The product of dosage compensation in female mammals is the inactive X chromosome (Xi). Xi facultative heterochromatin is organized into two different types, one of which is defined by histone H3 trimethylated at lysine 9 (H3K9me3). The rationale for this study was to assess SET domain bifurcated 1 (SETDB1) as a candidate for maintaining this repressive modification at the human Xi.IL1RAPL1 gene that is part of a common chromosome fragile site that is frequently deleted or rearranged in patients afflicted with intellectual disability and other neurological ailments. Centrally located within this interval is a powerful enhancer adjacent to an ERVL-MaLR element. In the absence of SETDB1, the enhancer is reactivated on the Xi coupled with bidirectional transcription from the ERVL-MaLR element. Xa deletion of the enhancer/ERVL-MaLR resulted in loss of full-length IL1RAPL1 transcript in cis, coupled with trans decompaction of the Xi chromosome territory, whereas Xi deletion increased detection of full-length IL1RAPL1 transcript in trans, but did not impact Xi compaction.Here, we show that loss of SETDB1 does not result in large-scale H3K9me3 changes at the Xi, but unexpectedly we observed striking decompaction of the Xi territory. Close examination revealed a 0.5 Mb region of the Xi that transitioned from H3K9me3 heterochromatin to euchromatin within the 3′ end of the These data support a critical role for SETDB1 in maintaining the ERVL-MaLR element and adjacent enhancer in the 3′ end of the IL1RAPL1 gene in a silent state to facilitate Xi compaction.The online version of this article (10.1186/s13072-018-0218-9) contains supplementary material, which is available to authorized users. The XX/XY chromosome mode of sex determination necessitates the need to compensate levels of X-linked gene expression between the sexes. In mammals, this is achieved through the process of X chromosome inactivation (XCI), whereby one of the two X chromosomes in females is rendered transcriptional silent early in development . Gene siIL1RAPL1) gene that transitioned from a H3K9me3-defined region of heterochromatin to euchromatin. Centrally located within this interval we identified a powerful enhancer element adjacent to an ERVL-MaLR that is reactivated from the Xi. Through characterization of these elements and their deletion from either the Xa or Xi, we reveal complex cis and trans effects that influence IL1RAPL1 transcription and chromosome compaction.By generating SETDB1 knockouts (KO) in human cells, we reveal that while SETDB1 is not required to maintain chromosome-wide H3K9me3 at the human Xi, its loss results in decompaction of the Xi chromosome territory. The most obvious chromatin change in the Xi corresponds to a 0.5-Mb interval embedded within the 3′ end of the large interleukin-1 receptor accessory protein-like 1  to assemble a pair of previously reported active transcription activator-like effector nuclease (TALENs) that target exon-2 Fig.  or changIL1RAPL1 gene showed gain of H3K4me2 and a corresponding reduction in H3K9me3 for the same region  shows a general nuclear distribution with obvious underrepresentation at nucleoli and the territory of the Xi that appears as a hole in the staining pattern within which is a single intensely staining focus  shows a . This siIL1RAPL1 that shows chromatin change in the absence of SETDB1 falls within a common fragile site (FRAXC) on the X chromosome [IL1RAPL1 is involved in regulating neuron development –35, and romosome , indicatIL1RAPL1 were selected for use in RNA FISH experiments , IL1RAPL1 signal associated with the XIST cloud (interpreted as originating from the Xi) and two IL1RAPL1 signals   within the 3′ transcript [rs140261831, rs113985890, rs190632360 and rs144991617: SNP-A through D, respectively   flanking the enhancer and bidirectional promoter with the RNA-guided Cas9 nuclease in an attempt to delete the interval [These data indicate that loss of SETDB1 results in the derepression of the Xi enhancer and bidirectional promoter embedded within the 3′ end of interval . NumerouTo further characterize these mutants, we initially assessed expression levels of IL1RAPL1 from the 5′ and 3′ ends by qRT-PCR. Interestingly, loss of the enhancer element from the Xa resulted in compete loss of 5′ transcript that initiates transcription from a promoter located greater than 1 Mb upstream. This indicates that transcription from the 5′ promoter is dependent upon the enhancer element. Interestingly, loss of the enhancer element from the Xi resulted in an increase in expression from the 5′ end that surprisingly corresponds to upregulation from the Xa in trans (see below). With the exception of one Xa enhancer mutant, expression from the 3′ end decreased, regardless of whether the enhancer was deleted from the Xa or Xi Fig. a. In ordSETDB1 loss in human cells does not result in chromosome-wide changes to H3K9me3 bands along the Xi, although a role in establishing these territories during XCI similar to what has been described in mouse cannot be ruled out . HoweverIL1RAPL1 gene. H3K9me3 was lost coupled with gain of H3K4me2 and broad acquisition of H3K27Ac throughout the interval. The highest peaks of H3K27Ac and H3K4me2 reside near the center of the chromatin change interval, and the DNA underlying these peaks correspond to a powerful enhancer adjacent to an ERVL-MaLR element that promotes bidirectional transcription. While transcription in the reverse direction produces a novel long noncoding RNA (lncRNA), the forward transcript has the potential to generate a protein isoform of IL1RAPL1 that lacks part of the N-terminus. Both transcripts are abundantly transcribed from the Xi in SETDB1-KO clones and appear interspersed with the XIST cloud. Given the central role of SETDB1 in silencing endogenous retroviral elements [Alongside the decompaction phenotype, we observed that loss of SETDB1 triggered a large-scale chromatin state transition on the Xi within an approximately 500 kb interval located in the 3′ end of the elements –48, we iIL1RAPL1 results in intellectual disability [Interestingly, mutations in or loss of SETDB1 have been reported in cancer , 55. Bassability  and the sability , 56–61, sability , autism sability , 63 and sability . The linsability .The reactivation of the powerful enhancer coupled with adjacent bidirectional expression on the Xi upon SETDB1 loss raised the possibility that this could be mechanistically linked to the Xi decompaction phenotype. Deletion of the enhancer from the Xi in the absence of SETDB1 mutation did not result in Xi decompaction, but deletion of the enhancer from the Xa did, which was coupled with detection of 3′ bidirectional transcripts from the Xi only. How this trans-chromosomal effect is mediated is unclear but was not a phenomenon restricted to the Xa deletion impacting the Xi, because cells with the enhancer deleted from the Xi showed increased expression on the Xa from the 5′ end of the gene. A summary of our findings is outlined in Fig. IL1RAPL1 locus on the Xi allele. Through targeted deletion of the ERVL-MaLR element and enhancer, we reveal unexpected cis and trans effects impacting chromosome compaction and transcription of IL1RAPL1 isoforms. In light of these new data, further investigation of these findings in the context of genomic instability, neurological disorders and expression changes in brain tumors is warranted.Collectively we reveal a novel critical function for SETDB1 in ensuring the proper compaction of the human Xi chromosome territory through the silencing and seeding of heterochromatin at an ERVL-MaLR LTR and adjacent enhancer in the 3′ end of the hTERT-RPE1 (RPE1), a female telomerase-immortalized human retinal pigment epithelial cell line, was obtained from Clontech (C4000-1). This product is no longer available through Clontech but can be obtained directly through the American Type Culture Collection (ATCC). Human female embryonic kidney cell line 293 (CRL-1573) was obtained from ATCC. Cells were maintained according to the supplier recommendations. Transfection of plasmid DNA into 293 cells was performed using Lipofectamine 3000 (Cat. No. L3000008) according to the supplier instructions (ThermoFisher Scientific). TALEN expression plasmids and targeting constructs were introduced into RPE1 cells using a 4D-Nucleofector (Lonza) using SF Cell Line 4D-Nucleofector reagent (Cat. No. V4XC-2024), as were gRNA/Cas9 expression constructs.TALEN pairs previously reported to actively cut the human SETDB1 locus were assembled using the FLASH procedure essentially as described . Activithttp://crispr.mit.edu) and cloned into pSpCas9(BB)-2A-GFP (pX458) as described [Sequences flanking the putative IL1RAPL1 enhancer core were used to identify suitable gRNAs using the Optimized CRISPR Design , as were rabbit polyclonal antibodies to histone H3 trimethylated at lysine 9 (Cat. No. 07-523), histone H3 dimethylated at lysine 4 (07-0030), anti-acetyl-lysine (06-933) and mouse monoclonal antibodies to HP1β (Cat. No. MAB3448). Alexa-Fluor-conjugated secondary antibodies were obtained from ThermoFisher Scientific. Rabbit polyclonal antibodies to SMCHD1 were obtained from Bethyl Laboratories Inc. (Cat. No. A302-872A). Rabbit antibodies to β-actin were obtained from Cell Signaling Technology (Cat. No. 4970). Rabbit polyclonal antibodies to SETDB1 were obtained from Cell Applications Inc. (Cat. No. CY1112).Protein extracts, polyacrylamide gel electrophoresis, Western blotting and detection were carried out as described previously .All qRT-PCR was performed on a CFX96 (Bio-Rad) using an EvaGreen 2X qPCR mastermix  as described previously . StandarChIP-Seq to H3K4me2, H3K27Ac and H3K9me3 was performed through the full-service procedure provided by Zymo Research according to their established protocols. This included the ChIP assay, library preparation, Next-Gen 50 bp single-end sequencing, bioinformatics and all quality control steps. Three independent frozen pellets of 5 million formaldehyde cross-linked cells for each sample were provided to Zymo Research for the ChIP procedure. In brief, cells were cross-linked with formaldehyde at 1% final concentration for 7 min at room temperature. ChIP assays were conducted using the Zymo-Spin ChIP kit  following the manufacturer’s instructions. Sonication was performed at high power setting for 40 cycles  using a Bioruptor Plus , yielding a modal fragment size of < 600 bp. Antibodies used in ChIP assays included: anti-H3K4me2 (Abcam ab7766), anti-H3K9me3 (Abcam ab8898), anti-H3K27ac (Abcam ab4729) and normal rabbit IgG (Millipore 12-370 or Diagenode C15410206). Approximately 10 µg of chromatin from RPE1 or SETDB1 cells was used in each ChIP assay with 4 µg of antibodies. IgG negative control was included with each assay. DNA libraries were prepared by Zymo Research Epigenetics Services and were sequenced on a HiSeq sequencer. ChIP-Seq reads were aligned to human reference genome GRCh37/hg19 by Bowtie  with at https://genome.ucsc.edu/index.html). Sashimi plots were generated using the function provided by the Integrative