pathway, which provide new insight into the link between ethylene and salt stress [BBX18 (AT2G21320), SOBIR1 (AT2G31880) and CPNB2 (AT3G13470) via NRS1, NRS2 or MybSt1 in Arabidopsis, respectively. The function of BBX18 and SOBIR1 in Arabidopsis response to stress has been confirmed. Heat stress responsive gene BBX18 plays a negative role in thermotolerance [BBX18 displayed both increased basal and acquired thermotolerance in the transgenic plants, while over-expression of BBX18 reduced tolerance to heat stress in transgenic lines [ANAC074 was down-regulated gradually by heat (37 °C) treatment compared with the normal condition (22 °C) , which suggested ANAC074, as an potential upstream regulator of BBX18, is likely to play an important role under heat stress. In addition, SOBIR1 (AT2G31880) participates in regulating biotic stress response including Magnaporthe oryzae [SOBIR1 in biotic stress has been confirmed and whether ANAC074 also plays a role in biotic stress requires further studies.Some NAC transcriptional factors are involved in biotic or abiotic stress response through the ethylene signaling pathway. For example,  pathway . An et at stress . In thisolerance . Under-eic lines . At the e oryzae  and funge oryzae . The funSince the NACRS motif, containing the “CACG” core sequence, has been identified to be recognized by a lot of NAC TFs ,19,20. OArabidopsis [Arabidopsis NAC family was divided into ten major polytomies by collapsing branches with bootstraps <40% [Researchers identified 117 NAC genes in bidopsis . On the aps <40% . To studaps <40% . Some NAAll in all, the knowledge of NACs that can bind to MybSt1, NRS1, and NRS2 besides NACRS will contribute to revealing the function of NACs in depth.cis-regulatory elements bound by ANAC074.A random short DNA sequence insertion library was constructed according to Ji et al. as the prey library . The ORFThe pHIS2 plasmids were rescued from the positive clones identified by Y1H analysis ,24, and HIS3 gene in pHIS2. These pHIS2 constructs were respectively co-transformed with pGADT7-ANAC074 into Y187 yeast cells to study their bindings to ANAC074 using Y1H [The MybSt1-binding element “GGATA”, ARR1AT motif “NGATT”, and LTRE-1 motif “CCGAAA” were predicted in these insertion sequences. Three tandem copies of “GGATA”, “TGATT”, and “CCGAAA” were respectively cloned into MCS to drive sing Y1H ,24.TTAGTTTCGGG” and “CCCTGAATCTCGGG”, were used in serial deletion analysis. Three tandem copies of these deletions were cloned into pHIS2, and respectively reacted with ANAC074 using Y1H analysis [The sequences containing novel DNA motifs were serially deleted to determine the exact DNA sequence recognized by ANAC074. Since the two flanking sequences of the random insertion sequence may also be part of novel DNA motifs, three bases of each flanking sequence together with the insertion sequence, i.e., “CCCanalysis ,24. The GCGTT”, “TAGGG”, “GGGGG” ; “TCATC”, “GACGC”, “TCCGA” ; “TTATA”, “GTCGA” and “TTCGC”  were cloned into pHIS2 vector (see The two novel motifs were designed as NRS1 and NRS2 (NRS: NAC Recognized Sequence). Three tandem copies of the NRS1 sequence “TAGTT”, NRS2 “GAATC” and the MybSt1 motif sequence “GGATA” together with their respective mutants “GGGGG”, NRS2 “GAATC” and its complete mutant “TCCGA”, the MybSt1 motif “GGATA” and its complete mutant “TTCGC” were respectively fused to the minimal 35S promoter (−46 to +1) to drive the GUS reporter gene   were tiled on Murashige & Skoog  agar medium. The procedure of transformation followed the manual for the Biolistic PDS-1000/He Particle Delivery System (Bio-Rad). For coating particles with DNA, 50 μL gold powder suspension with a diameter of 1 μm (60 mg/mL), 5 μL plasmid DNA (1 ug/uL), 50 μL CaCl2 (2.5 M), and 20 μL spermidine (0.1 M) was added and then proceeded as follows. Vortex for 10 min, then let sit for 1 min. Spin down briefly (~10 s) to settle loose gold and abandon supernatant. Wash once with 140 μL 70% ethyl alcohol, then spin down briefly. Wash once with 140 μL of 100% ethyl alcohol, then resuspend gold