Exosomes participate in intercellular communication by transporting functionally active molecules. Such cargo from the original cells comprising proteins, micro-RNA, mRNA, single-stranded (ssDNA) and double-stranded DNA (dsDNA) molecules pleiotropically transforms the target cells. Although cancer cells secrete exosomes carrying a significant level of DNA capable of modulating oncogene expression in a recipient cell, the regulatory mechanism is unknown. We have previously reported that cancer cells produce exosomes containing NANOGP8 DNA. NANOGP8 is an oncogenic paralog of embryonic stem cell transcription factor NANOG and does not express in cells since it is a pseudogene. However, in this study, we evaluated NANOGP8 expression in glioblastoma multiforme (GBM) tissue from a surgically removed brain tumor of a patient. Significantly higher NANOGP8 transcription was observed in GBM cancer stem cells (CSCs) than in GBM cancer cells or neural stem cells (NSCs), despite identical sequences of NANOGP8-upstream genomic region in all the cell lines. This finding suggests that upstream genomic sequences of NANOGP8 may have environment-dependent promoter activity. We also found that the regulatory sequences upstream of exosomal NANOGP8 GBM DNA contain multiple core promoter elements, transcription factor binding sites, and segments of human viruses known for their oncogenic role. The exosomal sequence of NANOGP8-upstream GBM DNA is different from corresponding genomic sequences in CSCs, cancer cells, and NSCs as well as from the sequences reported by NCBI. These sequence dissimilarities suggest that exosomal NANOGP8 GBM DNA may not be a part of the genomic DNA. Exosomes possibly acquire this DNA from other sources where it is synthesized by an unknown mechanism. The significance of exosome-bestowed regulatory elements in the transcription of promoter-less retrogene such as NANOGP8 remains to be determined. Glioblastoma multiforme (GBM) is the most aggressive and the least curable of all the malignant tumors of the central nervous system, with a 5-year survival of 6.8% and mean survival of fewer than 18 months , 3. The Research suggests that extracellular vesicles play a crucial role in imparting therapy resistance to the CSCs in GBM , 5. ExosNANOG, a homeobox transcription factor, is the crucial regulator of embryonic stemness and pluripotency of mammalian cells , 14. OurPrevious work from our lab has identified differential DNA sequences of NANOG/NANOGP8 exons and 3’ UTRs in GBM exosomes . The molIn the current study, we analyzed the NANOGP8 DNA 5’ UTR in CSC-secreted exosomes for the presence of core promoter elements and the transcription factor binding sites (TFBS) and compared with the genomic counterpart. Viruses, such as human papillomavirus (HPVs), human cytomegalovirus (HCMV), and neurotropic Epstein-Barr virus (EBV), are implicated in oncogenesis , 31. HPVhttps://blast.ncbi.nlm.nih.gov/Blast.cgi). A concise account of the steps followed in the NANOGP8 DNA sequence analysis is given in Fig 1. The transcription levels of NANOGP8 were checked with quantitative PCR of the cDNA template. To confirm the identities of NANOGP8, RFLP analysis was conducted on the PCR products of cDNA as well as gDNA. The nucleotide sequence analysis (GENEWIZ®) of each clone was plugged into NCBI’s VacScreen tool to eliminate the pCR4TOPO-TA vector sequence contamination that would skew the results of the analysis (https://www.ncbi.nlm.nih.gov/tools/vecscreen/). To remove the possible NANOG/NANOGP8 intron, exon, and UTR sequences from the promoter element analysis, the non-vector exosomal DNA sequence of each clone was compared with NANOG and NANOGP8 using NCBI’s Basic Local Alignment Tool (BLAST). The details of NCBI reference numbers used in comparative BLAST analyses are given in Table 1.The exosomal and the genomic DNA was PCR amplified and the PCR products were cloned in a sequencing vector. The clones selected on Ampicillin were Sanger-sequenced and the sequences were analyzed using the Basic Local Alignment Search Tool (BLAST: 2 and the reaction was stopped with serum containing NT2 media (recipe below). The cells were then passed through a cell strainer using NT2 media to facilitate cell passage. Collected flow through was centrifuge at 4°C, at 4000g for 5 minutes, and the cell pellet was washed with NT2 media. After 2 washes, the cell pellet was finally resuspended in 10 ml of NT2 media and transferred to tissue culture treated T-75 flask for overnight incubation at 37°C with 5% CO2. The following day, floating spheres of cells were transferred to a non-treated (suspension culture) T-75 flask containing NSC media. After 2 weeks of incubation, the adherent cells were trypsinized and passaged for propagation. The GBM neurospheres in suspension culture, containing a mixed population of cancer cells and CSCs, were subjected to immuno-separation using CD133, a stemness marker, to collect CSCs  x 40 cycles. We measured relative NANOGP8 expressions using the 2-ΔΔCT Livak-method and performed statistical analysis one-way ANOVA followed by post-hoc Dunnett’s test employing GraphPad Prism (v9.3.0) statistical program.Total RNA was extracted from all three types of GBM cells as well as NSCs using the Direct-zol RNA extraction kit , following the manufacturer’s protocol. 2.5μg of RNA was synthesized to cDNA with oligo(dt), using the SuperScript™ III First-Strand Synthesis System (Invitrogen) Fisher scientific catalog # 18080051). RNA was incubated with Oligo(dt) at 65 ˚C for 5min, followed by an RNA conversion reaction that proceeded as follows: 25˚C for 10 min, 50˚C for 50 min, and 85˚C for 5 min. For qPCR, 125 ng of cDNA was amplified in 20 μL reactions using Fast SYBR Green Master Mix . NANOGP8 expression was measured using NANOGP8 specific primers (qNANOGP8 F: 5’-GTCTTCTGCTGAGATGCCTCACA-3’ and Reverse: 5’-CTTCTGCGTCACACCATTGCTAT-3’ Position on NANOGP8 gene- GenBank Accession: 388112: 418–802). Using High-Performance GoTaq® G2 DNA Polymerase (Promega), the PCR reactions were set up as follows: Pre-denaturation: 94˚C -7 minutes,  x 35 cycles, post extension: 72˚C -10 minutes). The amplified PCR product was digested with AlwNI at 37˚C for 5 minutes and run on 2% TEA-agarose gel.We amplified the total cDNA of the cell samples using NANOG/NANOGP8 primers  following the manufacturer’s protocol. 1 μg of amplicon DNA was digested with restriction enzymes AlwNI  with Fast Digest buffer. The digestion reaction was incubated at 37° C for five minutes and electrophoresed on 3% Agarose gel in 1X TAE buffer.Table 1, we confirmed the presence of NANOG DNA in the exosomal clones as well as genomic DNA clones. Upon confirmation of the NANOG or NANOGP8 gene sequence, the upstream sequences were compared with NCBI-reported sequence data. This was done to check their identity to the reported promoter region of NANOG and the upstream region of the NANOGP8 gene. For comparison of exosomal and genomic upstream/regulatory regions, 1000 nucleotides upstream of the 5’UTR were added to the BLAST. Because NANOG possesses an annotated promoter, the NANOGP8 upstream sequences were compared with the promoter region’s genomic sequence. None of the upstream sequences matched with the annotated promoter region of NANOG or NCBI reported 5’ upstream region of NANOGP8.Using the NCBI information given in http://www.bioinformatics.org/yapp/cgi-bin/yapp.cgi). This tool scanned the nucleotide sequences for canonical promoter elements and detected the TATA box, to identify transcription-start-site (TSS), downstream promoter element (DPE), and an initiator element (INR). YAPP also identified synergistic combinations of the core promoter elements found in upstream sequences. YAPP algorithm calculates matrix similarity score