Genomics Viewer (IGV_2.3.97).RNA-Seq was performed through the services provided by Novogene. Triplicate total RNA extracts for each clone were prepared using the Nucleospin RNA Plus kit from Macherey–Nagel (Cat. No. 740984). For each sample, 6 μg of total RNA was provided to Novogene who then performed quality control, removed rRNA and prepared strand-specific libraries before generating at least 60 million 150 bp paired-end reads per sample. Adapter sequences were removed using Cutadapt ; Fastq sAll Sanger sequencing was performed using the services provided by Eurofins Genomics, and sequences were edited and visualized using Sequencher 5.4.6 (Gene Codes Corporation).A DNA fragment of 810 bp encompassing the core conserved putative enhancer region of IL1RAPL1 and a larger fragment of 2613 bp were amplified with primers that incorporate a 5′ SalI and 3′ BamHI site before cloning into pMiniT2.0 . Clones were verified to be free of PCR-induced mutations before subcloning into the SalI-BamHI sites of pGL4.23  downstream of the luciferase expression cassette. As an enhancer control, the SV40 enhancer fragment of pGL3.control  was excised with XbaI and BamHI and subcloned into XbaI-BamHI cut pGl4.23. A DNA fragment of 529 bp encompassing the ERVL putative promoter element was amplified and cloned into pMiniT2.0. Clones were verified to be free of mutations by sequencing, and the insert was excised using XhoI and subcloned into XhoI cut and de-phosphorylated pGL4.10 . Clones were isolated that contained the ERVL element in either orientation. Dual luciferase assays were carried out as described previously . Empty vAll BAC clones were obtained from ThermoFisher Scientific. Clones were cultured according to the supplier recommendations and DNA isolated using the NucleoBond Xtra BAC isolation kit from Macherey–Nagel (Cat. No. 740436). BAC clone information is given in Additional file Images were collected on a Delta Vision pDV. Images were de-convolved with softWoRx 6.5.2  and compiled into images using Adobe Photoshop CC 2018 (Adobe Systems). For nuclear volume, de-convolved D3D files were used with Model-2D polygon finder, selecting a rectangle box around the DAPI image with a minimum perimeter setting of 300. 3D object builder was used to generate 3D objects followed by the Measurements function to generate a table of 3D measurements. H3K27me3 Xi volumes were measured as above setting the minimum perimeter to 30. The Xi to DAPI ratio was calculated in Microsoft Excel before importing into JMP pro 12 for statistical analysis of volume measurement. Typically, 40 nuclei are assessed for each sample.t tests were performed using the QuickCalcs function in GraphPad software. JMP Pro 12 was used for the statistical analysis of Xi volume measurements and luciferase assay data. Means of all pairs were compared using the Tukey’s honest significance test.Unpaired Additional file 1. TALEN-assisted targeting of the SETDB1 gene. (a) Inverted ethidium bromide-stained agarose gel image showing the results of the Surveyor assay. 293 cells were transiently transfected with mammalian expression constructs expressing a previously reported active TALEN pair targeted to exon-2 of human SETDB1 [b) Schematic representation showing the relative position of exons 1–3 of SETDB1. Gray-shaded intervals represent repetitive DNA sequences, whereas white regions are unique. The location of the left and right homology arms (LHA and RHA) are shown in pink above the genomic locus. Upward- and downward-facing black arrow heads indicate the binding location of the top-strand binding and bottom-strand binding TALENs. Inward-facing black arrows represent the location of oligonucleotide primers used for the Surveyor PCR. The location of exons 1–3 is shown as black boxes (exons) joined by black lines (introns) below the map. (c) Schematic representation of a successfully promoter trap targeted SETDB1 locus. The blue shaded interval represents the integrated pSEPT vector cloned between the LHA and RHA that contains the promoterless neomycin cassette. Inward-facing arrows show the approximate location of oligonucleotide primers used to amplify between the pSEPT vector and genomic locations up and downstream of the homology arms that are used for screening of clones by PCR. Beneath the map is a schematic showing the splicing of exon-1 to the neomycin cassette followed by transcription termination by a polyadenylation signal located in the pSEPT vector.n SETDB1  (SETDB1)n SETDB1  , right homology arm integration (bottom) and the presence or absence of the TALEN cut site (middle). Molecular weight markers are in the first lane of each gel, and the sizes of fragments in kb are indicated to the left. PCR results for independent clones are shown in each lane and labeled above. Negative controls of RPE1 genomic DNA and water are shown at the far right of the gels. Successfully targeted clones generate a 1512 bp product for the left side and a 2183 bp product for the right side. The TALEN cut-site PCR generates a 269 bp product if the interval is intact or only targeted at one SETDB1 allele. Clones 3, 6 and 40 were selected for further analysis. All three are positive for the correct left and right targeting and are negative for the cut site PCR.Additional file 3. Nuclear volume measurements. Graph shows the results of nuclear volume measurements (based on DAPI volume) in RPE1 alongside the three SETDB1 mutants, S3, S6 and S40 (x-axis). DAPI volumes are indicated on the y-axis. Each black dot indicates a measurement made for individual nuclei in each sample. The green diamonds show the mean  and 95% confidence interval between the apexes of the diamond. The width of the diamond is proportional to the sample size with wider diamonds indicating more measured nuclei. Circles on the far right show all pairs Tukey–Kramer 0.05 p value spread with significance presented by the angle of intersection between circles. None of the SETDB1 mutants have nuclear volumes that are significantly bigger than parental RPE1.Additional file 4. Validation of the SETDB1 mutant-specific H3K4me2 Xi band within the 3′ end of the IL1RAPL1 gene. (a) Schematic map of the IL1RAPL1 gene locus. The horizontal line represents introns bisected by vertical lines corresponding to exons. The solid black bar beneath the map indicates the approximate extent of chromatin change observed by ChIP-Seq  Top panel shows representative examples of metaphase Xi in SETDB1 mutant S40 showing the distribution of H3K4me2 by indirect immunofluorescence (red) merged with DAPI (Blue). White arrow heads indicate the location of the novel H3K4me2 band observed in the mutant clones. The second row shows the hybridizing BAC probe signal (green) merged with DAPI (blue), whereas the last row shows a merge of all three. (c) Top panel shows an example of the H3K4me2 indirect immunofluorescence pattern (red) merged with DAPI (Blue) on the Xa alongside the Xi. The Xa-specific translocation of chromosome 10 at Xq28 results in a substantially longer Xa relative to the Xi, facilitating the ability to readily distinguish the two chromosomes. White arrow heads indicate the presence of the H3K4me2 band on the Xi but not Xa. The second panel shows the hybridization pattern for BAC probe 426F14 (Green) merged with DAPI (Blue). The bottom panels show a merge of all three.Additional file 5. Genotyping results for transcripts originating from the 3′ end of IL1RAPL1. (a) Schematic map of the 3′ end of IL1RAPL1. The gray bar represents the genomic locus and black vertical lines indicate exons. The relative location of SNPs A–D is indicated above the genomic map. Below the genomic map is a representation of the IL1RAPL1 primary transcript followed by the relative position of BAC clones. (b) Inverted ethidium bromide-stained agarose gel image showing RT-PCR results with primers spanning SNPs A, B, C and D. Each gel shows RT-PCR results for a negative control water sample, cDNA prepared with reverse transcriptase (+RT) and without reverse transcriptase (− RT) for parental RPE1 and SETDB1 mutant S40. (c) DNA sequence traces for SNPs A, B, C and D in RPE1 genomic DNA, genomic DNA isolated from a somatic cell hybrid in which the RPE1 Xa is the only human chromosome present, and cDNA samples from RPE1 and SETDB1 mutant S40. The downward-facing black arrow heads at the top indicate the location of the SNP that appears as two merged traces in samples containing material from both alleles.Additional file 6. List of oligonucleotides used in this study. All oligos were obtained from Eurofins Genomics LLC. Oligo names are given in column-1, the corresponding sequence is in column-2, and what the oligo was used for is listed in column-3.Additional file 7. Validation of successful deletion of the IL1RAPL1 enhancer and the impact on allele expression by SNP genotyping. (a) Ethidium bromide-stained agarose gel images showing PCR results from individual clones exposed transiently to the gRNA pair flanking the enhancer locus. Successful deletion of the enhancer interval is indicated by the presence of a PCR product. Parental RPE1 and water are negative controls, whereas the “Pool Sorted” sample represents a pool of FACS sorted RPE1 cells exposed to the gRNAs as a positive control. (b) Dropped metaphase chromosome spreads for the indicated clones showing the hybridizing FISH signals for Xp control BAC probe ZFX (Red) and a probe generated from the enhancer deleted region (Green) merged with DAPI (Blue). The Xa and Xi are indicated. (c) DNA sequence traces for SNPs A and C in RPE1 genomic DNA, genomic DNA isolated from a somatic cell hybrid in which the RPE1 Xa is the only human chromosome present, and cDNA samples from RPE1, SETDB1 mutant S40 and four independent enhancer mutants. The downward-facing black arrow heads at the top indicate the location of the SNP that appears as two merged traces in samples containing material from both alleles.Additional file 8. List of BAC clones used in this study. BAC clone names are given in column-1 and the corresponding gene content is listed in column-2Additional file 9. IL1RAPL1 RNA FISH data for the BACs indicated in the cells listed. Xi expression is defined as a single associated with the XIST RNA cloud. Each column represents data from replicate experiments. Therefore, a clone with BAC data in two columns indicates that the experiment was done twice, whereas that in four columns indicates the experiment was done on four occasions