pellet in 48 μL 100% ethyl alcohol. Vortex well before spreading the coated gold out on each flyer. The transformation conditions were 9 cm target distance, 1100 psi helium pressure, and 2 times for the number of bombardments. Transfer to a MS agar medium after bombardment, incubate at 24 °C in the dark for 2 days before examining GUS activity. GUS activity levels were determined according to Jefferson [Each of these reporters was co-transformed with effector into tobacco leaves using a particle bombardment method . Firstly, detached leaves from tobacco  happen to be the target genes of ANAC069 [The sequences of MybSt1, NRS1, and NRS2 were searched using the Patmatch program on Tair  to find  ANAC069 . We specAT4G17500  containing NRS1 “TAGTT” (pHIS2-NRS1p+), without “TAGTT” (pHIS2-NRS1p-) or with NRS1 complete mutant “GGGGG” (pHIS2-NRS1pm) driving HIS3 gene, were respectively generated as reporter vectors  containing NRS2 “GAATC” (pHIS2-NRS2p+), without “GAATC” (pHIS2-NRS2p-) or NRS2 complete mutant “TCCGA” (pHIS2-NRS2pm) driving HIS3 gene were respectively generated as reporter vectors  containing MybSt1 “GGATA” (pHIS2-MYBp+), without “GGATA” (pHIS2-MYBp-) or MybSt1 complete mutant “TTCGC” driving HIS3 gene were also respectively generated as reporter vectors (see The pHIS2 harboring the truncated promoter of AT4G17500 (same as used in Y1H) containing NRS1 “TAGTT” (pCAM-NRS1p+), without NRS1 (pCAM-NRS1p-) or NRS1 complete mutant “GGGGG” (pCAM-NRS1pm), were respectively generated (see AT1G74930 (same as used in Y1H), which contained NRS2 “GAATC” (pCAM-NRS2p+), without “GAATC” (pCAM-NRS2p-) or NRS2 completely mutant “TCCGA” (pCAM-NRS2pm) were respectively fused to the minimal 35S promoter (−46 to +1) to substitute 35S promoter to drive GUS as reporter vectors (see AT4G11280 (same as used in Y1H) containing MybSt1 motif “GGATA” (pCAM-MYBp+), without “GGATA” (pCAM-MYBp-) or MybSt1 complete mutant motif “TTCGC” (pCAM-MYBpm) were also respectively generated.  into tobacco leaves using the particle bombardment method , respectively. GUS activity levels were determined according to the method of Jefferson . Three iArabidopsis thaliana in the Columbia ecotype background. The 3-week-old transgenic plants were used for ChIP assay following the procedures of Haring et al. [To confirm the binding of ANAC074 to NRS1, NRS2, and MybSt1 actually occurs in plants, ChIP analysis was performed. The coding regions of ANAC074 without the termination codon were ligated in frame to the N-terminal of GFP driven by 35SCAMV promoter to generate the ANAC074::GFP fusion gene, and transformed into g et al. . BrieflyTo compare the binding activity of ANAC074 to NACRS, MybSt1, NRS1, and NRS2, the effector construct pROKII-ANAC074 was co-transformed with each reporter constructs pCAM-NRS1, pCAM-NRS2, pCAM-MybSt1 or pCAM-NACRS into tobacco leaves using particle bombardment method . The empty pCAMBIA1301 was transformed and used as positive control. Three independent experiments were performed.p < 0.05).Statistical analyses were carried out using SPSS 16.0  software. Data were compared using Student’s t test. Asterisks indicate a significant difference compared with NACRS  at the site between SMART III Sequence and CDS III Sequence using the infusion method. Each of the 10 effector vectors containing NAC gene was co-transformed with reporter vectors pHIS2-NRS1-D4, -D7, -D6, -D8, pHIS2-NRS2-D4, -D5, -D6, -D8, or pHIS2-MybSt1 into yeast cells. The bindings of the 10 NACs to the elements harboring in pHIS2 were analyzed using the Y1H system ,24.In conclusion, we identified the MybSt1-binding element and two novel motifs NRS1 and NRS2, which are bound by NACs, and further showed that ANAC074 may be a regulator functioning upstream of ethylene synthesis genes and stress responsive genes via binding to the MybSt1, NRS1 or NRS2 element. Identifying the cis-acting element recognized by a transcription factor is crucial to parse its function, and the identification of the MybSt1, NRS1, and NRS2 motifs will be beneficial to future NAC gene function analyses.