for matches with consensus sequences to qualify as promoter elements. If each nucleotide in the sequence to be analyzed corresponds to the highest conserved nucleotide in the same position of the matrix, this perfect match receives a score of 1.0. Matrix similarity score >0.8 is considered good and, therefore, the optimum cutoff value in the analysis [For the core promoter element analysis, the upstream exosomal NANOG DNA sequences were plugged into YAPP Eukaryotic Core Promoter Predictor . This program identified the putative binding sites and the TF proteins that bind to them in DNA sequences. The app uses TFBSs defined by the TRANSFAC® eukaryotic TF database (version 8.3) [Transcription factors (TFs) fall under two major categories; general and specific. General TFs bind to the core promoter region to assist the docking of RNA polymerase for basal transcription, whereas the specific TFs regulate the transcription of an individual gene. We analyzed the exosomal clones for sequences recognized by the latter type. Using a virtual laboratory app, “PROMO” (version 3.2.0), we scanned the upstream sequences of exosomal NANOGP8 DNA for TFBSs (ion 8.3) , 42.. Therefore, it is inferred that GBM and NSC have a mixed population of the NANOGP8 transcripts. The difference in the PCR product sizes of the upper and lower band also implies that the distinct populations of NANOGP8 transcript have deletions/additions of about 44 nucleotides.The AlwNI-mediated digestion of cDNA PCR products in RFLP analysis confirmed the presence of NANOGP8 transcripts in NSCs and GBM cells. For both the cell lines, the RFLP analysis results were identical. In our samples, the PCR amplification of the transcript using the NANOG/NANOGP8 primer pair unexpectedly yielded two distinct product bands. The upper band was of expected size, i.e. 384 base pairs and the lower band was approximately 340 base pairs. Each band reamplified separately using the same primer pair, followed by a digestion with AlwNI, gave interesting results. The upper band showed partial digestion affirming the presence of a mixed population of NANOG and NANOGP8 transcripts. A complete digestion of the PCR products was observed in the lower band, indicating that the transcript belonged solely to NANOGP8 + GBM CSCs expressed NANOGP8 approximately 20 times more as compared to NSCs, while CD133- GBM cancer cells expressed only a negligible level of NANOGP8 . We also found that NANOGP8 expression in NSC was remarkably lower  than the expression in CD133+ GBM CSCs, but significantly higher  than CD133- GBM cells.qPCR mediated transcription analysis demonstrated that CD133+ GBM CSCs, we investigated the genomic upstream region of NANOGP8 to check for differential sequences that may have played a role in promoter-like activity in GBM and NSC cell lines. The amplified sequences were analyzed using Human BLAST. The genomic sequences from both the cell lines matched 100% with each other  and showed 99% identity with the reported sequences of the NANOGP8 gene (NC_000015.10), including its upstream region .Upon detection of upregulated NANOGP8 transcription in CD133.Because the restriction enzyme cutting sites for HpyF31 are present uniquely in the sequence of 5’ UTR and upstream regions of genomic DNA of NANOGP8 (positions 35085287 and 35085520), but not the parent gene NANOG or its other paralogues, a complete digestion with the RE confirmed that the amplification product belonged solely to NANOGP8 To analyze the exosomal NAOGP8 DNA and the adjoining upstream sequences, a pair of universal-forward primer and NANOGP8-reverse primer (position on mRNA NM_001355281.2: 690–707) was used on all the exosome samples. Bioinformatical analyses revealed that for both NSC and GBM, NANOGP8- flanking sequences had identities to multiple core promoter motifs, putative transcription factor binding sites (TFBS), and viral elements known to play a role in cancer.Fig 5 and S1A Table detail the promoter element analysis. S1B Table lists the synergistic combination matches of the sequences and the type of promoter motifs of the NANOGP8 promoter region.Upstream NANOG DNA sequences associated with NSC-derived exosomes revealed forty promoter elements with regulatory protein-binding motifs, depending on TSS locations anticipated by the YAPP tool. Out of these, four were TATA boxes and the rest DPE. Seven synergistic combinations of the TATA box and DPE were also detected. Thirty-eight motifs had a matrix similarity score of >0.9, indicating a near-perfect match to a consensus nucleotide sequence within promoter elements. - cancer cells and CD133+ CSCs. Fig 6 depicts the core promoter profiles. Out of twenty-four core promoters identified, five were INR, and the rest were DPE. No TATA box elements or synergistic combination matches of the sequences were detected.The GBM exosomes are collected from the suspension culture of GBM neurospheres containing a mixed population of CD133- GBM had three INR and nine DPE core promoter elements. However, there were no TATA box elements similar to the sample from GBM exosomes. One synergistic motif combining INR and DPE elements was detected. Fig 7 depicts the core promoter profiles and S3A and S3B Table details the sequences and the types of promoter elements.Out of twelve core promoters identified, the upstream of the NANOG exosomal DNA sequence from CD133- GBM showed identity to 20 nucleotides of human immunodeficiency virus HIV-1 isolate AE-Env_CR12_Apr10R from Thailand envelope glycoprotein (env) gene (JN388157.1) . The underlined nucleotides “AGATA” denote promoter element DPE present in the viral sequence . This result is an example of a possibility of retroviruses providing the promoter region with regulatory sequences.The upstream sequences of exosomal NANOGP8 DNA aligned with Human papillomavirus HPV (NC_027779), Epstein-Barr virus EBV (V01555), hepatitis B (KY417926), and Human T-cell leukemia virus type 1 or HTLV-1 (JX507077) showed no identity. The same exosomal sequences were also plugged into NCBI’s retrovirus nucleotide BLAST window to check for identities with a general retroviral sequence database. Exosomal DNA clone from CD133Unexpectedly, all the upstream sequences of exosomal NANOGP8 DNA showed identity to Alcelaphine Herpesvirus-1, also known as Malignant Catarrhal Fever Virus (NC_002531), in varying degrees. This virus is not reported to infect humans. Human herpesviruses such as Gammaherpesvirus  and Human herpesvirus 4 type 2 (NC_009334) share a significant identity Alcelaphine Herpesvirus-1. Therefore, the exosomal sequences matching with Alcelaphine Herpesvirus-1 were aligned with the three human herpes viruses. Although human herpes viruses are implicated in high-grade gliomas, no significant similarities were detected .