Nuclear factor erythroid-derived-2-like 2(NRF2) regulates its downstream genes through binding with antioxidant responsive elements in their promoter regions. Hyperactivation of NRF2 results in oncogenesis and drug resistance in various cancers including non-small cell lung cancer (NSCLC). However, identification of the genes and pathways regulated by NRF2 in NSCLC warrants further investigation. We investigated the global NRF2 genomic binding sites using the high-throughput ChIP-Seq technique in KEAP1 (Kelch-like ECH-associated protein 1)-mutated A549 (NSCLC) cells. We next carried out an integrated analysis of the ChIP-Seq data with transcriptomic data from A549 cells with NRF2-knockdown and RNA-Seq data from TCGA patients with altered KEAP1 to identify downstream and clinically-correlated genes respectively. Furthermore, we applied transcription factor enrichment analysis, generated a protein-protein interaction network, and used kinase enrichment analysis. Moreover, functional annotation of NRF2 binding sites using DAVID v7 identified the genes involved in focal adhesion. Putative focal adhesion genes regulated by NRF2 were validated using qRT-PCR. Further, we selected one novel conserved focal adhesion gene regulated by NRF2–LAMC1 (laminin subunit gamma 1) and validated it using a reporter assay. Overall, the identification of NRF2 target genes paves the way for identifying the molecular mechanism of NRF2 signaling in NSCLC development and therapy. Moreover, our data highlight the complexity of the pathways regulated by NRF2 in lung tumorigenesis. Lung cancer is the leading cause of cancer mortality in men and women around the world, and ~85% of all lung cancer cases are non-small cell lung carcinoma (NSCLC) . DespiteThe transcription factor NFE2L2 (nuclear factor erythroid-derived-2-like 2), also known as NRF2, is a master regulator of the antioxidant response pathway. Under homeostatic conditions, NRF2 binds to its inhibitor KEAP1 (Kelch-like ECH-associated protein-1). KEAP1 acts as a scaffolding protein for NRF2 and Cullin-3 E3 ubiquitin ligase (CUL3), which promotes the ubiquitin-mediated proteasomal degradation of NRF2 . Due to Chromatin immunoprecipitation followed by next-generation sequencing is an effective technique for the identification of specific transcription factor binding sites (TFBSs) . PreviouTo examine the genome-wide distribution of NRF2 binding sites in lung cancer cells, we performed ChIP-Seq of human A549 NSCLC cells. The ChIPed DNA was sequenced and mapped using an Illumina sequencer. The mapped reads were aligned using Bowtie version 0.12.7-1. From this, we identified a total of 8,619,855 aligned tags for control DNA , and 1,4We then used HOMER software  to perfoWe further annotated the genomic locations of 2,395 NRF2 binding sites using the ChIPSeek web tool . The annde novo motif discovery algorithm. Motifs were sorted based on p-values. As expected, the enrichment results for known motifs were strongest for the bZIP family TFBSs  of the target sequences contained the 12-bp consensus NRF2 ARE (ATGACTCAGCAA) among all TFBSs, with a p-value of 1e-1057  . TRAP anTo determine the binding pattern of the previously-known classic NRF2 target genes listed in review articles –27, we sOur ChIP-Seq data identified 2,051 coding and non-coding regions that represented 2,395 binding sites. We then identified the cell-signaling networks associated with these genes. To decode the networks associated with the NRF2 TFBS, we used the computational tool eXpression2Kinases (X2K). Notably, the X2K transcription factor enrichment analysis accurately revealed NRF2 as the top predicted transcription factor regulating the TFBS-encoded genes . The proTo achieve a more effective TFBS analysis of the functions and gene-regulatory mechanisms of NRF2, we analyzed our NRF2 ChIP-Seq data in combination with GSE28230 microarray data . BesidesTo investigate clinical correlations of the NRF2 TFBS with genes affected by genetic KEAP1 alterations (mutation and deletion) in LUAD patients, we carried out an integrated analysis of the two sets of genes using Venny . This coWe uploaded the list of 2,395 NRF2 TFBS genes into the functional annotation tool DAVID v7  to obtaiInterestingly, previously unrecognized NRF2-regulated pathways were identified among the nine KEGG pathways identified using DAVID v7 see . The newInterestingly, DAVID-KEGG pathway analyses of our ChIP-Seq genes identified a set of 31 candidate genes with 42 binding profiles involved in the focal adhesion pathway. Binding sites in the genes CAPN2, LAMC1, MAPK8, SHC1, SPDYA, and SPP1 were located in promoter-TSS regions and others were located distal to the TSS. Since the binding potency of NRF2 is very high in the promoter regions of genes, we selected those located in the promoter regions for TFBS validation.We next used qRT-PCR to assess the mRNA expression levels of all these genes in siNRF2-C27 and siGFP-C5cells, which are stable NRF2-siRNA knockdown and control A549 NSCLC cells, respectively. Five of the six genes – CAPN2, LAMC1, MAPK8, SHC1, and SPP1 – had reduced mRNA expression in the NRF2 knockdown cells, and SPDYA did not show any activity . Thus, tLAMC1 gene promoter, we performed in silico analysis using the TRAP web tool [LAMC1 binding site sequences across different species using ConTra v3 [LAMC1, we carried out ChIP assays in siNRF2-C27 and siGFP-C5 cells. As expected, the NRF2 knockdown reduced its binding to the ARE site in the HO-1 promoter . Interestingly, the transfectants resulted in a 2.4-fold induction of luciferase activity compared to the control pGL3-treated transfectants  regulates invadopodia and breast cancer invasion , promote therapy , 58. How [, 2019 , showed Interestingly, our functional annotation analysis of TFBS genes identified two other important pathways –the ErbB signaling pathway and fructose and mannose metabolism – which play major roles in oncogenesis. The ErbB family of receptor tyrosine kinases is often dysregulated and aberrantly activated in different kinds of tumors. These receptors are considered to be important specific targets for anti-cancer therapeutics due to their localization in the transmembrane region of the cell . NSCLC iGenetic alterations in KEAP1, NRF2, and CUL3 such as somatic mutations, copy number variations, and epigenetic changes are the major reasons for the gain-of-function and aberrant activation of NRF2-regulated pathways in human cancers and are often correlated with poor prognosis and survival , 66. TheAltogether, our results suggest the fundamental process of how NRF2 participates in the direct regulation of genes involved in invasion, migration, and metastasis in A549 NSCLC cells . However2, and passaged every 3–4 days. All media supplements for cell cultures were from Invitrogen (China).Unless otherwise stated, all chemicals were from Sigma–Aldrich Co., Ltd. , and all antibodies were from Santa Cruz Biotechnology . The A549  and MCF7 cell lines were purchased from the American Type Culture Collection (China). All cells were cultured at 37°C in 95% air and 5% COsiNRF2-C27, an A549-derived cell line stably expressing NRF2-siRNA, was developed in this laboratory and described previously . siGFP-C6 cells/dish then cross-linked with 1% formaldehyde and immunoprecipitation was performed overnight with Nrf2-specific antibody (Santa Cruz Biotechnology) and IgG as control following the instructions with the immunoprecipitation kit . The PCR primers for the GAPDH promoter (negative control) were provided in the kit. The PCR primers for the ARE site in the human HO-1 promoter were as described previously [Chromatin immunoprecipitation (ChIP) experiments were carried out as described previously . Brieflyeviously . To valiThe sequencing and generation of short DNA reads of immunoprecipitated samples were carried out at Genenergy Inc.  using an Illumina Hiseq 2000. Reads were mapped against the human reference genome hg19 with Bowtie 0.12.7-1 (bowtie-bio.sourceforge.net); a maximum of 2 mismatches were allowed in the seed. The ChIP-seq data of raw and aligned files have been deposited in NCBI Gene Expression Omnibus, accession number GSE141497.The significant peaks (NRF2 binding sites) of mapped reads were identified using the program ‘findPeaks’ from the freely-available software suite HOMER (Hypergeometric Optimization of Motif EnRichment)  with thede novo motif discovery as well as identification of the enriched sequence motifs within the ChIP-Seq peak regions. Enrichment of de novo motifs was determined by comparing all 2,022 target sequences with all 46,776 background sequences using the cumulative hypergeometric distribution algorithm. We further submitted the de novo motif matrix to the STAMP website to assess the similarity between known candidate motifs [We used the‘findMotifsGenome.pl’ program of the HOMER software package to perform e motifs .We uploaded the gene symbols identified from the ChIP-Seq results into the eXpression2Kinases (X2K) web tool  for tranThe Identification of putative NRF2 AREs in the 151-bp binding region of human LAMC1 was carried out using the TRAP web tool . This prWe used publicly-available microarray data from NRF2-knockdown A549 cells retrieved from NCBI Gene Expression Omnibus (accession number GSE28230)  to perfoThe ChIP-Seq TFBSs containing gene symbols were uploaded into the DAVID bioinformatics web tool  as descrIsolation of total RNA and qRT-PCR were performed as described previously , 71. Thevia the XhoI and KpnI restriction sites and verified by DNA sequencing.pGL3-LAMC1-151bp is a luciferase reporter plasmid with the 151-bp human LAMC1 promoter. The construct was made by amplification of the 151-bp DNA fragment from human LAMC1  (upstream to the TSS) by PCR with the forward primer 5'-ATGGTACCTGCTCCGGGACTAAAACTCA-3' and the reverse primer 5'-TACTCGAGTGTTCCTCGGCTTGCCTTTA-3'. The amplified DNA fragment was subsequently cloned into the pGL3-promoter vector  Renilla luciferase, was used as a control for transfection efficiency. The dual luciferase activity was determined using the Dual-luciferase Reporter Assay System (Promega) as described in the manufacturer’s instructions.The luciferase reporter plasmid pGL3-LAMC1-151bp was transiently transfected using Lipofectamine 2000 (Invitrogen) as described previously . The pRLStatistical comparisons were performed with unpaired Student’s t-tests. A value of p<0.05 was considered statistically significant.Supplementary FigureSupplementary TablesSupplementary Table 1Supplementary Table 2Supplementary Table 3Supplementary Table 4