HsMar1, a human DNA transposon, differs in presence or absence of endogenous HsMar1 copies. In the case where HsMar1 transposition is regulated, the number of repetitive DNA sequences issued by HsMar1 and distributed in the human genome makes HsMar1 a good candidate to regulate neighboring gene expression by epigenetic mechanisms.Both classes of transposable elements (DNA and RNA) are tightly regulated at the transcriptional level leading to the inactivation of transposition via epigenetic mechanisms. Due to the high copies number of these elements, the hypothesis has emerged that their regulation can coordinate a regulatory network of genes. Herein, we investigated whether transposition regulation of HsMar1 copy was inserted in HeLa (human) and CHO (hamster) cells and its genomic excision monitored. We show that HsMar1 excision is blocked in HeLa cells, whereas CHO cells are competent to promote HsMar1 excision. We demonstrate that de novo HsMar1 insertions in HeLa cells (human) undergo rapid silencing by cytosine methylation and apposition of H3K9me3 marks, whereas de novo HsMar1 insertions in CHO cells (hamster) are not repressed and enriched in H3K4me3 modifications. The overall analysis of HsMar1 endogenous copies in HeLa cells indicates that neither full-length endogenous inactive copies nor their Inverted Terminal Repeats seem to be specifically silenced, and are, in contrast, devoid of epigenetic marks. Finally, the setmar gene, derived from HsMar1, presents H3K4me3 modifications as expected for a human housekeeping gene.A recombinant active HsMar1 are not similarly regulated by epigenetic mechanisms. Old HsMar1 are generally detected as lacking epigenetic marks, irrespective their localisation relative to the genes. Considering the putative existence of a network associating HsMar1 old copies and SETMAR, two non-mutually exclusive hypotheses are proposed: active and inactive HsMar1 copies are not similarly regulated or/and regulations concern only few loci (and few genes) that cannot be detected at the whole genome level.Our work highlights that de novo and old The online version of this article (10.1186/s12863-019-0719-y) contains supplementary material, which is available to authorized users. Transposable elements (TEs) are mobile genetic elements representing a prevalent part of eukaryotic genomes, including human. They are known to display significant genetic consequences, promoting various types of mutation such as disrupting genes (upon neo-insertions) or inducing recombination between homologous sequences at divergent loci. Beyond these foreseeable consequences in view of TEs mobility and/or amplification, other more unexpected effects relying on TEs occurrence were illustrated during the two last decades. First, TEs possess their own regulatory sequences, and then could alter the normal expression pattern of neighboring genes. It has also been shown that amplification of various TEs family can provide new gene regulatory networks . FinallyThe genetic consequences of TEs moving around genomes via an RNA intermediate  were widely illustrated in human since this class of TEs represents 97% of human TEs and because some are still active and responsible for several genetic diseases. 0.3% of human TE insertions have been suggested for causing a disease, i.e. one insertion in every 20–100 live births, and approximately 96 new transposition events are directly linked to single-gene diseases . BecauseSleeping Beauty (SB) transposition is easier when the transposon is methylated [trans [Less is known about the genetic consequences of TEs moving around genomes via a DNA intermediate  that represent 3% of human TEs. Studies in other mammalian species suggest that transposons are also controlled by epigenetic mechanisms, potentially differently than retro-transposons. For instance, thylated , 12 and d [trans . This coSeveral groups seek to understand how TEs silencing takes place upon genome invasions and exaptation of elements. This requires developing new biological models to mimics TE invasion in a naive context, to address de novo insertions. For the human L1 retro-element, this was done by studying pseudo-founder transgenic mice and their progeny . De novomariner elements were amplified 45 My ago and the HsMar1 sub-family is probably the only one to display a current quite active copy. The “modern” human genome contains about 250 defective HsMar1 copies , beside a domesticated copy, which codes (together with a histone-methylase gene) a chimeric protein called SETMAR [HsMar1 transposase, except for the ability to cleave the first DNA strand upon excision [HsMar1 transposition around the human genome. Next to the full-length copies, the human genome also contains thousands of miniature HsMar1  and solHsMar1 active copies provides opportunities to perform de novo insertions in either naive genomes (non-human) or HsMar1-containing genomes (human). Doing so, we demonstrate that de novo HsMar1 insertions in HeLa cells (human) undergo rapid silencing by cytosine methylation and affixing of H3K9me3 marks, whereas de novo HsMar1 insertions in CHO cells (hamster) are not repressed and enriched in H3K4me3 instead of H3K9me3 marks, and transcriptionally active. The overall analysis of HsMar1 old endogenous copies indicates that neither full-length endogenous inactive copies nor TIRs seem to be specifically silenced; in contrast, they are devoid of epigenetic marks. Taking into account the putative existence of a network associating HsMar1 old copies and SETMAR, two non-mutually exclusive hypotheses are proposed: active and inactive HsMar1 copies are not similarly