- GBM exosomes revealed identity to HIV-1 strain GS66 LTR repeat region. In standard BLAST, the upstream sequences of exosomal NANOGP8 DNA of NSC matched with virus integration sites for MuLV, Human T-lymphotropic virus 1, and various isolates of HIV-1 . GBM exosomal DNA clone had a match with HIV-1 isolate HK_JIDLNBL_S071 from Switzerland nonfunctional gag protein (gag) gene, complete sequence; and nonfunctional pol protein (pol) gene, partial sequence . CD133- GBM exosomal clone showed only one 20-nucleotide extended identity with HIV-1 isolate AE-Env_CR12_Apr10R from Thailand envelope glycoprotein (env) gene, complete cds as shown in Fig 8(A).Upstream sequences of exosomal NANOGP8 DNA of NSC, GBM and CSC showed identities to retrovirus gene sequences, retroviral LTRs, and their integration sites. NSC exosomes showed identities to Human Immunodeficiency Virus -1 (HIV-1), and Murine leukemia virus Graffi GV-1.4 (MuLV)—a gammaretrovirus, and its xenotropic integration site , 45. TheFig 11 shows the details of the alignment.Using Multiple Sequence Alignment Viewer 1.21.0, the details of the viral identities were further analyzed. Importantly, exosomal DNA showed SINE-Alu elements in the sequences upstream of NANOG. Alu element inserted near a gene potentially influences its regulation with oncogenic implications . Fig 11 - GBM, a few are unique to each cell type. It is important to identify the significance of each of these TFBS because, in GBM, the upregulated TFs with binding sites in exosomal sequences may have a crucial role in NANOGP8 transcription [Table 2 summarizes the TFs for which the binding sites were found in the exosomal DNA.Though some TFBS are shared by NSC, GBM as well as CD133cription . Table 2As mentioned earlier, exosomal DNA clones of NSC had a unique pattern of NANOG and non-NANOG sequences alternating with each other. The non-NANOG sequences are considered for promoter analysis and mentioned as the first and second upstream sequences of exosomal NANOGP8 DNA. Binding sites for TFs MBF1, a member of the mediator protein family that connects general TF to gene-specific ones affecting the individual TF’s function and enhancing transcriptional efficacy was detected in the first upstream sequence of NSC. A binding site for another TF, EFII, was also present. The second upstream sequence of NSC exosomal DNA was found to have TFBS for signal transducer and activator of transcription proteins (STAT) family members. Their presence is significant because, through the conserved DNA binding domain, STAT TFs are the major players in transcription control in normal cells as well as CSCs, including GBM , 49. Ano- GBM cells. AP-1 is a transcription factor complex comprising hetero- or homodimerized leucine zipper proteins of FOS and JUN subfamilies [The TFBS analysis of the upstream sequences of exosomal NANOGP8 DNA in GBM revealed a binding site for a solitary TF, myocyte enhancer factor 2A (MEF-2A). The presence of the binding sequence for this TF in exosomes secreted by GBM proliferating cells is compelling because MEF-2A is reported to promote stemness in gliomas, though limited research has found it to be through SOX2 transcription . Cheng efamilies . Where cfamilies , 56. AP1families . c-Fos afamilies . Leukemifamilies . MuLV sefamilies . Considefamilies . Given tfamilies . Incidenfamilies . Nuclearfamilies .- cells. Yes-associated protein (YAP1) is a transcriptional regulator in the Hippo tumor suppressor signaling pathway. YAP binds to the promoters of many stemness genes, consequently regulating their expression [YAP1 and POU2F1 binding sites were found in NSC as well as GBM exosomal DNA. Where the YAP1 binding site was detected in proliferating cells’ exosomes, the POU2F1 binding site was found in the exosomes secreted by the differentiating CD133pression –67. Simipression . NANOGP8pression , 70. TFB+GBM-gDNA clones have 100% sequence identity . Therefore, the following promoter element analysis, TFBS analysis, and retrotransposable/retroviral inundation analysis results shown for the CD133+GBM-gDNA clone are valid for NSC as well.To compare the upstream sequences of exosomal NANOGP8 DNA with the nuclear DNA, we amplified the gDNA using NANOGP8 specific primers as described earlier. NSC and CD133Fig 12). Along with several core promoter elements like TATA, INR, and DPE, a singular synergistic combination comprising TATA and INR motifs was detected , STAT, YY1, NF-AT2, Elk-1, AP-1 , GR, POU2F1, E2F, c-Ets-1, and c-Ets-2 found in exosomal sequences are also detected in gDNA. The oncogenic and stem cell-related significance of the TFs has already been discussed in exosomal DNA analysis. TFs NF-AT1, NF-AT4, and NF-AT3 belong to the nuclear factor of activated T cells family that has a regulatory role in other cell types besides T lymphocytes. NFAT proteins are reported to regulate metastasis-related genes . IRF-1 . Especially, c-Ets-1 and c-Ets-2 TFBS were found in the sequences of exosomal DNA and CD133+ GBM gDNA sequences, respectively. Though the binding sites for many other TFs have been detected in the exosomal sequences, c-Ets-1 TFBS warrants special mention and further analysis. Not much research literature is available for the regulatory region analysis of the NANOGP8 gene. For the first time, Park et al. have analyzed transcription factor Fli-1 as a direct regulator of NANOGP8, identifying its putative binding site and functionality. Friend leukemia integration 1 (Fli-1), a proto-oncogene, is an ETS transcription factor with a E26 transformation-specific (ETS) binding domain that recognizes the core consensus sequence of GGAA/T in the promoter region of the target gene [Some of the TFBSs found in the upstream of NANOGP8 were common in exosomal as well as gDNA clones from get gene . TFBS foget gene . Exosoma+ CSC, we used CD133 antibody for the immunocapture of CSC from the mixed population of GBM cells [+ (undifferentiated) to a CD133- (differentiated) state, NANOGP8 expression decreases significantly. Since GBM cells used in our analysis originated from the same subject, samples were genetically homologous. An epigenetic modification of the upstream region of the gene is most likely responsible for the changes in NANOGP8 expression. The role of exosomal NANOGP8 DNA remains to be elucidated in the alteration process.Glycoprotein antigen CD133 is expressed by normal as well as cancer stem cells . It is eBM cells . In our GBM and NSC cell lines in our study have identical genomic sequences of the NANOGP8 upstream region. However, corresponding regions of their exosomal DNA shared no identities with the genomic sequences or the sequence reported by NCBI. Consequently, the core promoter element, viral sequence inundation, and the putative transcription factor binding site (TFBS) analyses of NANOGP8 exosomal DNA yielded different outcomes for each cell line. The exosomal DNA of NSC had a unique pattern. The BLAST analysis revealed that, unlike GBM, NSC exosomal DNA had NANOG/NANOGP8 sequences punctuated with non-NANOG stretches. The dissimilarities between exosomal DNA sequences and comparable genomic sequences suggest that exosomal NANOGP8 DNA with its upstream region is possibly acquired from a source other than the cell’s genome. An unexplored import mechanism may account for the existence of NANOGP8 DNA in the exosomes. The following paragraphs discuss the possible role of exosomal NANOGP8 regulatory sequences in transcription of this gene.We have detected regulatory sequences comprising multiple core promoter elements and TFBS flanking the exosomal NANOGP8 DNA. The transcription factors (TFs) having binding sites in exosomal DNA are implicated in oncogenic regulation. The same region of exosomal DNA has also revealed cancer-related retrovirus sequences containing long terminal repeats (LTRs) known to modulate cellular oncogene expression . Therefo+ GBM gDNA sequences, respectively. This important discovery suggests a probability of a minimal promoter region of NANOGP8 for other transcription factors as well [The role of a limited promoter with minimal promoter elements is also possible in the transcriptional regulation of NANOGP8 exosomal DNA. Not much research literature is available for the regulatory region analysis of the NANOGP8 gene. For the first time, Park et al. have analyzed transcription factor Fli-1 as a direct regulator of NANOGP8, identifying its putative binding site and functionality. With a serial deletion of the promoter region, they have identified a “minimal promoter region” of NANOGP8 DNA that is needed for the Fli-1 to interact. The ETS transcription binding site in the minimal promoter region is essential for transcriptional control of NANOGP8 by Fli-1. Friend leukemia integration 1 (Fli-1), a