The aim of this study was to analyze potential influences of polymorphisms within the regulatory region of the bovine SIRT6 gene on carcass quality traits. Expression analyses suggested that SIRT6 gene is predominately expressed in kidney, compared with other tissues. In 535 indigenous Chinese beef cattle, two novel single nucleotide polymorphisms (SNPs) were identified within the promoter region of the SIRT6 gene.Association analysis indicated that G allele of the c.-1100 A > G had a positive effect on fat deposition, and the Hap4/4 diplotype had more favourable results than other dipoltypes with respect to the evaluation of carcass quality traits. Furthermore, promoter activity associated with the Hap3 haplotype was measured at higher levels than the Hap1 haplotype, which would be in agreement with the previously described association analysis.The SIRT6 promoter variants significantly affect transcriptional levels and subsequently significantly influence bovine intramscular fat content. SIR2 and its homologs, termed sirtuins, are members of the class III nicotinamide adenine dinucleotide-dependent deacetylase family . Seven hPrevious studies demonstrated that SIRT6 gene could deacetylate histone H3K9, and modulate the expression level of associated metabolic genes . More spThrough modifying transcription factor binding sites, sequence variation within promoter and other regulatory regions of gene may impact expression level, and influence phenotypes . PreviouAll animal experiments were conducted according to the guidelines established by the regulations this work was performed at a farm in the Department of Animal Sciences and Technology at Qinghai University, China. Ethical approval for this study was obtained from the Ethical Committee of Qinghai University. The procedures were approved by the Ethical Committee of China Animal Care Qinghai University.As is shown in Fig. -△△Ct method [In this study, two housekeeping genes were used: β-actin AY141970.1) and GAPDH (NM_001034034). The qPCR was conducted by the Applied Biosystems 7500 Fast Real-Time PCR System  with the SYBR® Premix Ex TaqTM kit . The oligonucleotide primers used for the qPCR were designed with Primer Premier 5.0 software  , were obAs is presented in Table ApaLI and AgsI restriction enzymes were utilized to digest PCR products for genotype. The electrophoresis on a 2.5% agarose gel was employed to identify the digested products, which were stained with ethidium bromide.Two SNPs were detected in the promoter region of the SIRT6, named c.-84 C > T and c.-1100 A > G, respectively. Based on the sequence information, the KpnI site (CGGGGTACC) and reverse primer including a BglII site (GGAAGATCT). Use of the dual-luciferase reporter assay standard procedure, the activity of firefly luciferase activity and Renilla luciferase were observed in 3 T3-L1 cells. All experiments were performed in triplicate and repeated twice.Previous methods were adopted to culture 3 T3-L1 cells . A DNA fYijk = u + Gi + Sj + Ak + eijk, where Yijk were the phenotypic observations; μ was the averaged values, Gi was the fixed effect of genotype, Sj was the random effect of sire, Ak was fixed effect of age, and eijk was the residual effect.Evaluation of linkage disequilibrium (LD) was conduted by the HAPLOVIEW software (Version 3.32). The general linear model was used for the evaluation of SNP-phenotype association. The equation was as follows: t test.All values were presented as the mean ± SE. The difference between groups (gene expression levels of qPCR between tissues and relative luciferase activities between different constructs) was analyzed with the two-tailed As shown in Fig. ApaLI and AgsI restriction enzymes were utilized to digest PCR products for genotype.Sequencing of PCR products from genomic DNAs revealed two polymorphisms within the SIRT6 promoter, named c.-84 C > T and c.-1100 A > G, respectively Fig. . Based oP < 0.05). Whereas, genotypes of the other SNP in the promoter region of SIRT6 had no significant correlation with fat deposition.As shown in Table r2 were used for the assessment of the relationship between the c.-84 C > T and c.-1100 A > G. The values of D’ and r2 were 0.147 and 0.021, respectively. Previous studies stated that, LD was strong enough when the r2 values were beyond 0.33 [The estimated values of D’ and ond 0.33 . TherefoP < 0.05) in Qinchuan cattle. The results suggested that the diplotype Hap1/4 and Hap4/4 in fat deposition were prior to other combinations.Four haplotypes were detected and termed as Hap1 to 4 Table . Among tP < 0.05).In this study, four haplotypes were cloned, and then luciferase reporter (named pGL3-Hap1 to 4) were constructed to analyze the effect of various haplotypes on the activity of transcription. Those plasmids were transfected in 3 T3-L1 cells and to detect the transcriptional activities. Figure The intramuscular fat, one of the four mammalian adipose tissue , was locThe results of qPCR revealed that bovine SIRT6 gene mRNA was ubiquitously expressed, in agreement with the previous observations such as mice  and humaNowadays, growing observations indicate that genetic variations in the promoter region can influence economical traits in livestock . Both g.ci value > 85). No differences existed in transcription factors at the c.-84 C > T locus, consistent with the results showing that genotypes of c.-84 C > T did not affect fat deposition in Qinchuan beef. The prediction suggested that, c. -1100 A and c. -1100 G could in sequence bind in three and four cis-acting elements  might influence fat deposition as a result of alteration in SIRT6 transcriptional activity. This may contribute to deep insights into genes associated with the adaptation and specialization of beef cattle breeds in China.


C and WGCW overlapping hot motifs  on both DNA strands. Single mutations occur on 70% of transcribed DNA clones. Mutations are correlated over a 15 nt distance in multiply mutated clones, suggesting that deaminations are catalyzed processively within a stalled or backtracked transcription bubble. Site-by-site comparisons for biochemical and human memory B-cell mutational spectra in an IGHV3-23*01 target show strongly favored deaminations occurring in the antigen-binding complementarity determining regions (CDR) compared to the framework regions (FW). By exhibiting consistency with B-cell SHM, our in vitro data suggest that biochemically defined reconstituted Pol II transcription systems can be used to investigate how, when and where AID is targeted.Activation-induced deoxycytidine deaminase (AID) initiates somatic hypermutation (SHM) in immunoglobulin variable (IgV) genes to produce high-affinity antibodies. SHM requires IgV transcription by RNA polymerase II (Pol II). A eukaryotic transcription system including AID has not been reported previously. Here, we reconstitute AID-catalyzed deamination during Pol II transcription elongation in conjunction with DSIF transcription factor. C→T mutations occur at similar frequencies on non-transcribed strand (NTS) and transcribed strand (TS) DNA. In contrast, bacteriophage T7 Pol generates NTS mutations predominantly. AID-Pol II mutations are strongly favored in WR Activation-induced deoxycytidine deaminase (AID) plays a central role in the generation of antibody (Ab) molecules that bind with high-affinity to invading antigen (Ag) molecules, a vital step in defending against infection in eukaryotes . AID is Escherichia coli occurs primarily on the NTS . In the absence of other factors, AID has been shown to deaminate dC solely on single-stranded DNA (ssDNA) ,11. Prev the NTS ,14. Presin vivo shows no strand bias. There are roughly equal numbers of mutations on NTS and TS DNA of IgV in normal mice (ung−/−) deficient mice  bi-directional transcription at IgV and IgS regions ; (ii) reIn contrast to T7 Pol, transcription elongation of eukaryotic Pol II is regulated by promoter-proximal pausing and subsequent release of paused Pol II into an elongation phase . At leasbona fide mammalian Pol II transcription model system or how Spt5 modulates AID recruitment or influences deamination during IgV or IgS transcription.AID interacts directly with Pol II , and AIDIGHV3-23*01, which is the most commonly used variable region during normal immune responses  and purified by 12% denaturing polyacrylamide electrophoresis.IGHV3-23*01  were amplified by PCR and cloned into Pvu II sites of M13mp3 phage vector and verified by Sanger DNA sequencing. Circular ssDNA M13mp2 (100 nM) containing the TS or NTS sequence was prepared from purified M13 phage and annealed with two 20 nt primers  in 1 ml of Pvu II restriction buffer. Following Pvu II restriction digestion at 37°C for 2 h, digested DNAs were separated by preparative 10% denaturing polyacrylamide gel electrophoresis. ssDNA bands corresponding to 203 nt TS and NTS strands were cut out and DNAs were extracted, concentrated to 100 nM in 10 mM Tris (pH 8.5) and stored at −20°C.To minimize mutation background associated with chemical DNA synthesis, TS and NTS ssDNA sequences of A carboxy (C)-terminal hexa-His-tagged GST-AID variant  was exprHuman Pol II was purified from nuclear extract derived from a HeLa cell line that stably expressed the RPB9 subunit, essentially as described . To gene32P using USB Optikinase (Affymatrix) was used to monitor RNA transcript synthesis. Complete formation of transcription EC was accomplished with addition of the non-templated NTS strand (100–200 fmol) and incubated for another 10 min at 30°C. When present, 50 ng DSIF (284 fmol) was added to EC and incubated for 3 min followed by the addition of 20 ng AID (425 fmol). Transcription was initiated by addition of all four rNTP (500 μM) and incubated for 1 h at 30°C. For AID deamination in the absence of Pol II, scaffold RNA:TS:NTS was assembled in the same manner as described above, without Pol II prior to addition of AID. Reactions were terminated by twice extraction with phenol:chloroform:isoamyl alcohol (25:24:1). The extracted DNA was desalted using Biospin column P6 (Biorad). The excess of unannealed ssDNA (TS and NTS) was eliminated by treatment with 10 units of exonuclease I (NEB) at 37°C for 30 min. Exo I was removed using Monarch PCR and Reaction cleanup kit (NEB) and DNA was eluted in 20 μl of 10 mM Tris (pH 8.5).Human Pol II elongation complex (EC) was reconstituted on the scaffolded RNA:TS:NTS bubble substrate similar to what has been described for bovine Pol II ,44. For Following Exo I treatment to remove ssDNA, NTS and TS strands in Pol II transcription reactions were subjected to next-generation sequencing analysis using Maximum Depth Sequencing (MDS) . MDS seq2-3′), using T4 RNA Ligase 2-truncated KQ (New England Biolabs) at 16°C for 16 h. Ligated RNA transcripts were annealed to a NG-8 primer (5′-CAA GCA GAA GAC GGC ATA CGA GAT TCA AGT GTG ACT GGA GTT CAG ACG T-3′) and reverse transcribed using NEBNext First Strand Synthesis Enzyme mix (New England Biolabs) in a thermocycler . The product cDNAs were amplified 30 cycles  by Taq DNA polymerase (Promega) using NG-8 and NG-RNA-F (5′-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T TAT ATG CAT AAA GAC CAG-3′) as PCR primers. The RNA-seq library was purified using AMPure XP beads (Beckman Coulter) and sequenced on a MiniSeq system.The Pol II transcription reaction mix including AID (see above) was extracted twice with phenol:chloroform:isoamyl alcohol (25:24:1) to remove Pol II and AID followed by incubation with 2 units of DNAse I (New England Biolabs) for 15 min at 37°C. DNAse I was removed by extracting twice with phenol:chloroform:isoamyl alcohol (25:24:1), and RNA transcripts were desalted using a Bio-Rad Biospin column P6. RNA transcripts were ligated to a 5′-adenylated, 3′-blocked ssDNA adaptor  and (TS = 46.8 × 10−5 ± 8.0)  relative to the NTS (30.2 × 10−5), although this average difference is not statistically significant (P = 0.098), and can be attributed principally to a cluster of mutations occurring in a region containing 6 consecutive C residues on the TS  in the average NTS mutation rate over the entire IgV region  is virtually identical to another subset of clones with one IgV mutation . Both