In jawed vertebrates, oligodendrocytes (OLs) are the myelin-producing glial cells responsible for ensheathment of axons within the central nervous system and are also crucial for remyelination following injury or disease. Olig2 is a crucial factor in the specification and differentiation of oligodendrocyte precursor cells (OPCs) that give rise to mature, myelin-producing OLs in the developing and postnatal CNS; however, its role in adulthood is less well understood. To investigate the role Olig2 plays in regulating gene expression in the adult OL lineage in a physiologically-relevant context, we performed chromatin immunoprecipitation followed by next generation sequencing analysis (ChIP-Seq) using whole spinal cord tissue harvested from adult mice.We found that many of the Olig2-bound sites were associated with genes with biological processes corresponding to OL differentiation , myelination and ensheathment , as well as cell cycle and cytoskeletal regulation. This suggests Olig2 continues to play a critical role in processes related to OL differentiation and myelination well into adulthood. Wrapping axons in myelin sheaths has evolved in jawed vertebrates, gnatostomes, and some invertebrates as a way to increase nerve conduction velocity, enabling more rapid and efficient flow of information within the nervous system ,2. In thDuring embryonic development, OPCs located within the ventricular zones (VZ) of both the brain and spinal cord produce OLs that subsequently relocate and distribute throughout the CNS. In the spinal cord, OLs are derived from both ventral, and to a lesser extent, dorsal sources within the VZ beginning around embryonic day 12.5 and 15, respectively –9. NeuraOL lineage progression is regulated by several stage-specific transcription factors like Olig1, Olig2, Sox10, Nkx2.2 and Nkx6.2 –21. In aOlig2 is a basic helix–loop–helix (bHLH) transcription factor expressed at all stages of OL lineage development and is a major determination factor controlling the specification and differentiation of OPCs ,18. OligOur current understanding of Olig2 involvement in CNS myelination and remyelination in the adult CNS remains incomplete in part because the vast majority of transcriptional targets of Olig2 have been identified using purified cells in culture or from embryonic and postnatal CNS tissues as opposed to adult tissue. Identification of genome-wide direct transcriptional targets of Olig2 in the adult CNS tissue would not only improve our understanding of Olig2 function in OPCs and mature, myelinating OLs but would help us better understand and possibly manipulate myelin production and regeneration following spinal cord injury or demyelinating diseases. To this end, we sought to map the genomic loci targeted by Olig2 in the adult mammalian CNS in vivo using ChIP-Seq from spinal cord tissue from adult mice. We subsequently chose a subset of selected ChIP-Seq targets to validate through independent qPCR experiments (ChIP-qPCR). We also identified enriched motifs, and used in-silico analyses to identify critical pathways and functional networks that were overrepresented among the genes proximal to the target sites in the ChIP-Seq dataset. Our findings provide a new resource and important insights into the gene targets of Olig2 involved in proliferation differentiation and maintenance of neuroglia, especially oligodendroglia, in the adult CNS.Animal usage and manipulations were performed in accordance with the University of Miami Institutional Animal Care and Use Committee and Public Health Service animal care and use guidelines.™ Chromatin Immunoprecipitation System manual  according to manufacturer’s instructions with only a few modifications (see below). Briefly, dissected spinal cords were immediately weighed, cleaned, and minced with a razor blade in ice cold PBS. Minced tissue was then collectively homogenized by passing repeatedly through 18G and 21G needles before crosslinking chromatin with formaldehyde at a final concentration of 1% at room temperature for 10 minutes. Glycine was added to a final concentration of 0.125 M to quench formaldehyde crosslinking, and cells were then pelleted and washed with ice-cold PBS. Cell pellets were resuspended in Lysis Buffer plus Protease Inhibitors at a concentration of 1x10^6 cells / 50 μl. Nuclear lysates were then sonicated using a Branson® 150 Sonifier , yielding chromatin fragments of 50–300 bp. Dynabeads® Protein A/G  were coupled to either 5 μg of Polyclonal Rabbit anti-Olig2 antibody  or Rabbit IgG  by mixing end-over-end for 4 hours at 4°C prior to introduction to lysates. Antibody-conjugated beads were then combined with 500 μl of lysate (~1x10^7 cells) plus 500 μl of Dilution Buffer in 1.5 mL LoBind Eppendorf tubes  and mixed by end-over-end rotation overnight at 4°C. Approximately 1% of the total sheared chromatin was set aside and served as Input Control. Beads containing Protein–DNA complexes were subsequently washed with IP Buffer, formaldehyde crosslinking reversed by heat treatment, and the DNA eluted using a 12 Tube Magnetic Separation Rack . Here, we note that the Magnify™ protocol is designed for use with 0.2 uL tubes, so in all subsequent steps involving end-over-end rotation we instead placed each 1.5 mL LoBind tube on its side and applied gentle agitation using a platform rocker. This small change to the protocol ensured that the beads remained submerged in solution throughout. For all steps involving bead collection via magnetic rack, 1.5 mL LoBind tubes were placed in the rack in such a way as to concentrate bead collection toward the bottom of the tube, again to keep them submerged in solution. Specifically, foam padding was added at the base of the rack to keep the tubes slightly elevated. Input Control samples were processed at the reverse crosslinking step in parallel with the ChIP’d samples. IgG IP’d DNA was analyzed by qPCR (see below) but was not included in NGS analysis.6-week-old male C57BL/6 mice were terminally anesthetized  and perfused with only cold PBS just prior to removal of the spinal cord. A total of 5 cords were aggregated for each immunoprecipitation (IP) reaction. Tissue preparation and ChIP protocol closely adhered to steps outlined in the MAGnify® ChIP Sample Preparation Guide protocol , omitting only the gel purification step to maximize total yield of adapter-ligated library fragments. Input Control and ChIP’d libraries were prepared using recommended inputs of 100 pg/μl (50 μl), for a total of 5 ng per library prep. Libraries were indexed using TruSeq Adapters AR008  and AR001 . All QC steps  were evaluated using a Fragment Analyzer Automated CE system with High Sensitivity NGS Fragment Analysis Kit 1bp-6000bp . Libraries were sequenced at 2 x 50 bp read lengths using the Illumina MiniSeq Sequencing System and MiniSeq High-Output Reagent Kits .DNA libraries were prepared from total sheared chromatin (Input Control Library) and ChIP-enriched fragments (ChIP’d Library) using Illumina TruSeq ChIP Library Preparation Kit . For this experiment, we used a modified TruSeqhttp://www.basespace.com) This application utilizes the algorithms BWA [https://github.com/taoliu/MACS/; [http://homer.salk.edu/homer/ngs/peakMotifs.html; [https://sourceforge.net/projects/bamstats/). Samples for both ChIP’d, and Input Control libraries were used as inputs for MACS2 to improve spatial resolution (specificity) of ChIP-region binding sites.Sequence analysis was performed using BaseSpace Labs ChIP-Seq v.1.0.2 application  to idenfs.html; ) to idenhttps://docs.google.com/document/d/1lG_Rd7fnYgRpSIqrIfuVlAz2dW1VaSQThzk836Db99c/). The non-redundant fraction of the aligned reads was calculated as the fraction of distinct reads  divided by the total number of reads that aligned to exactly one location. The normalized strand cross-correlation coefficient and relative strand cross-correlation coefficient were calculated using phantompeakqualtools . These cross-correlation coefficients are relative measures of the enrichment of the ChIP data for peak-like distributions of reads  against both the human (hg38) and mouse (mm10) reference genomes to minimize spurious hybridization. Primers used in ChIP-qPCR are provided in Supplementary Materials. qPCR data were generated using an Applied Biosystems 7300 thermocycler and Rotor-Gene SYBR Green PCR Kit  according to manufacturer’s instructions. At least three independent ChIPs were generated and run in separate qPCR runs, the results are presented as means and presented in a box and whisker format using StatPlus . For between groups comparison a Student’s two-tailed t-test was used. Statistical significance was established when p < 0.05.ChIP was performed using a protocol similar to the one described above for ChIP-Seq with one minor change to the fragmentation protocol- the Branson 150 Sonifier settings were changed to power setting 4, 100% duty cycle for 5 × 30-s on 90-s off intervals, yielding chromatin fragments of 100–800 bp. Primer pairs were designed using primer-BLAST  to flanExpression of Olig2 in CNS oligodendroglia cells persists well into adulthood; however, it is unclear what role Olig2 transcriptional regulation plays in neuroglia, particularly OPCs and in mature OLs within the adult spinal cord, both under physiological and pathological conditions. Because of this lack of clarity, we sought to identify the genome-wide transcriptional targets of Olig2 in the adult spinal cord in vivo by performing ChIP-Seq analysis on whole spinal cord tissue harvested from healthy adult mice.Many factors contribute to variability among ChIP-Seq datasets, so to ensure reliability of our data, and in an effort to maximize site discovery and reproducibility, we endeavored to adhere to ENCODE working standards and protocols for conducting and reporting ChIP-Seq data for point-source factors such as a transcription factor . These mhttp://pipeline.lbl.gov/; [Homo sapiens to Danio rerio. Whereas peaks of other targets like transcription factors Nkx6.2  reveales Nkx6.2  and Klf9s Nkx6.2  were alsTo investigate whether any of the Olig2-occupied sites overlapped with genomic regions associated with known enhancer activity by comparing our ChIP-Seq peaks with the 900 enhancers that have consistent tissue-specific enhancer activity and were validated using transgenic assays . Indeed,In our analysis, the closest annotated gene to each Olig2-binding site was considered a presumed target. Several of the targets identified in our ChIP-Seq have been identified elsewhere as direct targets of Olig2, while many genes on our list are known to be expressed in oligodendroglia, including during myelination, but were not previously known as direct targets of Olig2 . For exaThe majority of Olig2-bound sites were