proto-oncogene, is an ETS transcription factor with a E26 transformation-specific (ETS) binding domain that recognizes the core consensus sequence of GGAA/T in the promoter region of the target gene. c-Ets-1 and c-Ets-2 TFBS were found in the sequences of exosomal DNA and CD133 as well .Fairbanks et al. reported that in the human genome, the NANOGP8 gene is inserted in reverse orientation in the long terminal repeat (LTR) region of the SVA element belonging to the SINE-VNTR-Alu (SVA) class of retrotransposons. They suggested that the core promoter elements residing in the LTR region may promote the transcription of the gene . HoweverTFs regulate the transcription not only by binding to their designated DNA sequences but also by physical interaction with other TFs. Detection of TFBS for the transcription factors that act synergistically is important because it helps to postulate their possible role in NANOGP8 transcription. The upstream sequences of exosomal NANOG DNA in our analyses are found to possess binding sites for such TFs. For example, NF-I and C/EBPβ are a part of a TF complex that shapes local chromatin architecture and are needed for chromatin accessibility maintenance in stimulation-induced TFs . SimilarThe role of various cancer-specific TFs for which the TFBS are detected in exosomal NANOGP8 DNA should also be analyzed. Out of over 2000 TFs in human transcriptome, there are only a few that control the transcription of oncogenes involved in cancer initiation and sustenance. Notably, NANOGP8 promoter sequences found in exosomal DNA do possess binding sites for almost all the oncogenic TFs identified by Mees group . For exaGenomewide analysis of core promoter elements suggests that a combination of two or three elements makes a more potent promoter as compared to a single weak element . AnalysiCpG island methylation in promoters is an important mechanism for epigenetic regulation via transcription silencing . The absWe have amplified the exosomal DNA with an indiscriminate strand status. The nucleotide sequences reported here may belong to an ssDNA or a dsDNA. Cheng et al have shown that a plasma membrane-associated transcription system exists in cells. The cytoplasmic DNA and the resulting transcripts are implicated in oncogenesis . If the It is important to underscore the possibility that the NANOG DNA found in the exosomes may belong to other NANOG paralogs besides NANOGP8. However, when transcribed due to the presence of regulatory elements, NANOGP8 is the only paralogue with the ability to make the oncogenic protein. We initially speculated that the insertions of proviral DNA fragments provide regulatory elements with protein binding abilities to the genomic NANOGP8 upstream region. We believed that theoretically, exosomal DNA with the regulatory elements flanking NANOGP8 integrate into the genome, providing auxiliary cis-acting transcriptional elements. Therefore, internalization of exosomes conveying promoter elements and viral sequences may possess the potential to instigate a normal cell to express the undesirable NANOGP8 protein thereby creating a cancer niche. One of the possible delivery vehicles for the proviral DNA inserts in exosomal NANOGP8 is exosomes, the nanovesicles transporting a cargo of retrotransposon elements and a repertoire of tattered retroviral DNA . In addiWe believe that the differential promoter sequences of exosomal DNA, combined with the microenvironment of a tumor, and the availability of oncogenic transcription factors, may have an essential role in NANOGP8 expression. Internalization of such DNA-carrying exosomes by a target cell may potentially convert a normal or a cancer cell to a CSC. Moreover, exosomal NANOGP8, along with the flanking regulatory region, possibly provides ancillary copies of the gene to create more transcripts resulting in cancer progression and metastasis. The participation of specific exosomal DNA sequences for NANOGP8 expression needs to be investigated to validate these theories.In addition to promoter elements, other regulatory elements in exosomal DNA, if inserted in upstream region of NANOGP8, although at a substantial distance, may confer the enhancer function to the region . BecauseThe exosomal DNA of NANOGP8 gene may have a promoter with differential sequences that can be activated depending on the tumor microenvironment and the availability of oncogenic TFs. Uptake and internalization of such exosomes may stimulate a normal or a cancer cell to become a CSC, even though their gDNA sequences remain identical. This study is only a snapshot of the promoter region showing regulatory elements in the exosomal DNA belonging to a NANOG family member. It is highly possible that there are more transcriptionally essential sequence motifs in the vicinity or farther away from the analyzed region that affect the NANOGP8 transcription. Nevertheless, studies to track the journey of NANOGP8 exosomal DNA through its unloading into a normal cell, and the subsequent fate leading up to its transcription are essential. They will help to understand the participation of exosome-associated promoter elements in cancer initiation, progression, and metastasis.S1 FiggDNA was PCR amplified using NANOGP8-specific reverse primer in 5’ UTR (NCBI Reference Sequence: NC_000015.10: 35085273–35085294). The forward primer sits in a sequence from the upstream region of the gene reported in the NCBI database (NCBI Reference Sequence: NC_000015.10: 35085802–35085783). The sequences from both the cell lines matched 100% with each other.(PDF)Click here for additional data file.S2 Fig(A) and (B). gDNA clones’ BLAST comparison with the reported sequences of the NANOGP8 gene (NC_000015.10) and the adjoining upstream region. gDNA was PCR amplified using NANOGP8-specific reverse primer in 5’ UTR (NCBI Reference Sequence: NC_000015.10: 35085273–35085294). The forward primer sits in a sequence from the upstream region of the gene reported in the NCBI database (NCBI Reference Sequence: NC_000015.10: 35085802–35085783). The sequences from (A) NSC and (B) CD133+ GBM showed 99% identity with the reported sequences of the NANOGP8 gene (NC_000015.10), and the adjoining upstream region.(PDF)Click here for additional data file.S1 TableA) The sequences and the types of promoter motifs of the NANOGP8 upstream region. (B) Synergistic combination matches of the sequences and the type of promoter motifs.((PDF)Click here for additional data file.S2 TableThe sequences and the types of promoter motifs of the NANOGP8 upstream region. No synergistic combination matches of the sequences was detected.(PDF)Click here for additional data file.S3 TableA) The sequences and the types of promoter motifs of the NANOGP8 upstream region. (B) Synergistic combination matches of the sequences and the type of promoter motifs.((PDF)Click here for additional data file.S4 TableA) The sequences and the types of promoter motifs of the NANOGP8 upstream region. (B) Synergistic combination matches of the sequences and the type of promoter motifs.((PDF)Click here for additional data file.S5 Tablehttp://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3). This program identified the putative binding sites and the TF proteins that bind to them in DNA sequences. The app uses TFBSs defined by the TRANSFAC® eukaryotic TF database (version 8.3).Using a virtual laboratory app, “PROMO” (version 3.2.0), the upstream sequences of genomic NANOG DNA were scanned for TFBSs ((PDF)Click here for additional data file.S1 Raw image(PDF)Click here for additional data file.S2 Raw image(PDF)Click here for additional data file.S3 Raw image(PDF)Click here for additional data file.S4 Raw image(PDF)Click here for additional data file.S5 Raw image(PDF)Click here for additional data file.