subsets containing a single mutation in IgV have essentially the same mutational profile as observed for the full ensemble of singly and multiply mutated IgV clones Figure . These dIGHV3-23*01 . On the TS, the fraction of clusters lying within a 1–10 nt window is ∼27%. Therefore, sizable numbers of mutational clusters occur on the NTS and TS within a range of ∼10 nt, which corresponds to the estimated size of a Pol II transcription bubble ,47.c between mutations occurring at any two sites i and j located on a single DNA clone. The statistical analysis is given in Methods. A value of c > 0 indicates that two mutations are positively correlated. We have calculated correlation lengths for AID of 15 nt on the NTS and 14 nt on the TS . The entire mutational distribution for the ensemble of transcribed IgV clones is shown in Figure s Figure . In clons Figure , which cs Figure , the TS IGHV3-23*01 ; CDR2 (nt 147–177), and three Ab structural framework regions, FW1 (nt 38–65); FW2 (nt 107–146); FW3 (nt 178–203) . The in vivo mutations were obtained from a subset of non-productive mutated IGHV3-23*01 clones, which in human circulating memory B-cells have not undergone Ag selection . Mutations at hot motif sites are favored in both CDR domains for B-cells  and for AID-Pol II . The overlapping AGCT hot motif in CDR2 (nt 144) is the most prominently mutated site in B-cells (asterisk *’). It also is mutated to a significant extent with AID – Pol II , although several other CDR hot motifs are mutated to a similar extent . Mutations at cold motifs (SYC) are barely detectable .The two CDRs contain large numbers of WR B-cells   and A:T template sites (‘gray’ bars). The average number of RNA transcripts with 3′-ends in CDR1 (2830 ‘counts’ per site) is significantly higher than the average number of transcripts for FW2 (487 ‘counts’ per site) . Thus, a higher density of Pol II paused or terminated transcripts in CDR1  appears to correlate reasonably well with higher AID-induced mutations , compared to a lower density of paused or terminated transcripts and lower mutation rates in FW2.To examine the relationship between Pol II pausing or termination and AID-catalyzed deamination, we have performed RNA-seq analysis of a Pol II transcription reaction in the presence of AID. The distribution of RNA transcripts having 3′-ends terminated at template positions at the 5′ side of IgV covering a portion of FW1 and the entire CDR1 and FW2 regions is shown in Supplemental Figure S9A. Since Pol II produces numerous short transcripts in the FW1 region just downstream from the scaffold bubble Figure , we havein vivo . The spatial profiles for singly and multiply mutated clones are virtually indistinguishable and contain mutations distributed throughout the entire 178 bp essively . Althougutations ,46,54. Tutations . Therefot Figure  are not t Figure . It has  in vivo .in vitro versus in vivo comparison of AID-initiated mutation spectra. For example, it is possible that the observed differences between the in vitro and B-cell spatial mutation distributions are wholly or partially caused by the absence of transcription factors, such as anti-pausing factor TFIIS/SII, in our ‘minimalistic transcription elongation’ system. A related consideration is the extent to which chromatin structure, as exists on Ig genes in vivo, modulates Pol II-dependent AID function. Given our development of biochemically defined, recombinant chromatin-templated Pol II transcription systems that mediate both accurate transcription initiation and subsequent TFIIS- and PAF1C-dependent elongation through an array of nucleosomes (in vitro transcription system have the potential to identify and characterize AID targeting and anti-targeting factors, with a broader goal of reconstituting SHM and CSR using purified proteins.This ‘simplest’ representation of a mammalian transcription elongation system, which includes just AID and Pol II in the presence or absence of DSIF, appears to share basic features in common with B-cell mutational spectra. Based on these results, it would now be advantageous to expand this transcriptional elongation system by including a Pol II promoter and transcription initiation factors ,71. The leosomes ,73, it sgkz821_Supplemental_FileClick here for additional data file.


Poly(ADP-ribosyl)ation (PARylation) is an important post-translational modification in which an ADP-ribose group is transferred to the target protein by poly(ADP-riboses) polymerases (PARPs). Since the discovery of poly-ADP-ribose (PAR) 50 years ago, its roles in cellular processes have been extensively explored. Although research initially focused on the functions of PAR and PARPs in DNA damage detection and repair, our understanding of the roles of PARPs in various nuclear and cytoplasmic processes, particularly in gene expression, has increased significantly. In this review, we discuss the current advances in understanding the roles of PARylation with a particular emphasis in gene expression through RNA biogenesis and processing. In addition to updating PARP’s significance in transcriptional regulation, we specifically focus on how PARPs and PARylation affect gene expression, especially inflammation-related genes, at the post-transcriptional levels by modulating RNA processing and degrading. Increasing evidence suggests that PARP inhibition is a promising treatment for inflammation-related diseases besides conventional chemotherapy for cancer. While the mechanisms of transcriptional regulation have been extensively and intensively investigated, increasing amounts of data indicate that expression is also tightly regulated at the post-transcriptional level. Because modulations in the stability of mRNAs provide rapid and flexible controls, inflammation-related genes , growth factors, and proto-oncogenes are more dependent on post-transcriptional regulation , 2. Pre-diseases –5; howevdiseases , 6. A ne+ as a substrate, PARPs catalyze the transfer of a negatively charged ADP-ribose polymer subunit to a target protein [+ and ATP) and amount of PAR, which has been investigated in recent studies [Using NAD protein , and thi protein . Several protein   and a nuclear localization sequence. The first two zinc fingers appear to play different roles in the recognition of DNA breaks and enzyme activation, and they are both required to stimulate the activation of PARP1 in response to DNA single-strand breaks, but only the first zinc finger is required for the activation of PARP1 by double-strand DNA breaks . A nucle, and Zn3ll death . Mild orll death –29. Undell death , 27.Fig.Over the past two decades, a growing amount of literature has revealed an important role for PARP1 in the regulation of gene expression. As an important nuclear protein, the altering of chromatin structure by modifying histones and acting as a component of enhancer/promoter-binding complexes are the two major mechanisms of PARP1 that affect transcription –32.Dcx), essentially priming the gene for activation. Once differentiation is induced, MEIS associates with PBX1, and rapidly and specifically recruits PARP1 to the Dcx promoter/enhancer. This recruitment is associated with PARylation and the dismissal of histone H1, thereby facilitating Dcx gene expression [A large number of reviews have comprehensively summarized that PARP1 can automodify itself or PARylate histones, thereby, altering the chromatin structure –33. The pression . The stuDrosophila, PARylation of CTCF-interacting protein CP190 promotes its interaction with insulator DNA-binding proteins, suggesting that PARylation regulates the ability of insulators to mediate contacts between distant sites in the genome [PARP1 is involved in the regulation of genomic DNA in the three-dimensional space. A chromatin insulator-binding protein CCCTC-binding factor (CTCF) has been shown to be PARylated by PARP1. For example, in e genome , 40. Zhae genome . More ree genome . Importae genome , 43. DatOn the other hand, free PAR generated by PARP1 has been shown to serve as a dynamic source of ATP, which is required for the activity of ATP-dependent chromatin-remodeling enzymes during cellular signaling . Free PAIl-1β and Il-18) mRNA expression [In addition to modulating transcription through alterations in chromatin structure, PARP1 also regulates the activities of enhancers and promoters either positively or negatively , 44. Nucpression .In response to DNA damage, activated PARP1 can influence the function and location of the p53 transcription factor. During DNA damage, super-activated PARP1 induces the PARylation of p53 . The E25In addition, C/EBPβ, the key pro-adipogenic transcription factor, is PARylated by PARP1 on its K133, E135, and E139 amino acids, which are present in a conserved regulatory domain. The PARylation of C/EBPβ inhibits its DNA binding and transcriptional activities, which influences early events in the differentiation of adipocyte precursors .New discoveries have demonstrated that many RNA-binding proteins (RBPs) are modified by PARylation, thereby regulating gene expression at the post-transcriptional level , 6. ThesAlternative splicing events play important roles in many cellular processes, including tissue-, cell type- and developmental stage-specific gene expression patterns in eukaryotes . AlternaAdditionally, PARP1 also plays a role in splicing by altering the RNA-binding dynamics of heterogeneous nuclear ribonucleoproteins (hnRNPs). In 2003, a proteomic approach demonstrated that hnRNPs can establish a strong noncovalent link with PAR. Specifically, the conserved domain located between RRM1 and RRM2 of hnRNPA1 is the PAR-binding motif . HnRNPs,Cajal bodies (CBs) are nuclear organelles in proliferative cells, which contain small nuclear ribonucleoproteins, small nucleolar RNPs, RNA pol II transcription factors and nuclear proteins . CBs areRemarkably, almost all human pre-mRNAs possess multiple cleavage sites and polyadenylation signals in their 3′-untranslated region (3′-UTR) . The genEukaryotic gene expression requires the export of protein-coding mRNAs and non-coding RNA molecules from the nucleus to the cytoplasm. The major classes of cellular RNAs are exported as RNA–protein complexes, which involves RBPs as well as receptor proteins that interact with the nuclear pore complexes . AlthougIP-10 mRNA induced by interferon (IFN)-γ is diminished in Parp1−/− mouse embryonic fibroblasts, suggesting that PARP1 regulates the IP-10 mRNA level by increasing mRNA stability, although the study did not dissect the precise molecular mechanism of how PARP1 influences the mRNA level of IP-10 [The amount of protein generated from any given mRNA depends not only on the rate of mRNA translation, but also on the rates of mRNA synthesis and decay. Thus, mRNA stability provides an additional crucial layer of regulation at the post-transcriptional stage of gene expression. Genetic deletions of PARP1 or pharmacological blockades of PARPs activity in mice result in defective inflammatory immune responses . Howeverof IP-10 .cis elements that are harbored in mRNA and the cognate trans