regulated or/and regulations concern only few loci (and few genes) and cannot be detected at the whole genome level.The availability of “reconstructed” HsMar1 element, a tool was designed to examine the first step of transposition, the excision. An excision cassette was composed of an active HsMar1 copy inserted in opposite orientation between the CMV promoter (pCMV) and the GFP coding sequence, thus preventing the expression of GFP. LoxP sequences were added at both HsMar1 ends. Upon HsMar1 excision , pCMV allows the expression of GFP in recombinant cells , the whole being enframed by PiggyBac ends. The excision cassette was introduced in HeLa and CHO genomes using PiggyBac transposition. Clonal recombinant cell lines were established and the presence of the excision cassette was controlled by PCR and sequencing. Each recombinant line contained at least one copy of the excision cassette , excision assays were performed using various amount of HSMAR-RA or CRE expressing plasmids. The empty pCS2 plasmid was used as control. Among the 22 established HeLa cell lines, none provided GFP expression when transfected with an HSMAR-RA expressing plasmid, and only eight provided low GFP expression when transfected with a CRE expressing plasmid. No GFP expression was detected in controls Fig. . In conttrans strongly supports the idea that the recombinant HsMar1 cassette is expressed in CHO, allowing the transcription and the translation of the transposase encoded by the recombinant copy. This was confirmed by RT-PCR . In contrast, HeLa cells used as control showed expression of HsMar1 transposase mRNA from endogenous HsMar1. Human cells contain about 250 full-length HsMar1 copies, all of them containing deletions or mutations preventing the production of an active transposase. The detection of HsMar1 mRNA resulted from the activity of its endogenous promoter, which is sufficient to drive the transcription of a remnant mRNA [The detection of GFP signals in the absence of recombinases (HSMAR-RA or CRE) provided in ant mRNA . This waHsMar1 excision was due to a lack of active transposase (HSMAR-RA) expression in HeLa cells. As expected, the active transposase is correctly expressed under our experimental conditions, in accordance with the amount of plasmid used for transfection  influences the capability of transposition of neo-inserted copies.The analysis of sequences surrounding the integration sites in HeLa cells did not show specific DNA elements as defined in ENCODE database, which could explain the absence of transposition in these cells. All together these first results strongly support that the genetic background of cells . Three regions within the nce Fig. a. In HeLGAPDH was used as an internal control, TAF7 as associated with H3K9me3 and EIF4α as associated with H3K4me3, both in HeLa and CHO cells [EIF4a matched the expected data. In contrast, TAF7 is not associated to H3K9me3. Since nothing is known about TAF7 regulation in hamster, we assume that it is different from that observed in human cells without impairing our analysis. Our results clearly indicated that both GFP and pCMV sequences had obtained H3K9me3 marks in recombinant HeLa cells, contrary to recombinant CHO cells. Thus, the inactivation of the HsMar1 cassette in HeLa cells is supported by both DNA methylation and H3K9me3 modification.DNA methylation is often sustained by histone post-translational modifications, especially with deacetylation of histones H3 and H4, loss of H3K4me3, gain of H3K9me3 and H3K27me3 , 21. We HO cells , 23. HeLHsMar1 copy added within genomes by recombination. We wanted to verify whether the old endogenous HsMar1 copies or TIRs behave as the recombinant copy does. The human genome contains 231 “full-length” HsMar1 elements, which vary in length between 1000 and 1300 bp. The ancestral reconstructed sequence is 1287 pb long, and the current genomic full-length copies are inactive since they accumulated various micro-mutations (insertions-deletions-substitutions). The 231 full-length elements display 80 to 95% identity with the ancestral sequence. Among them, 88 (about 39%) colocate within genes. We have also looked at HsMar1 TIRs (HsTIRs) that are short inverted sequences of 30 bp ending the transposon. They are known to act as target sequences for the transposase during the transposition process and, for the 5’TIR, to be part of the HsMar1 endogenous promoter [HsMar1 copies, those organized in miniature tandem repeats and solo TIRs. 3952 have conserved the ancestral length of 30 bp of which 39,9% colocate within genes. Other TIRs are shorter, but remain detectable up to 15 bp. If the endogenous copies are regulated as the recombinant copy does, the vicinity of genes evokes a potential role in regulating gene expression. Surprisingly, the 30 bp TIR sequences were well conserved by evolution for 45 My and even if only 6.2% are identical to the ancestral sequence, 67% of the changes relate to positions 24–25, which correspond to a CpG dinucleotide, suggesting that HsMar1 TIRs have been subjected to CpG shortage. Since mCpG are frequently deaminated to TpG on both strand, the evolutionary effect will be a progressive loss of CpG dinucleotides, and a concomitant replacement by TpA dinucleotides, precisely what is observed on HsTIRs (not shown). Small HsTIRs may equally be affected by mCpG. From ENCODE database, we have analysed the HsTIRs CpG methylation environment in the previously published HeLa-S3 genome : less mCpG are detected around HsTIRs than anywhere else in the genome.The results presented so far only concerned the recombinant promoter . The humHsMar1 promoter were