Plasmodium falciparum and highlight their cellular functions and possible mechanisms of action.In eukaryotic organisms, noncoding RNAs (ncRNAs) have been implicated as important regulators of multifaceted biological processes, including transcriptional, posttranscriptional, and epigenetic regulation of gene expression. In recent years, it is becoming clear that protozoan parasites encode diverse ncRNA transcripts; however, little is known about their cellular functions. Recent advances in high-throughput “omic” studies identified many novel long ncRNAs (lncRNAs) in apicomplexan parasites, some of which undergo splicing, polyadenylation, and encode small proteins. To date, only a few of them are characterized, leaving a big gap in our understanding regarding their origin, mode of action, and functions in parasite biology. In this review, we focus on lncRNAs of the human malaria parasite  Plasmodium falciparum is a member of this phylum, which is responsible for the deadliest form of human malaria . To. ToP. faderstood ,78,79,85ar genes ,86. Inteegulated . It has gulation .var lncRNAs transcribed by each var gene, additional ncRNAs were discovered within intergenic regions near var genes located within internal chromosomal clusters. Interestingly, these ncRNAs have a high GC-content that is uncommon in the P. falciparum genome that is extremely AT-rich (approximately 80%), particularly in the intergenic regions  during the late stages of IDC  ..P. falciP. falciparum. Strikingly, hundreds of unknown circRNAs were discovered using advanced strand-specific RNA-seq . It. Itcis opromoter . Furtherprinting . InteresA Pol II ,111. Thikaryotes ,112. It karyotes ,111. It Pfgdv1 functions by different mechanisms and regulates RNA stability and/or translation.In addition to these possible mechanisms, the act of lncRNA transcription could have a silencing effect, where the active transcription of an lncRNA interferes or blocks the transcription machinery of a nearby gene ,114. SucP. falciparum near members of virulent multigene families such as stevor, rifin, and var share many similarities with TERRA lncRNAs. TARE-lncRNAs were implicated in regulating the structural integrity of the telomeres and the genomic loci of these virulent genes, thus affecting their expression  are implicated in the maintenance of telomeres and telomerase function ,116. Thepression  90,120],120P. fand PfHP1 ,121–123.nd PfHP1 ,125. OneP. falciparum, splicing of pre-mRNAs is an important step in mRNA maturation, particularly because over half of the parasite’s genes contain introns [Plasmodium spp.At the posttranscriptional level, lncRNAs function as modulators of mRNA splicing, stability, and translation . Their i introns ,24. More introns ,129–131.6A. Consequently, the secondary structure of the mRNA transcripts could change and alter their interaction with RBPs or other regulatory factors, resulting in changes in their translation levels [P. falciparum contains an overrepresentation of RBPs and surprisingly high levels of highly structured mRNAs that could interact with lncRNAs to regulate mRNA stability and translation rate [6A modification in P. falciparum exceed those found in any other eukaryotes and are developmentally regulated [6A modification, one can question whether lncRNAs in P. falciparum play a role in regulating the rates of m6A levels and thus affect mRNA stability during the IDC.The regulation of mRNA stability, turnover, and translation in the cytoplasm are the final regulatory layers of gene expression. These interconnected processes directly affect the level of protein production and were found to be regulated by RBPs and cytoplasmic lncRNAs –134. lncn levels ,136. P. ion rate ,138. Furegulated . ConsideP. falciparum.Unveiling the specific interactions of lncRNAs with either DNA sequences, other transcripts, and/or proteins may provide important indications for their possible cellular function. Therefore, implementing novel methodologies such as ChIRP-seq  and RADIHistorically, lncRNAs were designated as RNA transcripts that are not spliced and do not contain open reading frames that are translated to proteins ,144,145.P. falciparum, including a few examples for splicing of lncRNAs, such as GEXP22 lincRNA, PfGDV1 antisense lncRNA, and antisense transcripts (P. falciparum further supports splicing of lncRNAs, since their circular structure is generated by splicing as demonstrated for the ARP_circRNA (P. falciparum is restricted, primarily due to the limitations in identifying these events.lncRNAs that undergo AS produce novel RNA sequences that could either function as ncRNAs or protein-coding transcripts ,151,153.nscripts , left [2_circRNA , right [Transcriptomic analyses and ribosome profiling experiments have shown that many lncRNA transcripts are associated with ribosomes and contain sORFs ,150,153.Drosophila [Drosophila implicated in regulation of endosomal maturation during phagocytosis [LncRNA-encoded small proteins have been shown to be involved in diverse cellular processes ranging from transcriptional regulation to cell division, DNA repair, and signaling pathways  154,157157–163. osophila , hemotinocytosis .P. falciparum at different stages of IDC [Plasmodium, were also demonstrated to have the capacity to encode small proteins in vitro and in vivo in other organisms [Plasmodium remain to be uncovered [Plasmodium.Approximately 160 small proteins corresponding to sORF regions of lncRNAs were recently identified in s of IDC . In addirganisms –167. Howncovered . ConsidePlasmodium spp., but surprisingly, very little is known about their biological functions and even less about the molecular and cellular mechanisms by which they act. Nonetheless, several ncRNAs were implicated in fundamental aspects of the parasite’s biology, such as regulation of antigenic variation and sexual commitment, emphasizing their potential roles as emerging key regulators of various biological processes in apicomplexan parasites and other eukaryotes.Evidently, regardless of their protein-coding purpose, many RNA molecules have different intrinsic regulatory functions. In the last couple of decades, numerous discoveries shed light on the important role of lncRNAs as regulators of cellular processes in many organisms. A relatively rich plethora of ncRNAs are transcribed by


Human intestinal epithelial organoids (IEOs) are a powerful tool to model major aspects of intestinal development, health, and diseases because patient-derived cultures retain many features found in vivo. A necessary aspect of the organoid model is the requirement to expand cultures in vitro through several rounds of passaging. This is of concern because the passaging of cells has been shown to affect cell morphology, ploidy, and function.Here, we analyzed 173 human IEO lines derived from the small and large bowel and examined the effect of culture duration on DNA methylation (DNAm). Furthermore, we tested the potential impact of DNAm changes on gene expression and cellular function.Our analyses show a reproducible effect of culture duration on DNAm in a large discovery cohort as well as 2 publicly available validation cohorts generated in different laboratories. Although methylation changes were seen in only approximately 8% of tested cytosine-phosphate-guanine dinucleotides (CpGs) and global cellular function remained stable, a subset of methylation changes correlated with altered gene expression at baseline as well as in response to inflammatory cytokine exposure and withdrawal of Wnt agonists. Importantly, epigenetic changes were found to be enriched in genomic regions associated with colonic cancer and distant to the site of replication, indicating similarities to malignant transformation.Our study shows distinct culture-associated epigenetic changes in mucosa-derived human IEOs, some of which appear to impact gene transcriptomic and cellular function. These findings highlight the need for future studies in this area and the importance of considering passage number as a potentially confounding factor.  This work describes cell culture–induced changes to DNA methylation, gene expression, and cellular function in human intestinal epithelial organoids. Globally, organoids lost DNA methylation with time in culture while DNA methylation also became generally more variable. This work suggests a shifted epigenetic profile in organoids cultured long term.Organoids are self-organizing, 3-dimensional structures that are derived from either pluripotent or somatic, organ-specific stem cells. They have been shown to closely mimic both anatomy and cellular function of the in vivo organ. Importantly, the ability to generate such organoids from human cells has turned them into powerful translational research tools with a wide range of applications, including the development of new therapeutics, testing of existing drugs, as well as the application of a personalized treatment approach in several diseases.5Among the most advanced human organoid models are mucosa-derived intestinal epithelial organoids (IEOs).,Another major advantage of human IEOs is the ability to keep them in culture over prolonged time periods . Indeed, prolonged culture periods also are required to sufficiently expand organoids before their experimental use. As a result, cellular cultures must undergo numerous rounds of passaging, a process during which organoids are dissociated into individual crypts that give rise to new organoids, thereby increasing their total number. The potential impact of prolonged in vitro culturing of a human mucosa–derived IEO remain largely unknown. Although previous studies have shown a high degree of genetic stability