factors. Among them, ARE and ARE-binding proteins have attracted the most attention. Up to 8%–10% of the genes in the human genome contain at least one putative ARE in their 3′-UTR [The regulation of mRNA stability involves both r 3′-UTR . AREs arr 3′-UTR , while HCxcl1, Cxcl13, and Il-1β [HuR is a member of the embryonic lethal abnormal vision family. Unlike other members  of the family, which have tissue-specific expressions, HuR is ubiquitously expressed in almost all types of cells , 85. HuRnd Il-1β . The mecnd Il-1β . Thus, tAlthough much of the focus has been on PARP1, studies over the past decade have begun to reveal the physiological and pathological roles of the other PARP family members, with many new and exciting functions being identified , 94–96. mRNA and microRNA (miRNA) form dense aggregated structures called stress granules to stop translation when cells are under stress conditions . Stress PARP12, a Golgi-localized single ADP-ribosyltransferase of the PARP family, functions in cell survival and anti-viral responses . The actPARP13, also known as ZC3HAV1 and zinc-finger antiviral protein (ZAP), is an RBP. PARP13 binds RNAs of viral origin during infection and then recruits RNA-decay factors, such as exosome complexes to degrade RNA . PARP13 PARP14, a member of the PARP family, is a pro-survival protein that has been reported as being involved in protecting lymphocytes from apoptosis , 104. AdInflammation plays critical roles in host defense responses and damage repair, and it is also a common underlying cause of many diseases and is recognized as a hallmark of many cancers , 107. OvCxcl1, Cxcl2, Cxcl13, and Il-1β) is influenced by PARP1–HuR signals (see above) [Pro-inflammatory factors, such as cytokines and chemokines, are key mediators of inflammation and host defenses, and their regulation is largely dependent on related transcription factors. As mentioned above, many transcription factors that are involved in the inflammatory process, such as activator protein-1 (AP-1), p53 and NF-κB, have been reported to be regulated by PARP1 , 118. The above) , which ae above) . The RNAe above) . Althouge above) , 61, 124PARP2/−− mice showed an attenuated neuronal inflammation and reduced spinal inflammatory cell infiltration and demyelination [Studies on other members of the PARP family in the inflammatory process have also emerged. The closest homolog of PARP1 is PARP2, which is able to PARylate itself or other target proteins . PARP2 hlination . Furtherlination , whereaslination . The RNAlination . By inhilination . In factlination . Additiolination , and PARlination  (Table 2PARPs modulate the molecular biology and biochemistry of stress responses at multiple levels. In addition to the important roles of PARP1 in DNA damage detection and repair, its roles in RNA biology, gene regulation, and cytoplasmic functions have now emerged Fig. . Althoug


GAL genes to perform accurate measurements of transcription and mRNA degradation in a set of mutants. This genetic analysis uncovered a link from mRNA decay to transcription elongation. We combined this experimental approach with computational multi-agent modelling and tested different possibilities of Xrn1 and Ccr4 action in gene transcription. This double strategy brought us to conclude that both Xrn1–decaysome and Ccr4–Not regulate RNAPII elongation, and that they do it in parallel. We validated this conclusion measuring TFIIS genome-wide recruitment to elongating RNAPII. We found that xrn1Δ and ccr4Δ exhibited very different patterns of TFIIS versus RNAPII occupancy, which confirmed their distinct role in controlling transcription elongation. We also found that the relative influence of Xrn1 and Ccr4 is different in the genes encoding ribosomal proteins as compared to the rest of the genome.Co-transcriptional imprinting of mRNA by Rpb4 and Rpb7 subunits of RNA polymerase II (RNAPII) and by the Ccr4–Not complex conditions its post-transcriptional fate. In turn, mRNA degradation factors like Xrn1 are able to influence RNAPII-dependent transcription, making a feedback loop that contributes to mRNA homeostasis. In this work, we have used repressible yeast  Gene expression was thought to be a linear process where each step came after the next without interfering in each other’s paths. However, abundant studies have made it evident that these pathways are interconnected and can influence one another (reviewed in ). The coA few transcription factors have already been found to be implicated in this cross-talk since they affect the fate of nascent mRNA. This process has been named mRNA imprinting , and theInterestingly, both of the main complexes involved in mRNA imprinting have been found to interact . In the RNAPII arrest and backtracking have been suggested to be important for transcription regulation of genes ,19. FurtCross-talk of transcription, mRNA degradation and translation has been mathematically modelled using a stochastic kinetic approach . Their rGAL1 gene can be rapidly repressed by adding glucose, allowing easy calculation of its mRNA half-life. Although changing carbon sources may have some overall influence on mRNA stability . Cell number was determined by counting the number of cells using a Neubauer chamber. Growth curves were done for each mutant by measuring ODurve see  for dataSCR1 using SYBR Green Premix Ex Taq (Takara) in a Light Cycler 480 II (Roche). Since SCR1 is an abundant and stable RNA that has a constant concentration during cell growth .Cells were grown to log phase and 10 ml was harvested by centrifuging for 2 min at 4000 rpm and flash freezing the cells in liquid nitrogen. Total RNA was purified using hot phenol–chloroform method as described previously  and revel growth , normaliGAL1p-YLR454w shortly after adding glucose , ribonucleotide mix , 0.1 M DTT and [α-32P]UTP (3000 Ci/mmol). The reaction was stopped by adding 1 ml of cold water and RNA was immediately extracted following the acid-phenol protocol. Slot-blotted membranes were elaborated as formerly described . After blocking with Tris-buffered saline containing 0.1% Tween 20 and 5% milk, the following primary antibodies were used: mouse monoclonal anti-HA 16B12 , mouse monoclonal anti-Rpb3  or mouse monoclonal anti-Pgk1 22C5D8 (Invitrogen). Finally, the membrane was incubated with peroxidase-conjugated goat anti-mouse IgG and proteins were detected using a chemiluminescence detection kit  and a ChemiDoc™ MP system (Bio-Rad).t-test was used to determine significant differences between biological samples. When correlating variables, a Spearman regression coefficient was carried out using a two-tailed significance test.All results were calculated and represented using Microsoft Excel. The statistics were carried out using SPSS. A Student’s https://CRAN.R-project.org/package=BayesVarSel) was used to perform a Bayesian analysis of the inclusion probabilities among some of the variables. We used the Bvs function of this package.The R package BayesVarSel  was used to program and run the model. SPSS in its latest version to conduct statistical analysis of the results both in experimental data and in in silico data obtained with the model.NetLogo in its latest version (currently 6.0.2) (www.broadinstitute.org/igv/). Normalized coverage tracks for genome browser visualization were generated with the functions bamCoverage and bamCompare from the deepTools2 suite. Coverage tracks from individual samples are expressed as reads per kilobase per million mapped reads (RPKM), whereas comparison tracks are expressed as the log2 of the number of reads ratio. Normalized average density plots around genomic features were calculated with the ngs.plot package  million reads. The quality metrics of the Fastq sequencing datasets were obtained with FastQC and visually inspected. After de-multiplexing, sequencing adapters were removed from raw reads with Fastx Clipper. Low-quality 3′-end of reads were trimmed with Sickle, and then PCR duplicates were removed with Fastx Collapser to ensure each fragment was represented only once. A second round of inspection with FastQC was carried out after the filtering steps to verify that the filters were applied properly. The remaining high quality sequences were mapped to the SacCer3 genome with Bowtie2, using default parameters. Resulting BAM alignment files were visually inspected with the genome browser IGV ( package . The ChI package . The ngs600 ∼ 0.7–0.8) were harvested, at least in triplicate. The whole final purified chromatin-enriched fraction P3 was resuspended in 20 μl of 1× Tris-Glycine SDS Sample Buffer and incubated for 5 min at 100°C, followed by spinning at 10 000 rpm for 30 s. This chromatin pellet was used for SDS-PAGE, followed by western blotting with different antibodies: anti-C-Myc (9E10 Santa Cruz Biotechnology), anti-Rpb3 , anti-Histone H3  or anti-phosphoglycerate kinase, Pgk1 . Histone H3 was used as a control of purified chromatin and Pgk1 as a control of cytoplasmic contamination.Chromatin-enriched fraction purification was performed according to the yChEFs procedure . BrieflyGAL genes can be especifically shut-off by adding glucose. We decided to use GAL1 as a suitable model to study the effect of mRNA degradation mutants on transcription elongation and vice versa. We studied the response of GAL1 to the genetic perturbation of mRNA decay and transcription elongation machineries by measuring parameters that define the different levels of the gene expression pathway. We studied mutants lacking Xrn1, the main cytoplasmic 5′-3′ exoribonuclease (rpb1-I756A), in which we have changed an amino acid involved in the interaction between the 3′ end of pre-mRNA and a conserved domain of RNAPII when this is backtracked  (We have previously described that cell volume influences nascent transcription and mRNA degradation rates . To testtes (GR) . Howevertes (GR) ; and acttes (GR) . This suThe correlations of cell volume with mRNA half-life . Properties of each agent are described in the corresponding flow charts (in silico mutant)  . Some ot mutant) . The evo mutant) .in silico experiments and calculated the mean values of five characteristic parameters of the system once it reached steady state mRNA levels, using the information provided by the monitors in the interface: number of mRNA molecules, mRNA half-life, total occupancy of the gene by RNAPII, occupancy by active (non-backtracked) RNAPII molecules and RNAPII speed. We repeated these experiments under conditions of no TFIIS, Ccr4–Not or Xrn1 molecules . We compared the values obtained with the in silico wild-type and calculated a data matrix reflecting the differences: 0 meant no significant difference with the wild-type using a Student’s t-test (P < 0.05), and +1 or -1 meant significantly increased/decreased values, respectively , and subtracting one point in those cases where a -1/+1 difference was found.The first model that we constructed (model 1) involved a positive action of nuclear Xrn1 molecules on transcription initiation, without subsequent action of this factor in elongation Figure . Regardiy