found methylated  appear more methylated than HsMar1 outside genes (blue line) but these differences were not statistically significant Fig. b. The meSETMAR, a fusion gene in which exon 3 is made of a full-length HsMar1 element lacking the 5’ TIR  are associated or not with H3K4me3 or H3K9me3 marks. Here, both histone modifications were searched within 20-kb windows around TIR sequences by 2 kb intervals . These results differed highly from randomly chosen control genes (79 and 48% respectively). All revealed that SETMAR promoter was marked by H3K4me3 but not by H3K9me3  is prevented in human cells while it works in cells naive for HsMar1 (CHO in our study). This clearly demonstrates that beyond the transposase expression regulation, the mechanism of the transposition itself is regulated for TEs transposing via a DNA intermediate in somatic cells. Inactivation of de novo HsMar1 elements in human cells is mediated by CpG methylation and deposition of H3K9me3 marks that are conventional for TEs inactivation [trans of conserved copies and raises two issues: how does the cell “recognize” the recombinant active copy and what is the regulation of the old, degenerated copies? An attempt to answer the first question could be provided by naive CHO cells since they are able to prevent HsMar1 excision, sustaining a role for piRNA acting in trans in human TEs regulation [Interactions between host-genomes and TEs have been studied during the two past decades regarding the potential of TEs to create regulatory networks via the coding of various regulatory elements that enables them to regulate genes expression , 24. Thectivated , it is ntivation . This megulation , 31; in gulation , 33. Thigulation , 35 and gulation . Beyond gulation .HsMar1. Overall approaches have indicated that both HsMar1 promoters and TIRs are hypo-methylated, probably the result of former methylations, followed by CpG shortage undergoing at position 24–25 of TIRs. In addition, endogenous HsMar1 display specifically low modifications of histone marks when compared to the whole genome (difference of a factor about ten), but respecting a prevalence of H3K4me3 over H3K9me3. Finally, the SETMAR gene display the same epigenetic regulation as others human housekeeping genes. The fact that HsMar1 has escaped host silencing after the primitive burst of amplification may appear counterintuitive, but many studies indicate that TEs are not as robustly silenced as commonly assumed  the cis-elements of the regulatory network, and SETMAR (after being domesticated) could be the actor of the regulation.In their recent publication, Chuong et al.  exemplifHsMar1 are differently regulated by epigenetic mechanisms. The mechanism that prevents the amplification of de novo insertions is highly efficient, suggesting at least an action in trans that may be supported by RNA interference. Old HsMar1, that could constitute the SETMAR network, are generally detected as lacking epigenetic marks, whatever their location relative to the genes. Despite the growing number of studies that have demonstrated SETMAR to be a main actor of genome stability in human cells, and the TIRs to be efficient binding sites, there are yet no data confirming that SETMAR may influence nearby gene expression. Future studies need to address this question, assessing this so promising network.Our work highlights that de novo and old HsMar1 excision cassette  in pMalc2 (New England Biolabs). The resulting fusion MBP-HSMAR-RA and HSMAR-RA were cloned in pCS2 plasmid. After PCR amplification, CRE ORF was cloned in pCS2.All constructs were controlled by sequencing (Eurofins MWG Biotech). DNA preparations to be transfected were performed with Nucleobond Xtra EF kit (Macherey-Nagel).Genomic DNAs needed for analyses were extracted and purified with Nucleospin Tissue kit (Macherey-Nagel).PCR primers used in the study are listed in Additional file HeLa  and CHO  cells were cultured as recommended by the Leibniz Institute DSMZ German Collection of Microorganisms and Cell Cultures. Transfections were performed at 60–70% cell confluence with JetPEI (Polyplus transfection). To prepare recombinant HeLa and CHO cell lines (containing the excision cassette), cells were transfected with 1 μg of mPB and pHsMar1. 48 h post-transfection, 1/10 of transfected cells were seeded in B10 plates with medium supplemented with 1 μg/ml or 10 μg /ml puromycin for HeLa and CHO cells respectively. After 2 weeks of selection, individual clones were selected and amplified.Cells were resuspended in Laemmli buffer. Proteins were separated in SDS-PAGE, transfered onto Hybond-ECL membrane , probed with MBP antibody (New Englands Biolabs) and revealed with HRP secondary antibody with ECL reagent .Excision assays were performed after 5 to 7 passages for individual established clones. 