over time,,,,DNA methylation (DNAm) is one of the main epigenetic mechanisms known to play a key role in regulating cellular function of human cells including the intestinal epithelium.Importantly, epigenetic instability in intestinal cells also has been seen in colon carcinogenesis, with substantial genome-wide DNAm changes observed in colorectal cancer.22Here, we set out to monitor global DNAm in human IEOs during prolonged in vitro culture and investigate the impact of associated epigenetic changes on cellular function. Based on the analyses of 173 human mucosa–derived IEOs, we have identified distinct culture-associated DNAm changes, some of which impact gene transcription and cellular function. This highlights the importance of considering culture duration in experimental design and interpretation of results.A). IEOs were harvested at various time points ranging from passage 1 (approximately 7–10 days) to passage 16 (approximately 4–10 months of culture duration), and genome-wide DNAm profiling was performed. First, we assessed DNAm profiles for any variation and potential association with specific phenotypes or donor characteristics. We therefore performed variance decomposition analyses. Briefly, principal component analysis (PCA) was performed to uncover variance of IEO DNAm profiles followed by testing for a potential correlation between observed DNAm differences and phenotypes (see the Methods section). As shown in Figure 1B, IEO DNAm was strongly associated with gut segment  and age. As reported previously by our group,Figure 1C and D). A significant correlation between DNAm and age  indicated that the biological age of donors also is retained in IEOs. Indeed, this was confirmed further by calculating the epigenetic ages = 0.52) , suggesting epigenetic age is maintained accurately over prolonged in vitro culturing. Interestingly, passage number also was found to be associated significantly with DNAm changes in Principal component (PC)1–3 and 6, indicating an impact of culture duration on epigenetic variation in IEOs . Plotting PC2 vs PC3 shows that passage number accounts for approximately 10% of variation observed in IEO DNAm profiles (rs = -0.82) .To examine the potential impact of prolonged in vitro culture on human IEO DNAm, we recruited a total of 46 children undergoing routine endoscopy and obtained mucosal biopsy specimens from the distal small bowel  and distal large bowel (sigmoid colon). Human IEOs were generated   and cultFigure 1G and H).To ensure that observed culture-associated DNAm changes were not a result of laboratory or sample cohort–specific culturing techniques, we tested this phenomenon in additional, publicly available cohorts previously generated by our group (cohort 2), as well as by an independent group (cohort 3) . In bothFigure 1Together, these results show that although the vast majority of IEO DNAm appears to be stable even during prolonged culture periods, approximately 10% of epigenetic variation is associated strongly with culture duration in both a large discovery as well as 2 additional validation cohorts.A). To identify directional DNAm changes, we performed differential DNAm analyses comparing low-  with high-  passage IEOs (see the Methods section).Having established a reproducible association between DNAm variance with culture duration , we next investigated directional changes of DNAm during prolonged in vitro culture. In general, there are 3 ways by which DNAm can change over time: (1) a stochastic occurrence of either gain or loss of DNAm , (2) loss of DNAm (hypomethylation), and (3) gain of DNAm (hypermethylation) A. To ideB). Furthermore, 17,352 CpGs (2.2%) showed loss of DNAm (hypomethylated) while 6414 CpGs (0.8%) gained DNAm (hypermethylated) over time  (B).Assessing DNAm changes in our discovery cohort 1, we identified 41,852 cytosine-phosphate-guanine dinucleotides (CpGs)  that showed significant heteroskedasticity with passage  B. Furthe > 0.15) B.C and D), as well as consistency in the magnitude of passage-associated differential DNAm (E and F).Directional changes in DNAm  associated with passage were validated by showing a major overlap of CpGs in both additional cohorts 2 and 3  C and D, ial DNAm E and F.Taken together, a subset of passage-associated directional DNAm changes were validated in 2 additional cohorts, further suggesting that this phenomenon occurs independently of the sample cohort and laboratory.A). IEOs were kept in culture for up to 12 passages (approximately 4 months) and subjected to a range of functional assays as well as genome-wide epigenetic  and transcriptomic profiling. As shown in Figure 3B, culture duration did not impact on the microscopic appearance of IEOs as distinct differences, such as a more budded appearance of small-bowel and cystic appearance of large-bowel/colonic IEOs were retained in high-passage organoids . Similarly, measuring IEO size over several days after passaging showed no difference in growth when comparing IEOs at low vs high passage within each gut segment (P < .05) .Having observed distinct DNAm changes associated with prolonged culturing of IEOs, we next aimed to investigate the potential impact of culture duration on cellular function and gene transcription. We, therefore, generated an additional cohort (cohort 4) of IEOs from small- and large-bowel biopsy specimens from 5 healthy individuals A. IEOs w,P < .05), suggesting that these cytokines impact on epithelial barrier function as expected (D and E). Importantly, culture duration did not impact the epithelial cell barrier because we did not observe any difference between high- and low-passage IEOs within each gut segment (P > .05) (D and E).Next, we aimed to test whether culture duration impacts barrier function of IEOs at baseline and in response to inflammatory cytokines. We therefore developed a modified forskolin-induced swelling assay, which has been used previously in the context of cystic fibrosis to test the function of cystic fibrosis transmembrane conductance regulator in gut organoids (see the Methods section).expected D and E. P > .05) D and E.A). Gene transcription was assessed on extracted RNA using RNA sequencing. As described previously, withdrawal of Wnt agonists led to decreased expression of intestinal epithelial stem cell marker LGR5 while expression of epithelial cell subset (MUC5B) and differentiation markers (FABP1 and FABP6) increased in a gut segment–specific manner . Furthermore, of 10,942 genes found to change expression in response to Wnt agonist withdrawal in low-passage IEOs, expression of 8968 also changed in high-passage IEOs . In total, 8% of genes that were found to change their expression in response to Wnt agonist withdrawal in low- but not high-passage IEOs are associated with at least 1 CpG showing passage-associated DNAm changes . As shown in Figure 4C, a major overlap was found between differentially expressed genes in response to IFNγ and TNFα comparing low- with high-passage IEOs and no microscopic changes were observed . Similar to transcriptional changes in response to in vitro differentiation, of the genes only changed in low-passage IEOs, 7% and 9% , were associated with at least 1 CpG showing passage-associated DNAm changes, indicating a limited impact of culture-associated DNAm changes on inflammation-induced gene transcription. However, a highly significant association between passage-associated DNAm and transcriptional changes was observed for several genes including EDAR and EIF4G1  binding sites, open chromatin, enhancers, and promoter flanking regions, but depleted in promoters and CCCTC-binding factor (CTCF) binding sitesFigure 6A). In contrast, CpGs gaining DNAm in culture were enriched in TF binding sites and promoter flanking regions, but also depleted in promoters and CTCF binding sites  , suggesting that the dysregulation of DNAm with passage occurred genome wide, but seemed to spare nonvariable unmethylated regions.As shown in Figure 6Pathways possibly affected by passage-associated DNAm changes were explored through enrichment of any gene ontology (GO) gene sets in the genes adjacent to passage-associated DNAm changes. Hypomethylated and heteroskedastic CpGs were enriched in similar GO gene sets, including adherens junction organization (GO:0034332) and cell–cell adhesion via plasma-membrane adhesion molecules (GO:0098742) , and theB). Moreover, CpGs that lost or gained DNAm in high-passage IEOs were enriched in cDMRs that lost or gained DNAm in cancer, respectively  . Heteroskedastic CpGs also were enriched in cDMRs hypomethylated in cancer  .Global loss and local gain of DNAm have been linked to various malignancies including colon, breast, and prostate cancers.Figure 6C).P < .001) (D). Interestingly, the same was true for heteroskedastic CpGs, while hypermethylated CpGs were located closer to origins of replication than expected by chance (P < .001) (D). Taken together, these results suggest that passage-associated DNAm changes in human IEOs share features of epigenetic changes observed in colon cancer and may be caused at least in part by fast cell turnover in vitro.It has been proposed in the context of cancer that late-replicating regions of the genome will have less time to remethylate CpGs on the daughter strand after DNA replication C.31 A re < .001) D. Intere < .001) D. Taken Since the establishment of human mucosa–derived IEO as powerful translational research tools just over a decade ago, the number of applications has continued to increase dramatically. Major new areas of interest include precision and regenerative medicine, drug discovery and development, as well as modeling of disease