associated with a protein-coding gene, comprising 89%, or 3736 of the 4,214 Olig2-bound targets, while non-protein-coding RNAs made up 11% of the total target list, or 448 targets, and pseudogenes, snoRNAs, rRNAs, and snRNAs each comprised less than 1% of the total list of bound targets . Of the To investigate whether certain DNA motifs were enriched in the Olig2-binding sites, we applied a motif-discovery algorithm, HOMER . AnalysiTo identify the major biological functions represented by the gene targets in our dataset, we looked for statistically overrepresented gene ontology (GO) terms related to biological processes using PANTHER . The ChFinally, we sought to validate the ChIP-Seq results by chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR). We included in these experiments an IgG “mock” IP condition, which served as an important negative control. For the validation, we selected 13 targets from the complete dataset, taking into account whether they had previously been identified as a direct target of Olig2 elsewhere, and whether they had a documented role in the physiology of the OL lineage. In addition, we utilized high, low, and mid-range Peak Score (PS) values, as determined by the ChIP-Seq analysis see , when maDespite its continued expression throughout the OL lineage well into adulthood, relatively little is known about how Olig2 functions in OL development and myelination in the mature spinal cord. In this report, we present findings from a ChIP-Seq analysis identifying direct in vivo targets of Olig2 in the adult mouse spinal cord. We found that Olig2 occupies both protein-coding and non-protein-coding targets, many with well-documented functional roles consistent with oligodendroglia physiology, in addition to numerous previously unreported targets. Moreover, de novo motif analysis revealed a binding motif that contained a canonical E-box that is strikingly similar to previously reported motifs recognized by Olig2. To our knowledge, ours is the first study to comprehensively identify genome-wide targets of Olig2 from adult mammalian spinal cord tissue.One reason that Olig2 might remain functionally relevant in adult OLs is to activate or repress genes necessary for structural remodeling of myelin during adaptation events. The two most overrepresented GO terms for biological process were myelination and axon ensheathment while microtubule family cytoskeletal proteins were the most enriched PANTHER protein class . IntraceIn the adult mammalian CNS, OLs continue to play crucial roles in providing both structural and metabolic support to the axons of neurons. The OLs transfer important energy metabolites, including glucose and lactate, which are important for survival of myelinated neurons, and act as anti-apoptotic and neuroprotective agents to help stave off inflammation and preserve the myelin sheath ,61. GO tAlong these lines, Olig2 could also function as a mediator of extrinsic cues, activating or repressing downstream effector genes of different signaling pathways. We found that the Pdgfa signaling pathway was overrepresented among our list of targets . Pdgf siThe identification of numerous cell cycle genes as in vivo targets of Olig2 as well as the overrepresentation of GO terms related to cell cycle regulation are consistent with a role for Olig2 in OPC proliferation in the adult. Among the list of Olig2 targeted cell cycle genes included the cyclin kinase Cdk4, and mini chromosome maintenance proteins Mcm4, and Mcm5, DNA helicases that are required for licensing the DNA for successful replication. It is possible that Olig2 is maintained in adult OPCs as a means to directly regulate their cell cycle, and because the aforementioned targets regulate the early phases of cell cycle, it could act as the gatekeeper of OPCs entering the cell cycle from G0.We identified several non-protein-coding targets of Olig2, including several miRNAs. miR-219 is one the most abundant miRNAs in mature OLs and is an essential factor in promoting OL differentiation, in part by suppressing cell cycle factors that promote continued OPC proliferation ,25. TherDespite the aforementioned advantages of using whole CNS tissue to detect physiologically relevant binding sites of TFs, one drawback of this approach is that it makes it difficult to differentiate between different cells types. While the importance of Olig2 in establishing the OL lineage is well known, we cannot rule out that some targets identified in our ChIP-Seq derive from a smaller population of Olig2 expressing astroglia –31. For Finally, in addition to its expression in adult oligodendroglia, Olig2 is a key driver of tumor growth and proliferation ,70, and In summary, our findings help clarify the role of OPCs and OLs in the adult mammalian spinal cord by elucidating the gene regulatory network of a major transcriptional regulator of oligodendroglia, Olig2, in an adult in vivo context. In this report, we have provided a novel and comprehensive list of direct binding targets of Olig2 in vivo in the adult spinal cord that will provide candidates for future studies aimed at understanding how adult OPC differentiate and how OL function is maintained during both physiological and pathological processes.S1 FigShown are conservation curves for the indicated organisms aligned to the mouse genome using the VistaPoint Alignment Tool (pipeline.lbl.gov) for target genes Nkx2.2 (A), Zfp536 (B), Nkx6.2 (C), and Klf9 (D). Below the VistaPoint curves are the tracks for Olig2 ChIP and Input regions from the corresponding genomic location generated using the IGV browser. A region was considered conserved if the conservation over that region met the default values for both the minimum conserved width (100 bp) and conservation identity (70%). Regions of high conservation are colored according to the annotation as exons (dark blue), UTRs (light blue), or non-coding (pink). Grey bar demarcates regions of strong conservation and corresponding Olig2 ChIP-Seq peak summit.(TIF)Click here for additional data file.S1 TableA complete list of all targets identified by Olig2 ChIP-Seq.(XLSX)Click here for additional data file.S2 TableGene Ontology Analysis of Olig2-bound target genes using PANTHER, categorized according to biological process (A), molecular function (B), cellular component (C), protein class (D), and pathways (E).(XLS)Click here for additional data file.S3 TableA complete list of ON-target (A) and OFF-target (B) primer pairs and their corresponding coordinates that were used in the ChIP-qPCR.(XLSX)Click here for additional data file.


KDM3A, JMJD1A or JHDM2A) controls transcriptional networks in a variety of biological processes such as spermatogenesis, metabolism, stem cell activity, and tumor progression. We matched transcriptomic and ChIP-Seq profiles to decipher a genome-wide regulatory network of epigenetic control by KDM3A in prostate cancer cells. ChIP-Seq experiments monitoring histone 3 lysine 9 (H3K9) methylation marks show global histone demethylation effects of KDM3A. Combined assessment of histone demethylation events and gene expression changes presented major transcriptional activation suggesting that distinct oncogenic regulators may synergize with the epigenetic patterns by KDM3A. Pathway enrichment analysis of cells with KDM3A knockdown prioritized androgen signaling indicating that KDM3A plays a key role in regulating androgen receptor activity. Matched ChIP-Seq and knockdown experiments of KDM3A in combination with ChIP-Seq of the androgen receptor resulted in a gain of H3K9 methylation marks around androgen receptor binding sites of selected transcriptional targets in androgen signaling including positive regulation of KRT19, NKX3-1, KLK3, NDRG1, MAF, CREB3L4, MYC, INPP4B, PTK2B, MAPK1, MAP2K1, IGF1, E2F1, HSP90AA1, HIF1A, and ACSL3. The cancer systems biology analysis of KDM3A-dependent genes identifies an epigenetic and transcriptional network in androgen response, hypoxia, glycolysis, and lipid metabolism. Genome-wide ChIP-Seq data highlights specific gene targets and the ability of epigenetic master regulators to control oncogenic pathways and cancer progression.The lysine demethylase 3A ( KDM3A  is crucial for gene regulation in a variety of biological activities such as spermatogenesis, metabolism, stem cell activity and tumor progression by demethylating mono- or di-methylated H3K9 [Methylation of histone lysine residues is a significant component of epigenetics and is associated with control of gene expression . SpecifiAR; Gene ID: 367) [PPARG, KLF2, ESR1, and HOXA1 [KDM3A activity is deregulated in several cancers –8. In prID: 367) . The abiID: 367) , 11. TheID: 367) . Once inID: 367) . In addind HOXA1 –17.KDM3A on the epigenome, we performed chromatin immunoprecipitation in combination with next generation sequencing (ChIP-Seq) of its binding and demethylation activity. We quantified changes of H3K9me1 or H3K9me2 marks, the two substrates of KDM3A, and mapped AR-binding in the CWR22Rv1 prostate cancer cell line in combination with knockdown of KDM3A. Alteration of H3K9 methylation marks mapped to genomic locations coinciding with AR binding pinpoints target genes and oncogenic pathways cooperatively regulated by KDM3A and AR.In order to further elucidate the impact of KDM3A in CWR22Rv1 cells showed minor effects on global levels of H3K9me1 or H3K9me2 by Western blot analysis . Since H3K9 methylation is a mark associated with the highly condensed heterochromatin state, we characterized the specific genomic regions associated with both H3K9 histone methylation marks. KDM events were functionally annotated by mapping bound regions to the human genome and by classifying them according to the nearest gene locus and relative position within coding regions. Promoter or transcription start sites (TSS) and transcription termination sites (TTS) genomic annotations are defined as being within ± 1000 bp of the gene-coding body. Intergenic regions were defined as the remaining regions outside the gene body. In the H3K9me1 ChIP-Seq experiment the intergenic regions were the most frequently found region with 21112 peaks (46.5%) followed by 21495 (47.7%) as intronic regions, 822 (1.8%) as exonic regions, 606 (1.3%) as promoter-TSS regions, 549 (1.2%) as TTS regions, 424 (0.9%) as 3′UTR regions, and 46 (0.1%) as 5′ untranscribed (UTR) regions  genes are reported as differentially down-regulated upon shRNA knockdown of KDM3A while 1002 genes are reported as up-regulated by KDM3A knockdown , contain both H3K9me1/2 marks Figure . While tn Figure . The comKDM3A shRNA knockdown   . Top hits included the androgen receptor, sterol regulatory element binding factor (SREBF), hypoxia inducible factor (HIF), activator protein 1 (AP1) complex of JUN/FOS, Krüppel-like factors (KLF), v-myc avian myelocytomatosis viral oncogene homolog (MYC), and forkhead box (FOX) families of transcription factors with significant enrichments and p-values below 1.0E-04  Figure . The dat) Figure . In detaa Figure . Transcr) Figure . Taken tKDM3A in CWR22Rv1 cells resulted in loss of KDM3A ChIP-Seq binding accompanied by specific, matched gain of histone lysine 9 demethylation  events, respectively, and 34614 peaks for KDM3A-matched AR binding. Such an experimental design allows one to distinguish between histone demethylase binding    Figure . Similar) Figure .KDM3A/AR knockdown . For example, androgen response and metabolic pathways showed incrementally higher enrichment with better defined input gene sets . Similarly, pathways of glycolytic and glycerophospholipid metabolism showed consistently higher enrichment with the lowest p-values in the set of 260 genes, indicating that KDM3A demethylation targets these pathways and causes an up-regulation of gene expression. Of all genes detected by both H3K9 demethylation and AR ChIP-Seq experiments as well as KDM3A and AR differential expression following knockdown , the AR response was the only significantly enriched pathway with a p-value of 5.29E-08. 