100,000 cells of recombinant HeLa or CHO were transfected with JetPEI and 150 or 1050 ng of pCS2-MBP-HSMAR-RA or 500 ng of pCS2-CRE, completed to 2 μg with pCS2 to prevent data variations due to transfected DNA amount variations. Control assays were performed with 2 μg of pCS2 or pCS2-GFP. The expression of GFP depends on its juxtaposition of the CMV promoter next to the GFP ORF which contains the GFP ATG. The in silico reconstitution of HsMar1-GFP fusion transcript did not allow the translation of GFP.GFP expression (for control or after excision) was observed by photonic microscopy 3–5 days later. The excision sites obtained after transposition or CRE recombination (HeLa or CHO recombinant cell lines) were amplified by PCR with e-primers using 50 ng of genomic DNA and FlexiTaq, according to the manufacturer (Promega). PCR products were cloned in pGEMT and sequenced by Eurofins MWG Biotech.HsMar1 TIRs were amplified using 50 ng of treated DNA and the m-primers designed by Methprimer  was treated with sodium bisulfite according to Epimark bisulfite conversion kit (New England Biolabs). Fragments adjacent to the 5′ and the 3′ recombinant GAPDH, TAF7 and EIF4a promoters, 5′ and 3’ HsMar1 adjacent sequences, using the c-primers  = 2-Δ ΔCt.Chromatin was extracted from recombinant HeLa and CHO cell lines according to Browne’s protocol . ChIPs HsMar1 TIRs and complete HsMar1 sequences were retrieved form hg38 assembly using Geneious software. We found 12,378 TIRs of which 3952 were 30 bp long, and 231 complete HsMar1 (length 1–1.3 kb). Intersect and Fetch Closest Feature tools available on the Galaxy plaform “Galaxeast” were used to analyse the association of full-length or HsMar1 TIRs with the epigenetic marks H3K4me3 and H3K9me3. HsMar1 TIR inside genes were defined as containing as least one base in common with transcribed genes, i.e. coding protein, miRNA and lncRNA genes. The transcripted genes were retrieved from gencodebasic-v27 UCSC database. HsMar1 TIR outside genes corresponded to TIRs excluded from the precedent criteria. To verify whether the association of the different histone marks with HsMar1 TIRs was random or not, 2000 random 30 bp sequences were extracted from hg38 reference human genome whatever their sequences and locations by an in home-made program written in R . The only constraint was that the number of random 30 bp sequences issued from each chromosome is proportional to the length of the chromosomes. These sequences were analysed as done for HsMar1 TIRs for their association with H3K4me3 or H3K9me3. 250 random genes were extracted with the Galaxy “random select lines” tool from the gencodebasic-v27 UCSC database.ENCODE ChIP-seq data were used for histone marks analysis (H3K9me3 and H3K4me3) of HeLa-S3 cells  . HsMar1 HsMar1 TIRs or shuffle TIRs were compared by a χ2 test. The distributions of histone marks around TIRs or full-length HsMar1 copies were compared by a χ2 test.The percentage of TIRs/histone marks association between Additional file 1:Data S1. Recombinant HsMar1 insertion sites in HeLa and CHO cell lines. Data S2. Inter-plasmidic active transposition of HSMAR-RA. Table S1. Primer list. (DOCX 123 kb)


Candida albicans, but it has not been experimentally demonstrated.The calcium/calcineurin signaling pathway is mediated by the transcription factors NFAT (nuclear factor of activated T cells) in mammals and Crz1  in yeasts and other lower eukaryotes. A previous microarray analysis identified a putative Crz1-binding motif in promoters of its target genes in CaCRZ1 was generated through CRISPR/Cas9 approach. Transcript profiling was carried out by RNA sequencing of the wild type and the inactivation mutant for CaCRZ1 in response to 0.2 M CaCl2. Gene promoters were scanned by the online MEME (Multiple Em for Motif Elicitation) software. Gel electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis were used for in vitro and in vivo CaCrz1-binding experiments, respectively.An inactivation mutant for Saccharomyces cerevisiae ScCrz1-binding motif [5′-GNGGC(G/T)CA-3′]. EMSA and ChIP assays indicate that CaCrz1 binds in vitro and in vivo to both motifs in the promoter of its target gene CaUTR2. Promoter mutagenesis demonstrates that these two CaCrz1-binding motifs play additive roles in the regulation of CaUTR2 expression. In addition, the CaCRZ1 gene is positively regulated by CaCrz1. CaCrz1 can bind in vitro and in vivo to its own promoter, suggesting an autoregulatory mechanism for CaCRZ1 expression.RNA sequencing reveals that expression of 219 genes is positively, and expression of 59 genes is negatively, controlled by CaCrz1 in response to calcium stress. These genes function in metabolism, cell cycling, protein fate, cellular transport, signal transduction, transcription, and cell wall biogenesis. Forty of these positively regulated 219 genes have previously been identified by DNA microarray analysis. Promoter analysis of these common 40 genes reveals a consensus motif [5′-GGAGGC(G/A)C(T/A)G-3′], which is different from the putative CaCrz1-binding motif [5′-G(C/T)GGT-3′] identified in the previous study, but similar to CaCrz1 differentially binds to promoters of its target genes to regulate their expression in response to calcium stress. CaCrz1 also regulates its own expression through the 5′-TGAGGGACTG-3′ site in its promoter.Video abstract Candida albicans is one of the most important human yeast pathogens. A previous microarray analysis identified a putative CaCrz1-binding motif in promoters of its target genes in C. albicans, but it has not been experimentally demonstrated. Using a new technology, RNA sequencing, we have identified 219 genes that are positively, and 59 genes that are negatively, controlled by CaCrz1 in response to calcium stress in this study. We have also revealed and demonstrated experimentally a novel consensus CaCrz1-binding motif [5′-GGAGGC(G/A)C(T/A)G-3′] in promoters of its target genes. In addition, we have discovered that CaCrz1 can bind to its own promoter, suggesting an autoregulatory mechanism for CaCRZ1 expression. These findings would contribute to our further understanding of molecular mechanisms regulating calcium homeostasis.Calcium ions regulate many cellular processes in both prokaryotes and eukaryotes, from bacteria to humans. Regulation of intracellular calcium homeostasis is highly conserved in eukaryotic cells. Gene expression in response to calcium stress is controlled by the calcium/calcineurin