pathogenesis.,,,Epigenetic mechanisms can alter cellular function in response to environmental changes. As one of the main epigenetic mechanisms in mammalian cells, DNAm is known to play a key role in regulating intestinal epithelial cellular function. In this study, we show distinct culture-associated DNAm changes in human mucosa–derived IEOs occurring over several months regardless of from which gut segment or human donor the IEOs were derived. Although the vast majority of CpGs tested was found to be highly stable, including those specific for age and anatomic location, approximately 8% of probes showed DNAm changes that were associated with culture. Although a proportion of methylation changes could be caused by limitations relating to the methodologies used, we were able to validate a large proportion of culture-associated DNAm changes across several IEO cohorts that were generated by independent groups. Our findings are in keeping with previous reports on directed and stochastic changes to DNAm with passaging of induced pluripotent stem cells.,A key question arising from our findings relates to the likely cause of DNAm changes observed in IEOs. Epigenetic changes occurring over time, or with age, generally are referred to as epigenetic drift. According to our current understanding, the main mechanisms involved in causing epigenetic drift include genetic variation, environmental factors, as well as stochastic changes as a result of inaccurate copying of methylation signatures during cellular divisions.Arguably the most important question we aimed to answer in our study is whether observed DNAm changes alter gene transcription or cellular function of IEOs. Reassuringly, we found that the overall impact of culture-associated DNAm changes in human IEOs was limited and did not impact cellular identity , epigenetic age, or broad cellular function such as growth and intestinal barrier function. However, performing simultaneous genome-wide transcriptional and epigenetic profiling of IEOs showed a subset of passage-associated DNAm changes that correlated with changes in gene expression at baseline, as well as in response to in vitro differentiation and exposure to inflammatory cytokines. The latter is particularly relevant and important for the studies using organoids as models to investigate related inflammatory conditions such as inflammatory bowel disease. Interestingly, we also identified highly significant gene expression changes at baseline and in response to treatment with inflammatory cytokines, which were not associated with DNAm changes. These changes could be caused by other epigenetic mechanisms such as post-translational histone modifications or expression of small noncoding RNAs and future studies are required to investigate this important question.Another key finding in our study was the observed global loss of DNAm in IEOs associated with prolonged in vitro culture, which is a recognized feature of various malignancies including colorectal cancer. Indeed, we identified several additional similarities including the overlap and directional methylation change between passage-associated DMRs with known cDMRs, as well as the significant proportion of heteroskedastic methylation changes. The latter has been linked to higher variability in DNAm of colon cancer compared with normal samples.We recognize the limitations of our study, including those associated with methodologies used to perform genome-wide DNAm analyses of IEOs. Although Illumina methylation arrays  are established and validated tools to comprehensively evaluate promoter regions, CpG islands, as well as other potentially relevant intergenic regions, they only cover 3% of the approximately 28 million CpG sites present in the entire human genome. Furthermore, because culture duration between passaging of IEOs varies we are unable to provide exact recommendations on when DNAm changes occur. However, it is clear from our results that the risk for epigenetic changes to develop is linked directly to culture duration. Indeed, our study includes IEOs that were passaged up to 16 times, relating to approximately 5 months in culture. Because organoids can be and frequently are cultured for substantially longer time periods , we would expect to see epigenetic and/or associated functional changes accumulating over time, further emphasizing the need to consider culture duration as a critical part of experimental design.In summary, our study identified distinct, culture-associated DNA methylation changes in human mucosa–derived IEOs that impact gene transcription and cellular function and share features of malignant transformation. Although global epithelial cell function was found to be retained, our findings highlight the critical importance of considering culture duration in the experimental design and interpretation of data derived from human IEOs.Intestinal biopsy specimens were collected from the TI and sigmoid colon from 52 children aged 1 to 16 years undergoing diagnostic endoscopy. This study was conducted with informed patient and/or carer consent as appropriate, and with full ethical approval (REC-12/EE/0482).,,,Human IEOs were generated from mucosal biopsy specimens by isolation of intestinal crypts and culturing in Matrigel  using media described previously.differentiation medium)   for an aHuman IEO growth was assessed using the Incucyte SX5  by imaging every 6 hours over 7 days for each passage. After 7 days, the images were analyzed using the Incucyte IEO analysis software, which allowed measurement of the IEO area over time. For each human IEO line and for each passage, 6 wells were imaged and analyzed to generate an average measurement of IEO growth. Comparisons between IEO passages were made using analysis of variance.Human IEO barrier integrity was evaluated by culturing the IEOs at early and late passages, as described earlier, from day 0 to day 4. On day 5, IEOs were collected from 48-well plates and transferred to 96-well plates, seeding 5–10 IEOs per well in 5 μL Matrigel and 100 μL growth medium. Using 3 wells per condition, human IEOs were cultured or in standard condition medium, or in vehicle control medium (+dimethyl sulfoxide), or in forskolin (5 μmol/L) medium in the presence or absence of IFNγ (20 ng/mL) or TNFα (40 ng/mL). The plates were placed in the Incucyte SX5 to be imaged every 2 hours for 48 hours. After 2 days, the experiment was stopped and images were analyzed to measure IEO area over time using the Incucyte IEO analysis software.At the end of each experiment, human IEOs were harvested and both DNA and RNA were extracted using the AllPrep DNA/RNA mini kit . DNA was bisulfite-converted using the EZ DNA methylation Gold kit .Genome-wide DNAm was profiled using the Illumina EPIC platform (Illumina), and deposited in ArrayExpress  under accession numbers E-MTAB-9748 and E-MTAB-11545. An overview of sample numbers can be found in Expression profiling was performed using RNA sequencing by Cambridge Genomic Services (University of Cambridge) and can be found in ArrayExpress under accession number E-MTAB-11548. The code for analysis is available at: redgar598.github.io/DNAm_organoid_passage.All authors had access to the study data and reviewed and approved the final manuscript.P value greater than .05 in 1% of samples. This filtering left 798,096 CpGs for analysis.DNAm data were processed using the minfi package,The association between DNAm and culture duration (quantified by passage) was investigated with PCA. The loadings of each PC were associated with technical and biological variables using analysis of variance for categoric variables or Spearman correlations for continuous variables. An association between DNAm and passage also was tested on an individual CpG level. Differential DNAm with increasing passage number was tested using linear models at each of 798,096 CpGs and significant heteroskedasticity in DNAm was tested with a Breusch–Pagan test.,,Publicly available data setsThe DNAm data were measured and processed as for cohort 1 . In the P values for differential and heteroskedastic CpGs, in each regulatory region, were calculated using 1000 randomly sampled lists of CpGs, to account for the underlying distribution of CpGs on the EPIC array. For hypomethylated and hypermethylated CpGs a change in DNAm of 0.15 of -0.15 was required. Therefore, the background of CpGs was modified to exclude those with a DNAm value >0.15 for hypomethylated CpGs and <0.85 for hypermethylated CpGs in the passage 1 IEO. These 223,695 and 295,469 CpGs, respectively, could never pass the threshold of change in DNAm and should not be included in the background CpGs list.Enrichment of CpGs that showed DNAm changes with passage in various genomic features was tested. Analyses were performed separately for CpGs showing hypermethylation and hypomethylation with increased passage as well as a heteroskedastic DNAm pattern. The differential and heteroskedastic CpGs identified were explored for enrichment in genomic regulatory features. The Ensembl Regulatory BuildP values were generated for CpGs in previously described cDMRs.Similarly, enrichment ,For each of the 42 validation samples, RNA was prepared with the Truseq mRNA library preparation  and sequencing was performed on NextSeq  75-cycle high output. RNA sequencing data were quality controlled using FastQC.To explore possible pathways affected systematically by passage, the CpGs differentially DNAm with passage were associated with genes, and these genes then were tested for enrichment in GO gene sets. A CpG was assigned to a gene based on proximity to a transcript from Ensembl Genes 99 GRCh37.p13 collected from BioMart.