7 genes, NDRG1, PTK2B, ACSL3, KRT19, INPP4B, NKX3-1, and MAF, with direct implication in AR signaling were present in all gene sets of the hierarchical enrichment analyses, thereby subject to KDM3A control by H3K9 demethylation, AR binding, and differential expression following knockdown  P Figure . The genP Figure . Such a P Figure . Enrichmnockdown . In the n , AR shRNA or lentiviral pLKO.1 control shRNA were purchased from Open Biosystems , and packaged in human embryonic kidney 293T cells  by calcium phosphate transfection. The supernatant containing lentiviral particles were collected 48 hours after transfection. CWR22Rv1 cells were transduced with the supernatant of lentiviral particles in the presence of polybrene (8 μg/ml) for 24 hours before replacement with the fresh growth media. Cells were analyzed at 48 hours post-transduction. The knockdown efficiency was confirmed by quantitative real time polymerase chain reaction (qRT-PCR) and Western-blot analysis.Lentiviral vectors encoding PPIA; peptidylprolyl isomerase A; Gene ID: 5478). The following primers served for ChIP-qRT-PCR validation of ChIP-Seq signal of H3K9me1/2-KDM around the AREs of AR target genes: KLK3 : 5′-TGGGACAACTTGCAAACCTG-3′; 5′-CCAGAGTAGGTCTGTTTTCAATCCA-3′; NKX3.1 : 5′-GGTTCTGCTGTTACGTTTG-3′; 5′-CTTGCTTGCTCAGTGGAC-3′; MYC : 5′-CCAGCGAATTATTCAGAA-3′; 5′-AATTACCATTGACTTCCTC-3′.Total RNA from prostate cancer cells was extracted using a mammalian RNA mini preparation kit  and then digested with deoxyribonuclease I . Complementary DNA (cDNA) was synthesized using random hexamers. Triple replicate samples were subjected to SYBR green  qRT-PCR analysis in an Eco system . Gene expression profiles were analyzed using the ΔΔCT method. qRT-PCR threshold cycle (CT) values were normalized using the housekeeping gene cyclophilin A (Whole cell lysates were harvested using radio-immunoprecipitation assay (RIPA) buffer composed of 50 mM trisaminomethane hydrochloride (Tris-HCl) pH7.5, 150 mM sodium chloride (NaCl), 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 0.1% sodium deoxycholate, 1.0 mM EDTA, 1.0 mM sodium orthovanadate, and 1x protease inhibitor cocktail. Lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and proteins transferred to a nitrocellulose membrane . The membrane was probed with ChIP-grade H3K9me1 , H3K9me2 , KDM3A , or ACTB  antibodies followed by a secondary antibody conjugated to fluorescent dye, and blots were imaged using the odyssey detecting system .Cells were crosslinked using 1% formaldehyde for 10 min at 298 K. Formaldehyde was removed and cells were incubated with 125 mM glycine for 5 min at 298 K. Nuclear extracts were collected and sonicated to obtain 300 bp chromatin fragments using the Covaris S2 ultrasonicator . 100 μg of chromatin was incubated with 5 μg of AR , 5 μg of KDM3A , 2 μg of H3K9me1 , or 2 μg of H3K9me2  antibodies overnight at 277 K followed by incubation with 30 μl of protein A/G beads for 4 hours. After four washes, crosslinking was reversed. Chromatin was digested with ribonuclease A followed by proteinase K. Then the DNA was purified using spin columns. The size of the DNA was confirmed by a bioanalyzer .The purified DNA library was sequenced using an Illumina HighSeq2000 at the Sanford-Burnham Medical Research Institute at Lake Nona, National Genome Library Core Facility. Sequenced regions were aligned to the reference human genome 19 using the Bowtie alignment program that utilizes an extended Burrows-Wheeler indexing for an ultrafast memory efficient alignment . Peak caDe novo motif analysis programs MEME and DREME identify frequently detected DNA sequences patterns and similarity matches of recurring ChIP-Seq sequences with DNA motifs of deposited studies in genomic sequence databases [Computational response element searching algorithms are able to estimate a sequence's likelihood in belonging to the response element of the query transcription factor using position site-specific matrix (PSSM) models where each position in the query transcription factor model gives each of the four letters in the DNA alphabet a score based on the probability of that nucleotide being found at that position . Summatiatabases , 70. Aftatabases  for indiatabases , 71.AR, or KDM3A shRNA for 48 h. Total RNA was isolated from cells, and 500 ng was used for synthesis of biotin-labeled cRNA using an RNA amplification kit . Biotinylated cRNA was labeled by incubation with streptavidin-Cy3 to generate a probe for hybridization with the GeneChip Human Transcriptome Array 2.0  or the genome-wide transcriptome Human HT-12 V4.0 . Four samples from two experimental groups (n=2 per group) were hybridized to the chip to obtain raw gene expression data, which was processed to obtain raw data in the form of expression intensities. Raw data was then exported for further processing and analysis using R statistical software version 3.1 in combination with the BioConductor oligo, affy and genefilter packages [KDM3A knockdown were selected and analyzed through the use of Ingenuity Pathway Analysis . Pathway enrichment of differentially expressed genes was determined by gene set enrichment analysis (GSEA) by pairing each gene with its expression value and ranking genes in descending order  [AR knockdown using GeneChip Human Transcriptome Array 2.0, platform GPL16686 , is deposited in the NCBI GEO database under accession number GSE86547. Acquired data of CWR22Rv1 cells with KDM3A knockdown using hybridization with genome-wide transcriptome Human HT-12 V4.0, platform GPL10558 , is deposited under accession number GSE70498.In order to quantify the transcriptomic effect of KDM3A or AR knockdown, a microarray profiling analysis was conducted on CWR22Rv1 knockdown cells. CWR22v1 cells were transduced with lentiviral pLKO.1 control shRNA vector, packages . The rawpackages . Qualitypackages , 74. Rawpackages . Genes wdge, MA) , 77. As dge, MA) . AcquireKDM3A) and shRNA(AR) knockdown (The Supplementary Information contains tables on genome-wide mapping, annotation, and overlap of H3K9me1/me2 demethylation ChIP-Seq and AR ChIP-Seq , gene senockdown , hierarcnockdown , and an nockdown .


NIPBL gene. NIPBL-AS1 is a long non-coding RNA transcribed upstream and antisense to NIPBL. By knockdown and transcription blocking experiments, we could show that not the NIPBL-AS1 gene product, but its actual transcription is important to regulate NIPBL expression levels. This reveals a possibility to boost the transcriptional activity of the NIPBL gene by interfering with the NIPBL-AS1 lncRNA.Cohesin is crucial for genome stability, cell division, transcription and chromatin organization. Its functions critically depend on NIPBL, the cohesin-loader protein that is found to be mutated in >60% of the cases of Cornelia de Lange syndrome (CdLS). Other mutations are described in the cohesin subunits SMC1A, RAD21, SMC3 and the HDAC8 protein. In 25–30% of CdLS cases no mutation in the known CdLS genes is detected. Until now, functional elements in the noncoding genome were not characterized in the molecular etiology of CdLS and therefore are excluded from mutation screening, although the impact of such mutations has now been recognized for a wide range of diseases. We have identified different elements of the noncoding genome involved in regulation of the NIPBL and NIPBL-AS1. Deletion of the enhancer using CRISPR genome editing in HEK293T cells reduces expression of NIPBL, NIPBL-AS1 as well as genes found to be dysregulated in CdLS.Further, we have identified a novel distal enhancer regulating both  NIPBL gene. NIPBL is critical for chromatin-association of the cohesin complex and has a dual role as transcription factor. The regulation of the NIPBL gene is of great interest since organisms are very sensitive to NIPBL levels. For instance, severely affected patients showed only ~65% and mildly affected patients ~75% of NIPBL mRNA levels. One case reports a CdLS phenotype with as little as 15% reduction of the NIPBL transcript. Further, in a number of patients with CdLS phenotype no mutations in known CdLS genes are found, raising the question whether mutations could also occur in gene-regulatory elements. Here we investigate the role of a long non-coding RNA NIPBL-AS1 originating from a promoter shared with NIPBL and observe a co-regulation by a distal enhancer that we have identified and that seems to be conserved between tissues. Deletion of the enhancer by CRISPR leads to reduced expression of NIPBL and NIPBL-AS1 but also of NIPBL target genes that were found to be dysregulated in CdLS patient cells. The lncRNA NIPBL-AS1 itself has no role for NIPBL expression, but its transcription reduces the expression of the NIPBL gene and vice versa, thus revealing an interesting mechanism for the fine-tuning of expression levels.The most frequent mutations in the human developmental disorder Cornelia de Lange Syndrome (CdLS) occur in the  Genetic information needs to be inherited without any changes over numerous cell generations and from parents to offspring. This process crucially depends on the cohesin complex that ensures genome stability during cell divisions, DNA damage repair and is involved in the three-dimensional organisation of the chromatin fibre in the cell nucleus ,2,3. TheNIPBL is the gene that is most frequently  found to be mutated in the human developmental disorder Cornelia de Lange syndrome  [SMC1A, SMC3, RAD21 and the cohesin regulator HDAC8 [ births) ,10 [11,1 births) ,14. Mutaor HDAC8 ,16,17,18NIPBL expression