Figure . A simile Figure . We reasin vivo and in silico results supported transcriptional contributions of mRNA degradation factors. Nevertheless, the highest score obtained was still 6 points short of the maximum possible score of 15. This indicated that this first model was far from fully explaining the transcriptional functions of Xrn1 and Ccr4-Not. Particularly relevant was the observation of RNAPII speed and active RNAPII. In all variants, these two parameters behaved the same, since the only restriction for RNAPII speed in this model was backtracking, which also resulted in lack of activity. However, in vivo results showed that ccr4Δ and dst1Δ caused a decrease in RNAPII activity without significant reduction in speed  . We testg/ml 6AU  49,50).,50.dst1Δreported  39,40).,40.dst1Δn tested . Howeverg/ml 6AU . These dhis drug .xrn1Δ showed 3.0 h doubling time in YPD at 30°C, versus 1.8 h of the wild-type. This growth defect was even more severe in the double ccr4Δ xrn1Δ mutant, with a doubling time of 8.6 h. These growth defects usually originate pleiotropic phenotypes that are only indirectly related to the genetic change. In order to exclude these possible side effects, we decided to analyse the effect of Xrn1 fast depletion with an engineered version of Xrn1 that is fused to an AID domain and become rapidly degraded in respond to auxin, a non-metabolized molecule. We treated an Xrn1-AID strain for 1 h with auxin and explored its effect on GAL1 mRNA synthesis and decay. We found a substantial increase in mRNA half-life and [mRNA], which indicated fast and effective degradation of Xrn1 after adding the drug. We also detected decreased density of active RNAPII molecules on GAL1, down to <20% of the untreated value, without a significant change in total RNAPII occupancy  and ribosomal proteins (RP) exhibited lower TFIIS/Rpb3 ratios than the rest of the genome Figure . In xrn1s Figure . In conte Figure . So, TFIΔ Figure . This isΔ Figure .xrn1Δ, we predicted that Xrn1 perturbation should not affect the activity of elongating RNAPII molecules in this gene category. We tested this prediction by quantifying levels of total RNAPII bound to several RP genes, by anti-Rpb3 ChIP, before and after depleting Xrn1-AID with auxin. We also measured the density of active RNAPII molecules on the same genes, by transcription run-on, in the same cultures. In contrast to GAL1 and GAL1p-YLR454w ,,63,GAL1,xrn1Δ would produce the higher recruitment of TFIIS that we detected in GAL1 and across the genome. In contrast, lack of Ccr4 did not produce such an effect in either GAL1 or our global analysis, but a general decrease of the TFIIS/Rpb3 ratios in most genes. This fits with the reported role of Ccr4 in facilitating the recruitment of TFIIS to elongating RNAPII .http://genome-euro.ucsc.edu/s/jorplan/xrm1_ChIP%2DseqData can also visualized in the UCSC browser: gkz660_Supplemental_FilesClick here for additional data file.


The organization of chromatin in the nucleus plays an essential role in gene regulation. About half of the mammalian genome comprises transposable elements. Given their repetitive nature, reads associated with these elements are generally discarded or randomly distributed among elements of the same type in genome-wide analyses. Thus, it is challenging to identify the activities and properties of individual transposons. As a result, we only have a partial understanding of how transposons contribute to chromatin folding and how they impact gene regulation.Using PCR and Capture-based chromosome conformation capture (3C) approaches, collectively called 4Tran, we take advantage of the repetitive nature of transposons to capture interactions from multiple copies of endogenous retrovirus (ERVs) in the human and mouse genomes. With 4Tran-PCR, reads are selectively mapped to unique regions in the genome. This enables the identification of transposable element interaction profiles for individual ERV families and integration events specific to particular genomes. With this approach, we demonstrate that transposons engage in long-range intra-chromosomal interactions guided by the separation of chromosomes into A and B compartments as well as topologically associated domains (TADs). In contrast to 4Tran-PCR, Capture-4Tran can uniquely identify both ends of an interaction that involve retroviral repeat sequences, providing a powerful tool for uncovering the individual transposable element insertions that interact with and potentially regulate target genes.4Tran provides new insight into the manner in which transposons contribute to chromosome architecture and identifies target genes that transposable elements can potentially control.The online version of this article (10.1186/s13059-018-1598-7) contains supplementary material, which is available to authorized users. The structural organization of the genome is regulated at different levels to establish a functional framework that facilitates cellular processes such as gene expression and programmed somatic recombination. Fluorescent in situ hybridization (FISH) studies revealed that chromosomes occupy discrete territories with very little intermingling between them. With the development of chromosome conformation capture (3C) techniques that rely on crosslinking of chromatin in close spatial proximity, additional levels of organization have been described. There are many different 3C-based variants, with Hi-C being the most comprehensive, in that it can potentially identify all pair-wise chromatin interactions in a given population of cells –5. The fA large portion of a typical mammalian genome is comprised of transposable elements (TEs) however they are typically ignored in high-throughput sequencing-based studies due to their repetitive nature , 18. As cis-regulatory elements such as those found at the 5′ UTR of long interspersed nuclear elements (LINEs) and long terminal repeats (LTRs) of endogenous retroviruses (ERVs) [cis-regulatory elements have evolved to control TE transcription but they can also influence expression of adjacent “host” genes [Recent studies have suggested that TEs might act as enhancers capable of influencing expression of endogenous genes , 27–32. s (ERVs) –36. Thest” genes , 38. In t” genes , 40. Mort” genes –44. In at” genes  observedt” genes . Therefo3C-based techniques have become instrumental in the identification of enhancers and the genes they regulate –50. HereCapture-4Tran uses biotinylated DNA oligonucleotides (oligos) to capture TEs in conjunction with surrounding uniquely mappable sequences, thereby enabling the identification of the interaction profile of a specific transposon copy, unlike the PCR variant of this technique. This provides a powerful tool for uncovering the individual insertion sites that interact with, and potentially regulate target genes. Here we designed an oligonucleotide that captures interactions involving human MER41 elements. We found that a significant fraction of MER41 interactions occur with promoters, STAT1 binding sites and histone modifications indicative of active transcription. We also observed that the loops are preformed and can be detected in the absence of STAT1 binding, prior to IFNγ induction, indicating that the contacts are not dependent on the presence of this transcription factor. Thus, 4Tran provides new insight into the role of TEs in shaping genome organization and regulating cellular processes in mammalian cells.To investigate different aspects of murine and human retrotransposon-mediated interactions, we used two variants of 4Tran. The first, based on circular chromosome conformation capture (4C-Seq), was called 4Tran-PCR , which are known to be particularly diverse, recent, and active in the murine genome . We selecis-regulatory elements recognized by transcription factors tend to occur [Primer pairs were selected based on the following criteria: location within the TE, number of integration copies that can be potentially captured and specificity to the transposon of interest. In addition, we selected baits located close to the 5′ and 3′ ends of TEs where to occur . It is ito occur . Since LTo test the primer pairs selected for each ERV, we performed 4C-Seq in mouse embryonic stem cells using DpnII and Csp6I as the primary and secondary restriction enzymes. As predicted, 4Tran-PCR primers located within sequences of endogenous retroviruses generated several bait-like profiles, instead of a single bait profile as in conventional 4C-Seq Fig. c. Since To determine if 4Tran-PCR can identify annotated ERV integration events, we selected preferred primer pairs based on their proximity to LTRs and their ability to generate efficient 4C signal amplification. As the mm10 reference genome from the Genome Reference Consortium is based on the C57/Bl6 strain, we performed 4Tran-PCR using template prepared from murine ex vivo derived splenic resting B cells from mice of this strain. B cells were chosen for their easy availability in our lab and because high-resolution Hi-C datasets that we could use as validations were restricted to B cells at the time of these experiments. Our first goal was to test whether the observed 4Tran bait profiles align with known ERV integration sites  database, which generated whole-genome sequencing of 18 different mouse lab strains [The Mouse Genomes Project  seems to be mostly guided by the propensity of large chromatin fragments with similar histone and DNA modification patterns to share the same physical space. Formation of compartment A, for example, results from the spatial clustering of genomic regions enriched for histone post-translational modifications associated with transcriptional activity, such as H3K4me1/3 or H3K27ac , 8. TherTo address this question, we used Hi-C and ChIP-seq data from a splenic murine lymphoma IgM+ B cell line, CH12 that is used as a model for B cells in the same developmental stage as those we use here . We perfWe next examined whether the local interactions of transposable elements are confined within smaller architectural structures such as TADs , 66. HerIdentifying interactions from individual TEs using 4Tran-PCR works optimally if insertion sites are well separated on the linear chromosome. However, 4Tran-PCR can confound the characterization of interactions from TE integrations in close proximity and it is thus best suited for analysis of TEs with relatively low copy number integrations. Under these circumstances, it can be assumed that interactions are captured from the nearest site, as by definition regions in the same domain interact at higher frequency than with the rest of the genome. A further drawback of 4Tran-PCR is that it is less efficient at amplifying interactions from families with highly divergent sequences, as it relies on the presence of two PCR primers and two restriction enzyme sites. To address these issues, we developed a second variant of 4Tran, Capture-4Tran.Capture-4Tran overcomes the problems related to repetitive sequence mappability by combining paired-end sequencing with biotinylated oligonucleotides that hybridize to the 5′ or 3′ end of a TE integration site. It can therefore be used to uniquely identify interactions from individual TE insertions. To test the feasibility of this approach, we first performed Capture-4Tran for the IAPEz family, which is one of the youngest and more homogeneous ERVs in the mouse genome. We performed a pilot experiment at low-sequencing depth using a probe based on the consensus sequence of the LTR that is most commonly associated with the IAPEz element, IAPLTR1a. As shown in Additional file We next tested if Capture-4Tran is able to identify interactions from slightly older TEs that are therefore less homogeneous in sequence, but may have greater potential for being coopted for adjacent gene regulation , 42, 67.In addition to the Capture-4Tran library, we sequenced a Hi-C library for comparison. Capture-4Tran yielded ~ 291 times more sequencing reads associated with MER41B-mediated interactions than the Hi-C approach (5 million unique contacts out of 19.0 million versus 55.0 thousand out of 61 million contacts in Hi-C). The reads generated a contact matrix that is clearly distinct from a Hi-C matrix in that TADs and compartments are no longer distinctly visible. Instead, strong horizontal and vertical lines stand out that are centered at the diagonal of the matrix and emanate from MER41 elements .  