signalling through the transcription factors NFAT (the nuclear factor of activated T cells) in mammals and Crz1  in yeasts and other lower eukaryotes. Extracellular calcium stress causes an increase in cytosolic calcium, which leads to the binding of calcium ions to calmodulin that triggers activation of the protein phosphatase, calcineurin. Activated calcineurin dephosphorylates Crz1 in the cytosol, which leads to nuclear localization of Crz1 and its binding to promoters of its target genes to regulate their expression. Saccharomyces cerevisiae, an increase in cytosolic calcium triggers the calmodulin/Ca2+ binding and activation of the protein phosphatase, calcineurin. Activated calcineurin dephosphorylates ScCrz1 in the cytosol, which leads to nuclear localization of ScCrz1 and its binding to promoters of its target genes, including the vacuolar calcium pump gene ScPMC1, the ER/Golgi calcium pump gene ScPMR1 and the ScRCH1 gene encoding the negative regulator of calcium uptake in the plasma membrane  is dependent on both calcineurin and CaCrz1 in albicans . Both mialbicans –36. Howealbicans .ThereforC. albicans strains and plasmids used in this study were described in Table C. albicans strain SN148 was used as the parent strain to construct the CRISPR inactivation mutant for CaCRZ1 through the CRISPR [Clustered Regularly Interspaced Short Palindromic Repeat)/Cas9] approach  as well as the repair DNA primers CRZ1-RFand CRZ1-RR containing 40-bp homologous regions flanking the SgRNA sequence , AGC codon (L24S), TCC codons   and lacZ_ORF_R(KpnI) from the plasmid pGP8 , which is different from the putative CaCrz1-binding consensus sequence [5′-G(C/T)GGT-3′] previously identified through DNA microarray [S. cerevisiae ScCrz1-binding motif [5′-GNGGC(G/T)CA-3′]  . Therefoe MoCrz1 , 50.CaUTR2 C(T/A)G-3′] from the MEME analysis described above, we found one putative CaCrz1 binding motif, the 5′-TGAGGCGTTG-3′ region in the complementary sequence of the 5′-C(− 342)AACGCCTCA(− 333)-3′ site in the promoter of one of the CaCrz1 target genes, C. albicans does not follow the universal genetic code, by translating the CTG codon into serine instead of leucine  in promoters of CaCrz1 target genes. In addition, CaCrz1 binds to its own promoter and shows an autoregulatory mechanism for Additional file 2: Figure S1. Construction and phenotypes of CRISPR mutants for CaCRZ1.Figure S2. Alignment between the amino acid sequences of the wild type and the codon optimized version of CaCrz1. Figure S3. Expression and purification of the codon optimized and His6-tagged CaCrz1 protein in bacterial cells. Figure S4. Differences between CaCrz1 and ScCrz1. Table S1. Primers used in this study.Additional file 3. Promoter analysis of 219 genes whose expression is positively regulated by CaCrz1#Additional file 4. Comparison of calcium-specific and Crz1-dependent genes in Saccharomyces cerevisiae and Candida albicans.


Leptospirainterrogans and genetic mechanisms controlling its virulence. Transcription is the first step in gene expression that is often determined by environmental effects, including infection-induced stresses. Alterations in the environment result in significant changes in the transcription of many genes, allowing effective adaptation of Leptospira to mammalian hosts. Thus, promoter and transcriptional start site identification are crucial for determining gene expression regulation and for the understanding of genetic regulatory mechanisms existing in Leptospira. Here, we characterized the promoter region of the L. interrogans clpB gene (LiclpB) encoding an AAA+ molecular chaperone ClpB essential for the survival of this spirochaete under thermal and oxidative stresses, and also during infection of the host. Primer extension analysis demonstrated that transcription of clpB in L. interrogans initiates at a cytidine located 41 bp upstream of the ATG initiation codon, and, to a lesser extent, at an adenine located 2 bp downstream of the identified site. Transcription of both transcripts was heat-inducible. Determination of LiclpB transcription start site, combined with promoter transcriptional activity assays using a modified two-plasmid system in E. coli, revealed that LiclpB transcription is controlled by the ECF σE factor. Of the ten L. interrogans ECF σ factors, the factor encoded by LIC_12757 (LA0876) is most likely to be the key regulator of clpB gene expression in Leptospira cells, especially under thermal stress. Furthermore, clpB expression may be mediated by ppGpp in Leptospira.There is limited information on gene expression in the pathogenic spirochaete  Leptospira interrogans is one of the many pathogenic species of the genus Leptospira and is composed of several strains that can cause leptospirosis in mammals, including humans. It has been reported that over 1 million human cases of severe leptospirosis occur worldwide each year, with approximately 60,000 deaths from this disease ATP  using polynucleotide kinase (Promega), denatured at 75 °C for 10 min, and annealed at 58 °C for 20 min. This was followed by the addition of extension buffer , 1 mM dNTPs, 20 U RNase-In (Promega), and 10 U of the AMV reverse transcriptase (Promega). The final reaction volume was 25 μL. After incubation at 42 °C for 30 min, 2.5 μg of RNase H (Promega) were added, and incubation was continued at 37 °C for 15 min. cDNA was precipitated in ethanol with 0.35 M sodium acetate (pH 5.5). The samples were then resuspended in a loading buffer  and resolved on a 7 M urea, 8% polyacrylamide sequencing gel, run in parallel with a sequencing reaction performed with the same labeled primer and pClpBLi plasmid