levels seem to be critical for developmental processes in human and mouse. In a cohort of severely affected CdLS patients only ~65% of NIPBL expression is observed while mildly affected cases showed ~75% expression; one case report describes a mild CdLS phenotype with as little as 15% reduction of the NIPBL transcript [Nipbl heterozygous knockout mice with a CdLS reminiscent phenotype, the levels of Nipbl expression are reduced by only 25–30%, suggesting a compensation by the intact allele [The actual anscript ,20,21. It allele . Furthert allele .NIPBL gene by identifying regulatory elements in the noncoding genome. Different studies have shown that a large number of potentially disease-causing mutations/variants in the genome are located outside coding regions in regulatory non-coding areas, highlighting the importance of the identification of these elements for the study and diagnosis of human genetic diseases [NIPBL  that afNIPBL promoter-associated antisense transcript of 5.3 kb, named NIPBL-AS1, in brain tissues from patients affected by Autism Spectrum Disorders (ASD) [NIPBL-AS1 and NIPBL showed a concordant expression in their hands as well as ours, we hypothesized that NIPBL-AS1 and NIPBL might be interconnected and that this could represent a gene regulatory mechanism.During the course of our project, a study aiming at analysing non-coding RNAs overlapping with known autism-related genes identified a rs (ASD) . Since Ncis but also in distant domains in trans  were shown to control genes located in their vicinity in iewed in ). These romoters ,30,31,32romoters . Alternaromoters ,38,39,40NIPBL gene were so far unknown. Here we identified an enhancer that stimulates expression of NIPBL as well as the lncRNA NIPBL-AS1.Other crucial gene regulatory elements are enhancers. In mammals, they have been found up to several hundred kilobases from the target transcription start site, as for instance the Sonic Hedgehog (SHH) enhancer that is located 1MB upstream of the gene . EnhanceNIPBL-AS1 is located upstream of NIPBL in a head-to-head orientation and encodes for a 5.3 kb lncRNA  or in the cytoplasm  [NIPBL-AS1 by fractionation of HEK293T cells into cytoplasmic and nucleoplasmic RNA. The RT-qPCR analysis of the cell fractions showed that NIPBL-AS1 is not retained in the cell nucleus as much as lncRNAs with nuclear functions like MALAT1 or XIST [NIPBL-AS1 is still present in the nucleus   we addre or XIST . However nucleus .NIPBL-AS1 acts directly on NIPBL expression, we depleted NIPBL-AS1 in HEK293T cells using Antisense Oligonucleotides (ASO), which activate the RNAseH pathway by forming DNA/RNA hybrids [NIPBL-AS1 RNA. ASOs are in particular suited to knockdown nuclear lncRNA but are also efficient for cytoplasmic lncRNA, so both pools can be efficiently depleted [NIPBL-AS1 gene (respectively ASO2 and ASO3) and one non-targeting control (ASO C). The transcript levels of NIPBL-AS1 (P132) and NIPBL  were assessed by RT-PCR/qPCR at 48 hours after transfection. Two intron-spanning primer pairs in the middle (P138) and at the 3’-end (P42) of the NIPBL gene that cannot discriminate between the different reported NIPBL splice variants [NIPBL-AS1 transcripts were significantly reduced by both ASOs (NIPBL-AS1 as high as 80% for ASO2 and 70% for ASO3. NIPBL transcript levels were not affected when we used a primer pair positioned more in the front part of the NIPBL gene (primer P138). When we used a primer spanning two of the last exons (P42) we observed a small but significant difference  of both genes are separated by 77 bp   as well as a control gRNA (GC) in distant gene desert on chromosome 5. Since it was shown that gRNAs targeting the non-template DNA strand have a higher gene silencing effect compared to those targeting the template strand [NIPBL-AS1 using two guide RNAs  targeting the non-template strand of NIPBL-AS1  targeting the non-template strand of the gene  . It has e strand , we recrBlock I, . To blocNIPBL-AS1 and NIPBL by ChIP-qPCR (for primer positions see NIPBL for Block I and NIPBL-AS1 for Block II).First, we analysed the wider promoter region of NIPBL and NIPBL-AS1 under the different blocks. When we transfected the cells with Block I, only 30% of the NIPBL-AS1 transcript was still detectable compared to the control guide RNA transfection (GC), indicating that NIPBL-AS1 transcription was significantly blocked  [NIPBL promoter (VP1), we observed contacts to two intergenic regions, namely R1 and R2, respectively at 130 kb and 160 kb upstream of the NIPBL promoter , also confirmed by two additional viewpoints inside NIPBL (VP2-3)  , initialpromoter . We focupromoter . Region  (VP2-3) . HoweverNIPBL promoter, we performed a higher resolution 3C-seq (using ApoI as restriction enzyme) using one viewpoint at the NIPBL promoter (VP4) and two viewpoints at R1 (VP5) and R2 (VP6) in HEK293T cells  showed contacts between this potential enhancer and the promoter of NIPBL and NIPBL-AS1 (or 5’end of NIPBL-AS1)  [NIPBL promoter to a potential distal enhancer next to the Slc1a3 gene     or R1_1 and R1_2 at the same time   . To dissd gRNA3) . In totad gRNA3) .NIPBL. RT-qPCR analysis showed a reduction of NIPBL transcript levels to in average 60% for both depletions and transcription of NIPBL-AS1 was also reduced  and one clone from D2 (D2_C35) (NIPBL promoter (VP4), R1 (VP5) and R2 (VP6) as viewpoints (NIPBL gene body.To investigate how the deletions D1 (including R1_1) and D2 (including R1_1 and R1_2) affect the long-range interactions of the (D2_C35)  using thewpoints . The D1 ewpoints  but the NIPBL/NIPBL-AS1 enhancer overlaps with the R1_1 region but both CTCF sites in R1 (CTCF#1 and CTCF#2) are required for the long-range interactions between R1 and NIPBL/NIPBL-AS1.We conclude that the BBX, ZNF695, GLCCI1) are downregulated in the larger deletion (D2) and two genes in the smaller deletion (D1)  . This fuNIPBL transcript levels were found to be reduced in CdLS patients with NIPBL mutations but the cause of this downregulation was unclear [NIPBL and NIPBL-AS1 transcript levels in cells from CdLS patients with a heterozygous truncation mutation of NIPBL [NIPBL transcripts in three patient LCLs with early truncating mutations in NIPBL (PT1-3) as well as in four control LCLs (C1-4)  or 24% (PT3) of mutant transcripts  without observing a significant change in NIPBL expression , transcribed from a bidirectional promoter shared with NIPBL. Both genes seem to be transcribed to comparable levels according to the RNA-seq signals  correlates strongly with active chromatin marks  while the second one (CTCF #2) shows only little correlation with those histone marks  that included CTCF#1 and CTCF#2 did not lead to further downregulation of expression. The enhancer of NIPBL/NIPBL-AS1 resides therefore in R1_1 and overlaps with CTCF#1.We also identified one distal enhancer of  call R1 . This rene marks . The delNIPBL/NIPBL-AS1 bidirectional promoter strongly contact the two CTCF binding sites within the R1 region. The motif orientation of these sites is consistent with the preferential motif orientation for loop formation. Both sites in R1 seem to be required for the long-range interactions of the NIPBL/NIPBL-AS1 promoter. This promoter-enhancer interaction seems to be conserved between different tissues and also across species  (NIPBL intragenic viewpoints (VP2-3) contact the promoter and the enhancer and vice versa the enhancer contacts the NIPBL gene body (VP5), we might eventually observe here a tracking of the promoter that is still in contact with the enhancer along the NIPBL gene together with elongating RNA Pol II, as recently described by Lee et al. [An interesting observation concerning the (188 kb) . Since te et al. , althougNIPBL gene and obtained functional insight on the regulation of the NIPBL gene expression. Although we did not observe a direct effect of the lncRNA for the NIPBL gene, we found that the transcription of the two genes is interconnected, suggesting a mechanism for the fine-tuning of NIPBL expression. With these experiments we have also demonstrated one possibility to boost the transcriptional activity of the NIPBL gene by interfering with the NIPBL-AS1 lncRNA. This could be further explored for other genes with a similar arrangement of lncRNA and gene, and eventually also be used to manipulate gene expression in patient cells.In summary, we have discovered very important features of the genetic context of the NIPBL/NIPBL-AS1. Given that even a modest reduction of NIPBL expression dramatically impacts on development, we suggest to include this non-coding genomic element into molecular diagnostics for CdLS.Moreover, we identified a distal enhancer controlling sense and antisense transcription at the bidirectional promoter of HEK293T cell line was cultured in DMEM supplemented with 0.2mM L-glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin and 10% FCS and was grown at 37°C and 5% CO2.HB2 cells  were cuLymphoblastoid cell lines derived from controls and Cornelia de Lange syndrome patients  were obtCells were harvested and total RNA was prepared using Trizol Reagent (Invitrogen). After chloroform extraction and isopropanol precipitation, pellets were dissolved in DEPC water. cDNA was generated by reverse transcription using oligo(dT)18 primer (Invitrogen), Superscript II Reverse Transcriptase (RT) (Invitrogen) and RNaseOUT Recombinant Ribonuclease Inhibitor (Invitrogen) according to the manufacturer’s instructions. The amounts of the different transcripts were compared by qPCR using SYBR Green and Platinum Taq Polymerase (Invitrogen) in CFX96 light cycler (BioRad) and specific primers. ΔΔCt method was used to calculate the fold change in gene expression using the housekeeping gene SNAPIN and the control sample for normalization.https://eu.idtdna.com/Scitools/Applications/AntiSense/Antisense.aspx?source=menu). Two ASO were designed against the 5’ and 3’ end of NIBPL-AS1 and one were designed against a non-targeting sequence to use as control. All the ASOs were modified with a phosphorothioate (PS) linkages that confer nuclease resistance.Anti-Sense Oligonucleotides (ASO) were designed followed the guidelines from Integrated DNA Technologies (IDT) (The following ASOs were used:ASO2 targeting the 5’ end of NIBP-AS1: G*C*C* C*T*T* C*C*C* T*C*T* G*T*G* T*A*A* T*T*CASO3 targeting the 3’ end of NIBP-AS1: T*G*T* G*G*G* T*T*T* C*T*G* G*T*G* T*T*G* T*G*GControl ASO non targeting sequence: A*T*A* T*T*T* C*C*A* C*G*C* C*A*G* C*C*A* G*AThe position of the phosphorothioate (PS) linkages is indicated as *.HB2 or HEK293T cells we transfected with 400 pmol of either ASO2, ASO3 or Control ASO (IDT) using Lipofectamine 2000 (Invitrogen) according to the manufacture’s instruction. Cells were harvested 48 hours