and focused on the most frequent contacts (Chicago interaction score > 10 and a linear distance less than 5 Mb from the MER41 anchor) and identified 377 interactions . In sum, our analyses suggests that multiple MER41 elements in the genome might function as regulatory elements, and that Capture-4Tran can be used to identify these elements as well as a list of target genes that could be further validated using other experimental approaches.A subset of genes known to be regulated by MER41 elements in response to IFNγ in HeLa cells such as AIM2  was not As many interactions do not involve STAT1 Fig. b, we alsMYPN gene, which encodes a protein specifically expressed in heart and skeletal muscle . In addition, we find that chromatin interactions of ERVs are mostly delimited by the organization of chromosomes into smaller, highly self-interacting domains and their chromosomal looping is largely restricted to these regions. Finally, we demonstrate that Capture-4Tran can be used to identify TEs that establish direct looping interaction with adjacent host gene promoters and thereby likely modulate host gene transcription.The choice between use of the PCR-based and the capture-based variants of 4Tran will depend on the biological question. 4Tran-PCR works best with younger TE families that have a moderate number (up to a few hundred) of integration events with high sequence similarity between different copies. It provides high-depth sequencing data at low cost, is easier to implement than Capture-4Tran, and can be used to characterize long-range TE contacts and boundaries of domains that restrict their local interaction. Capture-4Tran, on the other hand, can be used for analysis of younger TEs, as well as older families with high sequence divergence. Indeed, it bypasses most of the limitations of 4Tran-PCR, including small size and it is highly specific for the elements it captures. As a result, it can be used to study high copy number transposons such as SINEs, LINEs, and solo LTRs.A number of recent studies have focused on the role of transposons in gene regulation. In the absence of a chromosome conformation capture tool, TE influence has been examined in the limited context of the nearest gene. However, TEs can participate in both long- and short-range interactions and could potentially be involved in the regulation of multiple target loci. 4Tran therefore provides a tool for more precisely defining the extent of this control and identifying TE-mediated interactions with specific promoter regions.Capture-4Tran has a clear advantage over promoter Capture Hi-C , which eDrosophila contrasts with the findings from mammalian studies showing that pre-established loops connect enhancers and promoters even before gene activation in early development [Capture-4Tran results are consistent with a model in which a subset of MER41 elements act as interferon-inducible enhancers that contribute to STAT1-mediated transcriptional activation of interferon sensitive genes in response to pathogen infection. Our results show that MER41 elements function as a prototypical enhancer by making contact with the promoters of flanking genes. Furthermore, our analysis uncovers many direct MER41-gene associations that further support the notion that these primate-specific endogenous retroviruses have a substantial impact on gene regulation in human cells and represent an important force driving the regulatory evolution of the primate innate immune response. These experiments add to a growing body of evidence demonstrating two types of enhancer-promoter contacts: stable and dynamic. Stable contacts, such as the majority of enhancer-promoter contacts identified during a TNFα response, are formed prior to signaling . These aelopment . The facDespite major advances in the field since the development of chromosome conformation, there is still a lot to be learned about the role of nuclear architecture in gene regulation. In particular, our knowledge of the contribution of TEs in these processes is lagging behind because of an absence of tools amenable to their analysis. Here, we provide the first 3C-based approach for addressing this question. 4Tran is important for learning how TEs such as MER41 influence transcription of host genes, and this in turn provides new insight into the mechanisms by which TEs contribute to the evolution of gene regulatory networks. Furthermore, 4Tran can be used to study the impact of TEs in disease settings such as cancer where the methylation status of the genome is altered and transposons become activated , 81.It is well established that TEs represent an important source of genetic variation . These mAll mice used here were of wild-type inbred strains. Animal care was approved by Institutional Animal Care and Use Committee. Protocol number is 160704-02 (NYU School of Medicine). Young mice of less than 12 weeks were used in all experiments.4C-Seq material was prepared from the following murine cells: embryonic stem cells, embryonic fibroblasts, and splenic B cells. Starting material for each sample was 10 million cells. The 4C template material prepared from mouse embryonic stem cells was previously described , 83. TheFor each TE, 4C-Seq primers were designed to the consensus sequence obtained from Repbase. We then designed our algorithm to find all possible primer pairs based on the desired enzyme digestion. To be considered as a potential bait, the following criteria are required: 300 bp separating primary enzyme restriction sites and 150 bp between primary and secondary enzyme restriction site. For these locations a 20-nucleotide primer is designed that includes the primary restriction enzyme site and the second primer is designed using the default parameters of Primer 3. We used annotations from Repeatmasker based on the “-int” sequence for all repeats except RLTR4 where both the “-int” and solo LTR sequences were included since our probe was able to capture many solo LTRs.z-score was then defined for each window based on the mean and standard deviation across all windows in the genome. Finally, an FDR adjusted p value was calculated based on fitting to a normal distribution and enriched windows were defined based on p values smaller than 0.05. Windows separated by 100 kb were merged. Only those windows identified in both replicates were called as observed baits. To determine the location of predicted baits, we used the UCSC in silico PCR tool. One of the primers for each bait was input as the reverse complement and the lowest stringency mismatch option (15 nucleotides) was chosen. To determine the regions interacting most frequently with specific TE integration events  using bowtie2  as per tnts Fig. , we usedIFI6 gene was measured by reverse transcriptase quantitative PCR. Primers can be found in Additional file Murine B cell material was prepared as described in the section above. Human HeLa F2 cells were generous gifts from Cedric Feschotte and Edward Chuong. Cells were treated for 24 h with 1000 U/ml of recombinant human IFNγ . Following treatment cells were fixed with Formaldehyde for 4Tran-Capture as well as Hi-C. To verify that cells responded to interferon treatment, RNA was collected and expression of the Our approach was adapted from two published protocols. The Hi-C part of the protocol was performed mostly as described in . Ligatioensembl.org) with default parameters, without Repeatmasker filtering and allowing for up to 5000 hits. To detect fragments with high number of long-range interactions, each end of a mapped read-pair was first assigned to a restriction fragment in the genome using juicertools. Any read pairs that were within 100 fragments were removed. For the remaining interactions, the number of unique reads was counted and a quantile cut-off of 0.0005 was used to select the observed fragments with a high number of reads. This number was selected based on the percentage of DpnII fragments that Blastn predicts which are enriched by our oligonucleotide. Chicago [github.com/mirnylab/cooler) to process the data and HiGlass (higlass.io/) to visualize Capture-4Tran and Hi-C results. Normalization of the Capture-4Tran data was performed using DESeq2 on read counts in 1 kb bins overlapping by 500 bp in a 1 MB region around the bait. Normalized bedGraph files were used for visualization of data in IGV.Processing of Hi-C and Capture-4Tran reads was performed using Hicup . Reads w Chicago  was usedp value cut off of 0.01 and a log2 fold change of 2 was used to call significant interactions.Differential analysis for the 6026 interactions was performed using DESeq2. The input for the analysis was the counts in the interacting region for each sample (2 replicated per condition) and adjusted Paired-end reads were mapped separately using bowtie 2.0.1. Downstream analysis was performed using Homer default parameters to obtain the count matrix and PCA scores to determine A and B compartment composition.nih.gov/geo) and used as criteria a log fold change of 1 and an adjusted p-value of 0.05. To identify all genes that are potentially expressed in HeLa cells we used RNA-seq data  [To identify genes upregulated by interferon treatment in HeLa cells we used a microarray dataset  as this Hr IFNγ) . Single-Additional file 1:Supplementary Figures. This file contains all the supplementary figures for this paper. (PDF 4494 kb)Additional file 2:Table S1. List of 4Tran samples. Table S2. List of oligos used for 4Tran. Table S3. Integration differences between mouse strains with annotations of sequence variants from the Mouse Genome Project Database. Table S4. Differential interactions. Table S5. List of Potential Regulatory Interactions. (XLSX 151 kb)Supplementary Tables. This file contains 5 supplementary tables for this paper.