DNA  plasmid, alone or together with pAC-rpoE4, was introduced into E. coli MC4100ΔclpB mutant cells, and LiclpB expression was explored. Cells carrying p32σ-LiclpB (pGB2-ClpBLi) were used as a control for the heat-inducible expression. To detect ClpBLi in E. coli cultures, Western blotting was performed as described by [Li158-334 serum [2O2.The pClpBribed by  using anclpB in the pathogen L. interrogans. Primer extension analysis, in combination with promoter transcriptional activity assays permitted us to identify a major heat-inducible promoter located upstream of the clpB encoding sequence. Our results clearly indicate that the LiclpB transcription is under control of one of the σE-type factors, which have been also reported to regulate the biosynthesis of virulence factors in some bacterial pathogens. It has to be noted that ClpBLi is among the known leptospiral virulence factors. It is likely that the product of LIC_12757 plays a key role in clpB transcription in L. interrogans cells. Furthermore, we show that the ppGpp alarmone, which also allows expression of virulence genes in some bacterial pathogens, may mediate clpB expression in Leptospira cells. We believe that findings presented in this study help to improve our knowledge of regulatory mechanisms used by Leptospira and may also have an impact on the understanding of the leptospiral virulence mechanisms that allow this pathogen to adapt to its host organisms.To our knowledge, this is the first study providing insight into the transcriptional regulation of


Grade IV glioma, glioblastoma (GBM), is characterized by diffuse infiltration through brain parenchyma and invasion into the adjacent brain structures. Extracellular Matrix (ECM) is an important component of the tumor microenvironment; and a disrupted ECM is essential for GBM infiltration and invasion. The infiltrative phenotype contributes to GBM resistance to surgical removal and chemo/radiotherapy , therefovia mCpG-dependent KLF4 activity. To identify the transcriptional network regulated by KLF4-mCpG interactions, whole genome RNA-sequencing (RNA-seq) was performed, demonstrating significant transcriptional regulation of 613 genes—among which 82% (N = 500) were affected by KLF4 WT expression only. Among the 500 genes, 62% (N = 308) had upregulated gene expression, suggesting they were activated by KLF4 via a methylation-dependent mechanism. This was validated by comparing gene expression in cells treated with 5-aza-2′-deoxycytidine (5-Aza), a DNA methytransferase inhibitor. To identify KLF4-mCpG direct target genes, whole genome chromatin immunoprecipitation-sequencing (ChIP-seq) was performed and overlapped with the RNA-seq datasets. The study found 116 target genes directly activated by mCpG-dependent KLF4 binding, with the majority (72%) of these genes associated with highly methylated cis-regulatory elements, as revealed by the whole genome bisulfite sequencing. In addition to transcriptional transactivation, ChIP-seq analysis of one activating (H3K27ac) and two repressive (H3K9me3 and H3K27me3) histone marks demonstrated that KLF4 WT binding to mCpGs triggered histone modification and chromatin remodeling.In our prior study , we sougcis-regulatory regions of the UGDH gene. To determine whether UGDH expression was upregulated via a methylation-dependent mechanism, 5-Aza was administered and KLF4 binding to the cis-regulatory regions of UGDH was assessed. The study found a complete loss of KLF4 WT binding to UGDH cis-regulatory regions in the presence of 5-Aza, supporting the hypothesis that UGDH transcriptional activation was under the control of KLF4-mCpG interactions.The top 20 genes upregulated by KLF4-mCpG interactions, associated with migration and cytoskeletal reorganization, were selected for detailed functional analysis. Among them, cytosolic enzyme UDP-glucose 6-dehydrogenase (UGDH) is responsible for the production of UDP-α-D-glucuronic acid , an essein vivo studies with intracranial xenografts demonstrated that UGDH knockdown reduced tumor growth, migration, and the expression of critical ECM components (i.e. tenascin C and brevican).Two cellular systems, human GBM U87 cells and GBM neurosphere cells enriched for tumor-initiating cancer stem cells, were utilized to assess the impact of UGDH loss-of-function. Lentiviral transduction with UGDH shRNA dramatically knocked down UGDH expression and resulted in a marked reduction in GAGs, cell migration, and cell motility in both cell lines. Interestingly, hyaluronic acid supplementation (a key cellular GAG) rescued in migration inhibition in UGDH knockdown cells, suggesting that GAG reduction directly impacted migration. UGDH knockdown also resulted in decreased cell proliferation and clonogenicity—illustrated by a decreased ability to form colonies and neurospheres in GBM neurosphere models. Decreased UGDH expression caused a reduction in cyclin E and cyclin D1, leading to delayed G1/G0 to S-phase cell cycle transition. Most importantly, These studies highlight the importance of epigenetic mechanisms, i. e. DNA methylation, in gene activation and GBM malignancy including migration and proliferation. The work from Oyinlade reveals the UGDH enzyme is critical for ECM construction, which in turn affects the invasive and proliferative phenotypes of GBM. Future studies should be aimed at evaluating the anti-tumor potential of targeting UGDH in immunocompetent mouse models, either alone or combined with other therapeutic strategies. Furthermore, additional studies should seek to address whether these findings can be translated to other cancer models such as brain and/or bone metastases.