after transfection and cDNA was prepared as described above. ΔΔCt method was used to calculate the fold change in gene expression using the housekeeping gene SNAPIN and the control sample for normalization.http://cas9.cbi.pku.edu.cn/CasDesign) [The expression vectors for active Cas9 and catalytically inactive Cas9 were obtained from Addgene. The guide RNA were designed using the Cas9 design tool (sDesign)  and insesDesign)  followinNIPBL-AS1, two gRNAs  targeting the 5’ end of NIPBL-AS1 were used. To block the transcription of NIPBL, three gRNAs  targeting the 5’ end of NIPBL were used. As controls we used either a combination of guide RNAs on NIPBL that are not effective for RNA PolII blocking  or one guide RNA localizing outside of the locus (GC). The gRNA were designed using the web tool http://cas9.cbi.pku.edu.cn/CasDesign and cloned into a pCR-Blunt II-TOPO vector (Plasmid #41824 from AddGene) by Gibson Assembly (New England BioLabs) according to the manufacture’s instruction. HEK293T cells were transfected with the gRNAs vectors and the catalytically inactive Cas9 vector (dCas9) [To block the transcription of  (dCas9)  (PlasmidChromosome conformation capture sequencing was performed as previously described in . BrieflyTo deplete the candidate enhancers (R1_1 and R1_2) in region R1, three gRNAs  targeting the region were designed and cloned as described above. To delete R1_1 (5 kb deletion in R1 region), HEK 293T cells were transfected with 2ug of gRNA2, gRNA3 and the vector coding for the catalytically active Cas9  using Lipofectamine 2000 (Invitrogen) according to the manufacture’s instruction. To delete the entire R1 region including R1_1 and R1_2 (12 kb deletion), HEK293T cells were transfected with 2 μg of gRNA1, gRNA3 plasmids and the catalytically active Cas9. Cell were cultured in a puromycin-containing medium and single clones were picked and PCR analyzed for the presence of the corresponding deletion.Cells from different clones were harvested and lysed with lysis buffer .Genomic DNA was isolate using isopropanol and samples were analyzed by PCR. Primer sequences are listed in the Supplemental information.Clones positive for deletions D1 and D2 were grown, cells were harvested and cDNA was prepared as described above. HEK293T cells were used as control. ΔΔCt method was used to calculate the fold change in gene expression using the housekeeping gene SNAPIN and the Control sample for normalization.Fractionation of cells was performed as described  with addTo test whether the candidate enhancer has indeed an effect on the NIPBL promoter we cloned the luciferase gene under control of the NIPBL promoter  together with the putative enhancer elements (R1_1—(hg 19)chr5:36743873–36745877, R1_2 –(hg 19)chr5:36738465–36742009) or a similar sized control region in the pGL4.10 vector  with one of the following constructs: (1) empty pGL4.10 vector; (2) pGL4.10 vector containing R1_1 and NIPBL promoter; (3)(2) pGL4.10 vector containing R1_2 and NIPBL promoter; (4) pGL4.10 vector containing genomic DNA of the same size as the regulatory element. After 24 hours the cells were lysed and the activity of firefly and renilla luciferase determined with the Dual Luciferase Reporter Assay (Promega) in a TriStar2 LB Multidetection Microplate Reader . Genomic regions and primer sequences are given in the table. All measurements were verified in a minimum of three independent experiments and as triplicates in each experiment.The chromatin immunoprecipitation with anti-RNA PolII Ser5 and anti-PolII was performed as described .A DNA fragment of interest was PCR-amplified with a biotinylated primer as described . After dNIPBL—monoclonal rat anti-NIPBL, isoform A (long isoform) NP_597677  and isoform B (short isoform) NP_056199 SMC2 –rabbit polyclonal anti-SMC2 Tubulin—mouse anti-tubulin (Sigma)There are no ethical issues. All cell lines used in this manuscript were previously published .S1 FigNIPBL-AS1 as well as the control lncRNAs MALAT1 and XIST and the housekeeping genes SNAPIN and NADH with RT-qPCR .A) Fractionation of the RNA in HEK293T cells into cytoplasmic and nucleoplasmic fraction and detection of NIPBL-AS1 (B) and NIPBL (C) after antisense oligonucleotide (ASO) knockdown of NIPBL-AS1 in HB2 cells. Cells were transfected with either ASO2 or ASO3, targeting respectively the 5’ end or the 3’ end of NIPBL-AS1 and one non-targeting control ASO (ASO C). Transcript levels were normalized against the control sample (ASO C) and the housekeeping SNAPIN .B-C) Transcript levels of (PDF)Click here for additional data file.S2 FigNIPBL-AS1 and the NIPBL promoter region together with ChIP-seq data for RNA polymerase II, CTCF, the H3K4me3 histone mark and DNase hypersensitive regions in HEK293 cells (ENCODE). The locations of the different guide RNAs used for the CRISPRi blocks  as well as the primer used for ChIP-qPCR are shown.A) Overview of the NIPBL-AS1 is blocked (Block I).B-C) Enrichment of Ser5-phosphorylated initiating RNA polymerase  and general RNA Pol II  when transcription of NIPBL is blocked (Block II).D-E) Enrichment of Ser5-phosphorylated initiating RNA polymerase  and general RNA Pol II  when transcription of NIPBL-AS1 gene—right side: Block II primer AS7 in the NIPBL gene. ChIP-qPCR results are expressed as fold enrichment relative to the target region AS3 on each control (Block III) [The position of the guide RNA furthest into the gene body together with the ChIP primer are highlighted with blue boxes–left side: Block I primer AS3 in the ock III)  (average(PDF)Click here for additional data file.S3 FigNIPBL and NIPBL-AS1 promoter (VP1) detected by chromosome conformation capture (3C-seq) in the breast epithelial cell line HB2 using an BglII digest. The positions of the viewpoints are highlighted in yellow. Note that two viewpoints (VP2 and VP3) were positioned further into the NIPBL gene to validate the long-range interaction of the promoter (P) into the NIPBL gene body.A) Long-range chromosomal interactions of the region covering the B) Validation of interactions between the promoter region (P)  and two candidate regions R1 and R2 carrying enhancer marks  using the more frequently cutting enzyme ApoI in HB2 cells.C) CTCF ChIP sequencing track from HEK293 cells (ENCODE) and DNAse hypersensitivity. The orientations of the CTCF motifs as determined with JASPAR are shown below the track . The CTCF sites involved in the promoter-enhancer interaction are indicated with yellow triangles above the track.NIPBL and NIPBL-AS1 promoter region (P) and distal intragenic regions (R1 and R2) detected by 3C-sequencing analysis are highlighted with blue boxes.D) Histone modification profiles—H2A.z, H3K4me1, H3K4me2 and H3K4me3—of six different cell lines  are displayed as density graph in which black represents areas with the highest enrichment of the ChIP-sequencing signals. (PDF)Click here for additional data file.S4 Fighttp://promoter.bx.psu.edu/hi-c/view.php) (A-G) and in the CH12 mouse cell line (H). Interactions between the NIPBL promoter/NIPBL-AS1 and the potential enhancer in R1 are indicated by dashed lines. When available in ENCODE ChIP-seq signals for CTCF and different histone marks are shown. In GM12878 cells (A) also region R2 is shown and the interaction of R2 with the NIPBL promoter that is unique for this cell line is indicated with an arrow. Note that the potential enhancer in mouse cells (H) is positioned closer to the Slc1a3 gene than in human cells.Hi-C interactions maps at 5 kb resolution from seven different human cell lines  (maps ge(PDF)Click here for additional data file.S5 FigA) Location of the gRNAs  used to delete the potential enhancers R1_1 and R1_2. The ENCODE data for CTCF in HEK293 cell and histone marks  derived from six different cell lines  are shown to support that these regions are potential enhancers. Note that the combination of gRNA_2 and gRNA_3 will delete one CTCF binding site and the combination of gRNA_1 and gRNA_3 will delete two CTCF binding sites.(B-C) Schematic overview of the two different conditions used to create (B) a partial deletion of 5 kb  or (C) a full deletion of 12 kb . The primers used for genotyping of the clones and the respective PCR product sizes are shown.(D-H) Analysis of CRISPR edited clones with deletions D1 and D2. Genomic DNA of the clones was analysed with PCR primers specific for the deletions (for primer positions see B and C) and PCR products analysed on agarose gels. (D) PCR products in unedited HEK293T cells (Control). Note that primers P4-P8 give only in unedited cells a product of correct size. (E-H) Genotyping of clones obtained in two rounds of CRIPSR targeting. Clones D1_89 and D2_35 were obtained in the first round. In the second round four clones were obtained for D1 and three for D2, clones D1_63 and D2_103 are shown as examples. (E+F) Genotyping of D1 clones using one primer designed for a product unique for the D1 deletion  and primers designed to detect the intact genomic region (P6-P8). (G+H) Genotyping of D2 clones using one primer designed for a product unique for the D2 deletion  and primer designed to detect the intact genomic region (P4-P8).The expected product sizes are indicated on the agarose gel pictures in (E) and (G). The PCR products missing due to the deletions are indicated with boxes. The asterisks (*) indicate side-products of the PCR primers that become more prominent in the absence of the original templates.(PDF)Click here for additional data file.S6 FigNIPBL (#42 and #138) and one for NIPBL-AS1 (#132) .Transcript levels of the individual clones and the HEK293T cells used for genome edition were determined with two primers for (PDF)Click here for additional data file.S7 FigBBX, GLCCI1 and ZNF695 that were described as dysregulated genes in CdLS [Transcript levels of the genes  in CdLS  and prev in CdLS  were ana(PDF)Click here for additional data file.S8 FigNIPBL and NIPBL-AS1 transcripts. The lines were previously described [A) Details of control and CdLS patients lymphoblastoid cell lines (LCLs) used for analysing escribed ,20.NIPBL and NIPBL-AS1 in four controls and three CdLS patients. Two primer pairs for NIPBL and one for NIPBL-AS1 were used. Transcript levels were normalized against the housekeeping gene NADH. Note that transcript levels are reduced by only 30–40% in CdLS patients but the NIPBL-AS1 transcription is hardly affected.B) Transcript levels of C) The contribution of intact and mutated allele to the total RNA was determined in PT1 and PT3 by pyrosequencing to estimate the efficiency of nonsense-mediated decay. To visualize that the intact and mutant allele are transcribed at similar level nonsense mediated decay was blocked with cycloheximide.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.


Suppression of therapeutic transgene expression from retroviral gene therapy vectors by epigenetic defence mechanisms represents a problem that is particularly encountered in pluripotent stem cells (PSCs) and their differentiated progeny. Transgene expression in these cells, however, can be stabilised by CpG-rich ubiquitous chromatin opening elements (UCOEs). In this context we recently demonstrated profound anti-silencing properties for the small (679 bp) CBX3-UCO element and we now confirmed this observation in the context of the defined murine chromosomal loci ROSA26 and TIGRE. Moreover, since the structural basis for the anti-silencing activity of UCOEs has remained poorly defined, we interrogated various CBX3 subfragments in the context of lentiviral vectors and murine PSCs. We demonstrated marked though distinct anti-silencing activity in the pluripotent state and during PSC-differentiation for several of the CBX3 subfragments. This activity was significantly correlated with CpG content as well as endogenous transcriptional activity. Interestingly, also a scrambled CBX3 version with preserved CpG-sites retained the anti-silencing activity despite the lack of endogenous promoter activity. Our data therefore highlight the importance of CpG-sites and transcriptional activity for UCOE functionality and suggest contributions from different mechanisms to the overall anti-silencing function of the CBX3 element. In particular pluripotent stem cells are problematic in this respect, as these cells harbour strong intrinsic defence mechanisms against foreign and especially viral DNA7. Moreover, the differentiation of pluripotent stem cells towards more mature cell types involves genome wide epigenetic remodelling with a potentially detrimental impact on transgene expression. To evade this transcriptional repression so called ubiquitous chromatin opening elements (UCOEs) have been applied8. In previous studies, we and others have demonstrated the ability of UCOEs to establish stable expression of lentiviral transgenes driven by both viral and physiological promoters in murine and human pluripotent as well as multipotent target cells and thereof differentiated progeny13, thus demonstrating broad applicability of UCOEs.Although the design of lentiviral vectors has improved substantially over the last decade, even modern SIN-lentiviral vectors remain subject to positional effects that can lead to silenced or variegated transgene expressionHNRPA2B1-CBX3 housekeeping gene locus. This element contains two divergently transcribed promoters, the HNRPA2B1 promoter and the CBX3 promoter which are both spanned by a large stretch of non-methylated CpG-islands (CGI)13. A central 1.5 kb sequence containing the anti-silencing activity (A2UCOE) has been identified, which is associated with a permissive chromatin structure marked by hyperacetylation of histones H3 and H4, trimethylation of lysine 4 on histone H3 (H3K4me3), and the lack of H114. We have recently shown that a much smaller sequence comprising only the 0.7 kb CBX3 moiety of the A2UCOE (referred to as CBX3) can effectively protect SIN-lentiviral transgene expression in murine and human pluripotent cells as well as their hematopoietic derivatives15. The CBX3 prevented CpG methylation of a spleen focus forming virus (SFFV) promoter and reduced the presence of repressive chromatin marks (e.g. H3K9me3 and H3K27me3) while introducing active marks such as H3K4me3 and PhosPol2.The most commonly used UCOE is derived from the human 16 and the TIGRE locus T117, in single-cell derived mESC clones utilising recombinase mediated cassette exchange (RMCE).While mechanistically the anti-silencing function of the UCOEs has been attributed to the presence of non-methylated CGIs, a feature shared by all known UCOEs, up to now the anti-silencing properties of the UCOE have not been linked to specific structural features. To potentially identify such features within the CBX3 structure we have generated a number of CBX3-derived subfragments and analysed the functionality of these subfragments in the context of lentiviral vectors and a broad range of integration sites within murine ESCs and iPSCs as well as their differentiated progeny. In addition, we have investigated the relevance of the CpG-islands for the anti-silencing properties by employing two synthetic scrambled-CBX3 versions with either maintained or destroyed CpG-sites. Moreover, to explore the relevance of transcriptional activity for the anti-silencing function we examined the promoter activity of our CBX3 subfragments. So far all investigations assessing UCOE function have been performed in bulk cell populations with varying integration sites while a direct comparison of expression cassettes with and without UCOE in a defined integration site and chromosomal architecture has not yet been performed. Accordingly, it is not known if UCOEs can protect from silencing irrespective of the nature of the chromosomal integration site. Therefore, we now have examined the anti-silencing ability of the CBX3 at two defined chromosomal loci, the ROSA26To identify the structural attributes responsible for the anti-silencing function of the CBX3, we generated a total of six partially overlapping subfragments of the CBX3 element Table , Fig. 1AA murine ESC as well as an induced pluripotent stem cell (miPSC) line were transduced with an MOI of 10 and 30, respectively. By this procedure, similar vector copy numbers (VCN) of approximately 2–3 for mESCs and 1–2 for miPSCs were achieved for all vectors  of eGFP expression in transduced cell populations Table , indicat2. The in vitro differentiation of mESCs and miPSCs therefore represents a much more stringent system to analyse the potency of anti-silencing elements such as our CBX3 subfragments. To this end, the cells were subjected to undirected differentiation and the expression of eGFP in differentiated, SSEA-1 negative cells was assessed on day 8  as well as miPSCs  , no significant correlation was detected for CpG-site density (CpGs/100 bp) or GC-content (data not shown).Interestingly, these four CBX3 subfragments all contain a high number of CpG-sites and the amount of CpG-sites per fragment was found to positively correlate with its anti-silencing capacity (measured as the percentage of eGFP-positive cells following differentiation) based on data for mESCs (R01) Fig. . While aTo further assess the relevance of the CpG-sites within the CBX3 element, two scrambled versions of the CBX3 were designed. The first element was generated by random shuffling of the DNA sequence while retaining the CpG-sites  at their original position without changing the overall nucleotide composition (CscrCpG). The second element was designed by randomly shuffling the complete DNA sequence thereby destroying the CpG-sites  was calculated and used for further analysis. When MFIcorr/VCN was monitored over a 28-day period, relatively stable eGFP expression levels were observed for the individual constructs  to investigate the impact of a UCOE at two defined chromosomal sites. In particular, we integrated a cassette comprising the silencing prone viral SFFV promoter driving eGFP with or without the CBX3 UCOE at two defined chromosomal loci in mouse embryonic stem cells (mESCs); the ROSA26 and the TIGRE locus  fragment showed substantial anti-silencing activity in the pluripotent state, in differentiated cells two subfragments, CBX3(1-339) and CBX3(340-508), failed to show anti-silencing activity. The remaining subfragments CBX3(1-508), CBX3(85-508), CBX3(170-508) and CBX3(503-679) all showed activity in pluripotent as well as differentiated cells. These data indicate different mechanisms to be responsible for transgene silencing in the pluripotent state or during cellular differentiation. While transgene silencing in the pluripotent state is well documentedOf note, our data imply that the anti-silencing function of the CBX3 element cannot be mapped to a single region and thus emphasize the CBX3 element as a minimal UCOE version with almost complete functionality of the original 1.5 kb A2UCOE. While all subfragments displayed at least some level of activity, none of the fragments could completely substitute for the CBX3 in our experimental models. Nevertheless, the considerable activity of the CBX3(503-679) and the three subfragments containing the 170-508 bp sequence indicate the relevance of the central as well as the 3′ portion of the CBX3 in this context. In contrast, the rather low activity of the CBX3(1-339) subfragment and the similar properties of the CBX3(1-508), CBX3(85-508), and CBX3(170-508) subfragments suggest only moderate contribution, if any at all, from the 5′ end of the CBX3. We have not been able, however, to generate CBX3 subfragments lacking only the 5′ 170 or 339 bp to more formally test this hypothesis. Importantly, we demonstrated a significant correlation of fragment length and, even more important, overall CpG content with anti-silencing activity, further supporting the notion that the highly CpG-dense central and 3′ regions may be crucial determinants of CBX3 function.24, and their non-methylated CpG-sites have been identified as specific targets of various histone modifying proteins as well as DNA demethylating enzymes  that bears no resemblance to the CBX3 (or any other UCOE) besides maintained CpG-sites, has the ability to protect lentiviral transgene expression in pluripotent cells and thereof differentiated progeny. A completely scrambled CBX3 variant (CBX3scr) lacking the CpG-sites displayed minimal to no functionality. Although not completely equivalent in functionality to the CBX3 element, this ability of the CBX3scrCpG is quite remarkable, and strongly indicates the CpG-sites of the CBX3 as important structural determinants of its anti-silencing function. CpG-islands are detected in the proximity of 60-70% of gene promoters30. Synthetic CpG-islands have been shown to induce this bivalent state in ESCs if the synthetic sequence in addition to the CpG-dinucleotides is also enriched for G and C nucleotides31. This may imply that in addition to the CpG-sites also the overall GC-density of the CBX3 and thereof derived subfragments supports the overall function of these elements.Moreover, regions highly enriched in non-methylated CpG-dinucleotides (CpG islands) are associated with an open chromatin structure that particularly in pluripotent cells is marked by a bivalent chromatin containing both active as well as repressive chromatin marks18. On the other hand, the anti-silencing ability of the CBX3scrCpG element that lacked transcriptional activity indicates that CpG-site mediated anti-silencing can be independent of intrinsic promoter activity. However, since the synthetic element was less effective than the CBX3, it can be speculated that a combination of CpG motives and promoter activity might be important for full anti-silencing activity of the CBX3.Considering the impact of transcriptional activity on the chromatin status and the recruitment of binding factors, such as Cfp1, in the vicinity of CGIs, it becomes evident that the promoter activity of CBX3 may actually constitute a relevant factor for its anti-silencing function. In support of this notion we observed that CBX3 subfragments with higher endogenous promoter activity also tended to perform better with respect to their anti-silencing function. Along the same line, in the hands of other investigators a different CBX3 element devoid of transcriptional activity (0.7 kb intronic UCOE) displayed poor anti-silencing activityAdditionally, the CBX3(340-508) and the CBX3(1-339) subfragments, while sharing a low number of CpG-sites and a low promoter activity, differ markedly in their anti-silencing potential specifically in the pluripotent state. This clearly indicates the contribution of additional and as of yet undefined factors, such as for example the presence of specific transcription factor binding sites, to the anti-silencing potential of the CBX3.16; upon integration of heterologous promoters, variable expression patterns were observed suggesting that some of the promoters undergo substantial silencing33. The TIGRE locus was identified upon screening for sites supporting tightly regulated transgene expression17 in a broad range of tissues suggesting an euchromatic state.Up to now, most studies on UCOE functionality have been performed in polyclonal cell populations, which are characterized by a random panel of integration sites. So far, studies that allow the evaluation of UCOEs in a defined chromosomal context are lacking. Thus, it is not known if UCOEs can stabilize expression in all chromosomal sites prone to silencing and if they do that to a similar extent. To directly compare expression properties in the presence of absence of an UCOE, we now analysed the activity of the CBX3 at two defined chromosomal loci in mESCs; the ROSA26 and the TIGRE locus. The two loci were chosen because of their expression properties: The endogenous Rosa26 promoter is active in cells of all differentiation states36, thereby excluding the varying influence of random chromosomal sites.To facilitate efficient targeting of transgene cassettes into these sites we employed Flp recombinase mediated cassette exchange (RMCE). This method has been utilized for direct comparison of vector designs at defined chromosomal sites33. Interestingly, silencing of the SFFV promoter was less pronounced in the TIGRE locus. Of note, incorporation of the CBX3 into the transgene expression cassette increased the percentage of eGFP-retaining cells after differentiation in both loci, supporting the broad applicability of the CBX3.Silencing of the SFFV cassette in the Rosa26 locus is in line with previous studies indicating the loss of transgene expression from various promoters in this chromosomal locus37. These findings confirm our previous notion that not the chromosomal site as such but rather the combination of integration site and incoming promoter defines the expression properties38. Thus, the concept of universally ‘safe’ loci with predictable expression may need to be revised and assessed more carefully. In contrast to the ROSA26 locus, the TIGRE locus was more permissive to transgene expression particularly in pluripotent cells. Also this locus notably repressed the SFFV driven transgene expression during differentiation but the incorporation of a CBX3 resulted in significant improvement, rescuing expression in about 80% of cells. These data thus demonstrate that the CBX3 is able to alleviate transgene silencing at both the ROSA26 and the TIGRE locus, albeit at different levels.Transgene silencing at the ROSA26 locus demonstrated a gradual reduction of eGFP expression even in pluripotent cells despite the presence of the CBX3, and upon differentiation only a moderate, though significant, activity of the CBX3 was noted. This clearly indicates that the UCOE cannot completely shield the incoming cassettes from negative influences of the ROSA26 locus making this locus an unsuited site for integration of silencing prone promoters. Recently, another group evaluated various promoters upon RMCE based targeting into the AAVS1 safe harbour locus in hESCs. Interestingly, depending on the promoter, they encountered variable levels of transgene inhibition, both in pluripotent cells and during hepatic differentiationTaken together, our data indicate that the CpG-sites as well as the transcriptional activity significantly contribute to the anti-silencing function of the CBX3. Moreover, given the differential activity observed for individual subfragments in the pluripotent state and during differentiation, as well as the anti-silencing properties observed for the CscrCpG element despite its lack of transcriptional activity, our data suggest that different mechanisms are involved in the process of CBX3-mediated transgene stabilisation.39; referred to as SFFV.eGFP) to generate the vectors pRRL.PPT.CBX3(bp-bp).SFFV.eGFP.pre* referred to as CBX3(bp-bp).SFFV.eGFP. Insertion of the CBX3 element into the SFFV.eGFP by using the restriction enzymes XhoI and NheI gave rise to the vector pRRL.PPT.CBX3.SFFV.eGFP.pre* (CBX3.SFFV.eGFP). The CBX3(503-679) subfragment was generated by digesting the CBX3.SFFV.eGFP with XhoI and ApaI, filling the ends by using Klenow fragment (ThermoFisher Scientific) and subsequently self-ligating the vector to generate the construct CBX3(503-679).SFFV.eGFP.To generate the CBX3 subfragments, specific primers were designed and modified to include either an XhoI restriction site (forward) or NheI restriction site (reverse)  supplemented with heat-inactivated foetal calf serum , penicillin/streptomycin (1 mM) and L-glutamine (1 mM) . The cells were transiently co-transfected with lentiviral vector (5 µg), pcDNA3.GP.4xCTE , pRSV-Rev (5 µg), and pMD.G  in the presence of HEPES buffer (ThermoFisher Scientific) and chloroquine (Sigma Aldrich). 48 hours and 72 hours after transfection viral supernatants were harvested and concentrated by ultracentrifugation at 4 °C. Subsequently, viral titres were determined by transduction of the murine SC-1 fibroblast cell-line  in serial dilution and flow cytometric analysis of eGFP-expressing cells 3 days thereafter.Lentiviral supernatants were produced by transient co-transfection of 293 T cells using calcium phosphate precipitation according to the previously described protocol41, the miPSC line CD45.142 as well as the RMCE based mESC clones were cultured on mitomycin-C treated murine embryonic fibroblast (MEF) feeder cells in ESC medium  ). The medium for RMCE based mESCs was additionally supplemented with CHIR  and PD  to maintain their pluripotency. The cells were passaged every 2–3 days using Trypsin (Invitrogen).The mESC line CCE5 mESC or miPSC single cells were seeded per well on gelatine-coated 12-well plates containing standard ESC-medium supplemented with protamine sulfate  and lentiviral supernatant was added  for mESC or miPSC, respectively). 3–4 days thereafter the transduced cells were transferred onto MEF feeder cells with fresh medium and cultured as described above.For transduction, 1 × 1043, which harbour an RMCE compatible FRT-wt/FRT-F5 flanked expression cassette as well as a silent neomycin phosphotransferase expression unit at the ROSA26 locus43. RMCE compatible TIGRE ES cells carry a single copy of chromosome 19 derived BAC RP24-481E2 which was genetically engineered by integrating an RMCE-compatible FRT-wt/FRT-F5 flanked CMV-driven GFP cassette as well as a silent neomycin phosphotransferase expression unit in the tightly regulated (TIGRE) T1 locus17, which was designated as the TIGRE locus in this study. RMCE was induced in either G4B12 or TIGRE ESCs by co-transfection with FRT-wt/FRT-F5 flanked SFFV.eGFP or CBX3.SFFV.eGFP containing plasmids together with an FLP recombinase expression plasmid. G418-mediated selection was used to isolate correctly targeted isogenic subclones which were expanded individually44.The targeting of the ROSA26 chromosomal locus was performed by Flp recombinase mediated cassette exchange (RMCE) in G4B12 cells45 but without the addition of cytokines on day 5. In brief, mESCs or miPSCs were seeded on gelatine-coated 6-well or 12-well plates and cultured for 2–3 days without feeders. The cells were then moved into suspension culture to allow embryoid body (EB) formation. On day 8 the cells were harvested, dissociated using Collagenase IV (STEMCELL technologies), stained for SSEA-1 expression and analysed by flow cytometry.Undirected differentiation was carried out as previously described for hematopoietic differentiation40. In brief, cells were harvested and dissociated as described above and resuspended in phosphate buffered saline (PBS) (Invitrogen). The cells were then stained, washed with FACS buffer and analysed with a FACScalibur machine (Beckton & Dickinson). The raw data were then analysed with the software FlowJo (TreeStar).Pluripotent as well as differentiated mESCs and miPSCs were stained for stage specific-antigen embryonic antigen-1 (SSEA-1) expression using the Anti-Human/Mouse SSEA1-APC (eBioscience) antibody. Flow cytometric analysis was performed as described47. In brief, samples were analysed for the presence of the woodchuck post-transcriptional element (wPRE) and the polypyrimidine tract binding protein 2 gene . A linear plasmid containing both the wPRE and the ptpb2 sequence was used to generate a standard curve.Genomic DNA was isolated from transduced mESCs and miPSCs using the GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich). The vector copy number (VCN) was determined by quantitative PCR as was previously described15). The CBX3 sequence was randomly shuffled by using the DNA shuffling tool from the bioinformatics homepage (http://www.bioinformatics.org/sms2/shuffle_dna.html). To obtain the scrambled CBX3 with preserved CpG-sites (CscrCpG), the CpG-sites were extracted from their original position in the CBX3 sequence. The remaining sequence was then shuffled and the CpG-sites were subsequently reintroduced at their original position. The resulting sequence was then modified by shuffling specific areas to remove any newly formed CpG-sites, any identified start codons  finder from bioinformatics; http://www.bioinformatics.org/sms2/orf_find.html), as well as any recognition sites for the restriction enzymes XhoI and NheI (needed for subsequent cloning). The final sequence was analysed using the National Center for Biotechnology Information (NCBI) Nucleotide BLAST tool and no similarities to other sequences were identified. To obtain the fully scrambled CBX3 element, the same procedure was used without previously extracting the CpG-sites. Both of these scrambled CBX3 elements were then synthesized by Eurofins Genomics and subsequently cloned upstream of the SFFV promoter in an SFFV.eGFP third-generation SIN-lentiviral vector.The current study used the splice-site deleted version of the CBX3 element  was applied to perform Student’s t-test (α = 0.05) for a comparsion of two groups or one-way ANOVA with Tukey’s multiple comparisons test (α = 0.05) for a comparison of more than two groups. Error bars indicate the standard deviation (SD). Significance is indicated as follows: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.corr. Subsequently, the MFIcorr was normalized to the vector copy number (MFIcorr/VCN). For the correlation of promoter activity and anti-silencing activity, the data were adjusted to a linear scale by using the positive control (C-SG) as 100% and the negative control (SG) as 0%.Number of CpG-sites, length, GC-content, CpG-density, and the promoter activity of the UCOE elements were correlated with the anti-silencing activity by using linear regression analyses in Graphpad Prism. The anti-silencing activity was defined as the relative percentage of eGFP-expressing cells (with day 0 accounting for 100% activity) of cells transduced with SFFV-based lentiviral vectors following undirected differentiation. The promoter activity of the CBX3 subfragments was defined as their ability to directly drive eGFP expression (measured as median fluorescence intensity (MFI)). The MFI was corrected for background eGFP expression of the negative control (G) by subtraction from each sample (i.e. G = 0) thereby yielding MFIThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary material


Methanococcus maripaludis, the euryarchaeal archaellum regulator A (EarA) is required for the transcription of the fla operon, which is comprised of a series of genes which encode most of the proteins needed for the formation of the archaeal swimming organelle, the archaellum. In mutants deleted for earA (ΔearA), there is almost undetectable transcription of the fla operon, Fla proteins are not synthesized and the cells are non-archaellated. In this study, we have isolated a spontaneous mutant of a ΔearA mutant in which the restoration of the transcription and translation of the fla operon , archaella formation and swarming motility were all restored even in the absence of EarA. Analysis of the DNA sequence from the fla promoter of this spontaneous mutant revealed a deletion of three adenines within a string of seven adenines in the transcription factor B recognition element (BRE). When the three adenine deletion in the BRE was regenerated in a stock culture of the ΔearA mutant, very similar phenotypes to that of the spontaneous mutant were observed. Deletion of the three adenines in the fla promoter BRE resulted in the mutant BRE having high sequence identity to BREs from promoters that have strong basal transcription level in Mc. maripaludis and Methanocaldococcus jannaschii. These data suggest that EarA may help recruit transcription factor B to a weak BRE in the fla promoter of wild-type cells but is not required for transcription from the fla promoter with a strong BRE, as in the three adenine deletion version in the spontaneous mutant.In  In the third domain of life, the Archaea, the transcription machinery is composed of a multi-subunit RNA polymerase that shares homology to the eukaryotic RNA polymerase II, as well as two general transcription factors: the TATA-box binding protein (TBP) and transcription factor B (TFB) . The corAlthough Archaea use a eukaryote-like basal transcription machinery, the genome structure and its transcription regulation are more like that found in Bacteria. In Archaea, a cluster of genes is co-transcribed into a poly-cistronic mRNA under the control of a single promoter, which can be regulated by repressors and/or activators . TranscrMethanococcus maripaludis is a member of the phylum Euryarchaeota and a model organism for studies in Archaea. Here, the fla operon, encoding the components of the archaeal swimming organelle, the archaellum , followed by the fla-associated genes flaC-J   with shaking at 35°C (Escherichia coli TOP10 cells were cultured in Luria-Bertani (LB) broth or LB agar in the presence of 100 μg/mL ampicillin for plasmid selection at 37°C. Strains used in this study are listed in Table 1. (Mm900) , Mc. marA ΔearA,  and muta at 35°C . EscheriearA mutant was streaked three times for purity, and one colony was grown overnight and frozen as the stock culture at -80°C. Western blot analysis confirmed the cessation of FlaB2 expression in the ΔearA strain at this stage .Immediately after its generation, the Δis stage . The Δeafla promoter region spanning from -348 bp upstream of the TSS of the fla promoter to 162 bp downstream of the TSS from the ΔearA-sp strain was PCR amplified using primer pair P1-For/P1-Rev (Table 2) and washed ΔearA-sp cells as template  using Clustal Omega to detect the presence of any mutation  and washed ΔearA-sp strain cells as template. The PCR product was digested with BamHI and cloned into BamHI digested pCRPrtNeo  following a modified Gram negative bacteria RNA extraction protocol with an additional DNase treatment using a TURBO DNA-free Kit (Ambion) at 37°C for 30 min. Ten nanograms of total RNA from each extraction was converted into cDNA using an iScriptTM cDNA Synthesis Kit (Bio-Rad) with random hexamer primers. To detect the transcript level of flaB2, gene specific primers were constructed to amplify flaB2 and the slp gene that encodes the S-layer protein (the latter was used as the reference) . Analysthe TSS;  upstreamfla promoter region could have occurred elsewhere in the genome of ΔearA-sp that were solely, or partially, responsible for the restoration of FlaB2 production. To explore if the three adenine deletion detected in the fla promoter region in the ΔearA-sp strain alone would result in the restoration of expression of FlaB2 in the absence of EarA, a mutant which carried the same three adenine deletion mutation in the fla promoter region as that in ΔearA-sp, was generated from the original stock ΔearA strain that did not synthesize FlaB2. Since the size difference in the fla promoter region of ΔearA and the generated three adenine mutant would be only three nucleotides, we did not try to screen mutants by PCR analysis. Instead, we used western blotting to screen for FlaB2 production, since if the deletion of the three adenines was responsible for restoration of transcription of the fla operon, transformants bearing this deletion would be readily identified from transformants that had retained the wild-type seven adenine sequence in the BRE region. Western blotting of a random number of transformant colonies appearing on 8-azahypoxanthine plates identified both ones that did and did not synthesize detectable amounts of FlaB2. The sequence of the fla promoter of four colonies where FlaB2 production was detected and seven colonies in which FlaB2 production was not detected were determined. In each of the colonies in which no FlaB2 was detected by western blotting, a wild-type fla promoter sequence, i.e., with seven consecutive adenines in the BRE, was found. In each of the four colonies that were found to produce FlaB2, the fla promoter was identical to the wild-type sequence except for the three adenine deletion in the BRE (data not shown). One of the transformant colonies that produced FlaB2 and had the three adenine deletion in the BRE was designated Δ3A and studied further. As shown in Figure 3A, FlaB2 production in the ΔearA-sp strain was near wild-type levels. In contrast, in the Δ3A cells, the expression level of FlaB2 was lower than that from the ΔearA-sp strain. PCR analysis of the Δ3A cells confirmed that these cells still possessed the deletion in earA .It is possible that mutations other than the three adenine deletion in the earA-sp and Δ3A strains as demonstrated by the western blot results indicated that transcription of flaB2 was occurring in both mutant strains. A direct measure of the transcript level of flaB2 in these two mutants as well as control strains was obtained in qRT-PCR experiments . As expected, flaB2 transcripts were not detected in the ΔflaB2 strain and were barely detectable in the ΔearA strain. In contrast, the transcription level of flaB2 was increased over 4-fold and 2.5-fold in the ΔearA-sp and the Δ3A strains, respectively, compared to that detected in wild-type cells. The relatively higher transcription level of flaB2 in the ΔearA-sp cells compared to the Δ3A cells was consistent with production of FlaB2 in the two strains detected in the western blot. However, the production of FlaB2 in the Δ3A cells was lower than in wild-type cells even though flaB2 transcription was higher.Restoration of FlaB2 synthesis in the ΔflaB2 had been restored in the ΔearA-sp and Δ3A strains. To determine if the transcription and translation of the entire fla operon was restored in the two mutant stains resulting in assembly of archaella, cells were examined by electron microscopy. As shown in Figure 4, archaella were observed on the cell surface of both ΔearA-sp and Δ3A cells, as well as the wild-type cells, but not on ΔflaB2 or ΔearA cells.qRT-PCR and western blot analyses demonstrated that transcription and translation of earA-sp and Δ3A cells were functional, swarming motility assays were performed. Overnight cultures of ΔearA-sp, Δ3A, as well as Mm900, ΔflaB2, and ΔearA strains were inoculated onto semi-solid Balch medium III agar. After incubation at 37°C for 4 days Mm900, ΔearA-sp and Δ3A cells were clearly motile although the motility of the Δ3A cells was less than the other two strains . The non-archaellated strains, ΔflaB2 and ΔearA, remained at the inoculation spot, as expected. The swarming data are consistent with data from western blot, qRT-PCR, and EM analyses.To further determine if the archaella observed on Δfla operon in Mc. maripaludis via its binding to at least one of four consensus sequences located immediately upstream of the BRE and TATA box of the fla promoter. In a ΔearA mutant, transcription of the fla operon is barely detectable and cells are non-archaellated   , very siannielii  and featannielii . One of t of TBP , 2005. At of TBP . AnalysiMcc. jannaschii, a hyperthermophilic relative of Mc. maripaludis, two studies have identified promoter sequences on a whole genome basis (Mcc. jannaschii protein gene promoters that there was a greater variability in the BRE than in the TATA box (in vitro selected promoters (mostly for tRNA genes) in the earlier study. Examination of the BRE sequences in both protein-encoding genes and tRNA genes revealed that most had internal stretches of 3–5 adenines, far less than the seven adenines in the wild-type fla operon promoter. Interestingly, the fla operon BRE element has a G at position -1, the most commonly found base at that position in the strong tRNA gene BRE , while in protein-encoding genes the most common base at -1 is C  showing the strongest specificity determinants . There ame basis . The firme basis . These sme basis . It was TATA box . The ide -1 is C .fla operon promoter, with its stretch of seven adenines, does not show strong sequence identity to what may be considered strong BRE sequences as reported for Mcc. jannaschii. On the other hand, it is apparent that the three adenine deletion version found in the ΔearA-sp mutant much more closely aligns with BRE sequences found in strong tRNA gene promoters of Mcc. jannaschii . In addition, the mutated fla promoter in the ΔearA-sp strain shares high sequence identity with two studied promoter sequences in Mc. maripaludis, namely the nitrogen-regulated glnA and nifH promoters   suggest that the wild-type fla operon promoter even with EarA is not as strong as the three adenine deletion version in the absence of EarA.The wild-type version of the BRE of the gure 6A) , 1999. Bnditions . Both ni deleted . This incription , 1999. TSulfolobus solfataricus, transcription from the arabinose promoter is induced in the presence of arabinose, via an unidentified factor that binds to a consensus ara-box sequence located immediately upstream of the BRE and TATA box  T6 promoter . This activation is dependent on the weak BRE of the pf1089 promoter and is not observed if the pf1089 promoter BRE is replaced with the BRE from the strong gdh promoter  in the ΔearA-sp strain that could be revealed by comparison of the complete sequence of the ΔearA and ΔearA-sp strains.The appearance of the Δrn blots . It was earA mutant, indicating that in the spontaneous mutant the need for the transcriptional activator EarA for the transcription of the fla promoter was bypassed. Analysis of the DNA sequence in the fla promoter region from the spontaneous mutant revealed a three adenine deletion in the BRE region in the fla promoter. Sequence alignment showed that the mutated BRE in the fla promoter shares high similarity with BREs from strong promoters in methanogens, indicating that with this mutated BRE, the transcription initiation of the fla promoter could be conducted with components of the basal pre-initiation complex. We believe this is the first report of spontaneous mutation in the promoter region that overcomes the need for a transcriptional activator and it emphasizes the key role played by the BRE in promoter strength in Archaea.In this study, a spontaneous mutant with restored FlaB2 expression was isolated from a ΔConceived and designed experiments: YD, AB, CK, and KJ. Performed the experiments: YD, AB, CK, and KJ. Analyzed the data: YD, AB, CK, and KJ. Wrote the paper: YD, AB, CK, and KJ.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.