Cyp24a1, S100g, Trpv6, and Slc30a10. Other VD-regulated transcripts were unique to the SI crypt , villi , or colon , but this did not correlate with mRNA levels of the VDR. Furthermore, bioinformatic analysis identified unique VD-regulated biological functions in each compartment. VDR-binding sites were found in 70% of upregulated genes from the colon and SI villi but were less common in upregulated genes from the SI crypt and among downregulated genes, suggesting some transcript-level VD effects are likely indirect. Consistent with this, we show that VD regulated the expression of other transcription factors and their downstream targets. Finally, we demonstrate that compartment-specific VD-mediated gene expression was associated with compartment-specific VDR-binding sites (<30% of targets) and enrichment of intestinal transcription factor–binding motifs within VDR-binding peaks. Taken together, our data reveal unique spatial patterns of VD action in the intestine and suggest novel mechanisms that could account for compartment-specific functions of this hormone.1,25-dihydroxyvitamin D (VD) regulates intestinal calcium absorption in the small intestine (SI) and also reduces risk of colonic inflammation and cancer. However, the intestine compartment-specific target genes of VD signaling are unknown. Here, we examined VD action across three functional compartments of the intestine using RNA-seq to measure VD-induced changes in gene expression and Chromatin Immunoprecipitation with next generation sequencing to measure vitamin D receptor (VDR) genomic binding. We found that VD regulated the expression of 55 shared transcripts in the SI crypt, SI villi, and in the colon, including  VDR-mediated gene transcription is a multistep process that involves VDR binding to target genes at both active promoters and distal regulatory elements as well as recruitment of coregulatory proteins 2D3 target tissue 2D3 functions across the length of the intestine 2D3 rapidly stimulates calcium extrusion 2D3 action in the intestine are sparse. In addition, the diversity and complexity of 1,25(OH)2D3 signaling with respect to proximal–distal and crypt–villus axes have not as yet been evaluated when considering intestinal 1,25(OH)2D3 action.In addition to maintenance of calcium homeostasis, many other beneficial intestinal effects of 1,25(OH)xtrusion  but slowe villus . Meanwhie villus , 13. In i.e., RNA-seq, VDR ChIP-seq, ATAC-seq) to identify 1,25(OH)2D3-responsive target genes across the proximal-distal and small intestine (SI) crypt–villus axis. Our findings show that while a number of 1,25(OH)2D3-regulated genes are common across SI villus, SI crypt, and colon, the majority of 1,25(OH)2D3-regulated transcripts have compartment-restricted regulation patterns. Gene ontology (GO) and pathway analysis of the 1,25(OH)2D3-regulated transcripts from each compartment indicated regulation of unique biological functions, independent of calcium homeostasis, including regulation of RNA metabolic processes, tight junctions, metabolism of xenobiotics, lipid metabolic processes, and HIF1 signaling. However, not all VDR-regulated genes have VDR-binding peaks, suggesting that some transcript levels effects of 1,25(OH)2D3 are indirect and may be due in part to the contribution of other transcription factors. Our findings are the first to define 1,25(OH)2D3-molecular actions across the critical proximal–distal and crypt–villus axes that define the functional characteristics of the intestine and suggest novel mechanisms that may account for intestine compartment-specific functions of 1,25(OH)2D3.In this study, we used a series of complementary genomic tools (Lct) in the villus versus colon and a 189-fold greater expression of the colon marker, carbonic anhydrase 1 (Car1) mRNA, in colon versus villus , while expression of the intestinal stem cell marker Lgr5 was elevated significantly in crypts (+12.2 fold). Collectively, these compartment-level differences in transcript levels confirm the quality of our isolation procedure.We confirmed the quality of our isolation of the various intestinal segments in two ways. First, we visually examined the small intestinal villus and crypt preparations to confirm that they were pure 2 D treatment (B). 2 D across the three compartments at the 5% FDR , while 78% of the 1,25(OH)2D3-regulated transcripts were specific to just one compartment. Ninety three percent of the 55 common targets were upregulated by 1,25(OH)2D3 treatment. Similarly, more than 80% of 1,25(OH)2D3-regulated genes in the SI villus and colon were induced. In contrast, only 56% of SI crypt transcripts were upregulated.A summary of the impact of 1,25(OH)d Atp2b1 . In addireatment B. Figure2D3-differentially expressed gene list with data on 1,25(OH)2D3-regulated transcripts in the SI that was previously published by Lee et al. e et al. . Of the e et al. . Thirty 2D3-regulated gene list from each compartment for enrichment of GO terms and pathways 2D3-induced VDR-binding peaks in each intestinal compartment: 12,719 in SI crypt, 18,083 in SI villus, and 22,888 in colon. The ChIP-Seq signal was similar across compartments and VDR ChIP peaks averaged ∼ 1000 bp wide (A). Included in our VDR ChIP-seq peaks were the previously reported VDR-binding peaks in the Cyp24a1 gene ; the Trpv6 gene ; and Slc30a10 gene  2D3 treatment revealed regulatory sites that were either silent under basal conditions or under the ATAC detection limit in untreated mouse intestine (e.g.Slc30a10 gene).Using VDR ChIP-seq, we found many 1,25(OH) bp wide A. Includ +48 kb) . More th +48 kb) . In contB and An evaluation of the VDR-binding peaks for known transcription factor binding site motifs revealed that the VDR-RXR DR3 motif was the most enriched motif in all three compartments . Approximately, 70% of the 1,25(OH)2D3-induced transcripts in SI villus and colon had a VDR-binding peak. In contrast, only 52% of 1,25(OH)2D3-induced crypt transcripts had VDR-binding peaks, while even fewer 1,25(OH)2D3-suppressed transcripts had them . This suggests that the regulation of many genes, especially those induced in the crypt and suppressed in all compartments, were not direct 1,25(OH)2D3 target genes but may be a consequence of an upstream 1,25(OH)2D3-regulated event.We attributed the VDR-ChIP peaks to their nearest neighbor gene using GREAT in GSEA. This data was then used to identify the genes differentially expressed by 1,25(OH)ith them D. Approx2 D treatment could alter transcript levels independent of VDR binding to a gene regulatory region is that 1,25(OH)2D3 regulates the expression of other transcription factors that have 1,25(OH)2D3-independent downstream actions. Consistent with this hypothesis, bioinformatic analysis identified the protein class “transcription factors” as enriched in the 1,25(OH)2D3-regulated genes from crypt and colon (2D3 treatment (16 induced and six suppressed transcription factor genes had a VDR binding site). Of the 50 1,25(OH)2D3-regulated transcription factor messages, 18 had enrichment of their downstream target genes in our dataset nd colon . In addind colon , 50 were dataset .Figure 42 D treatment was due to differential binding of VDR to specific regulatory sites. Differential VDR binding was minimal between the small intestinal crypt and villus . In contrast, there were several hundred VDR peaks that were differentially enriched in either the colon or SI . As shown in Slc37a2, which has a VDR-binding site within an intronic enhancer in the SI that is absent in the colon, as well as Ptges, which has an VDR binding, intronic enhancer in colon that is lower in the SI .We next used Diffbind to compare VDR peaks across the three compartments to test whether compartment-specific regulation of genes by 1,25(OH)2D3 is a critical regulator of intestinal physiology that controls calcium absorption 2D3 in the mature small intestinal enterocyte related primarily to calcium absorption or protection against barrier dysfunction . However, only a small number of the 1,25(OH)2D3-regulated gene targets were common across the three compartments . Instead, the majority of 1,25(OH)2D3-mediated genomic events were distinct and compartment specific sorption , enhancesorption , 18, regsorption , and supsorption . Despitefunction , 21. In f debate , 13, 22.e crypts . We attespecific . Thus, ocrapings  or cultu2D3-induced transcripts could be explained by differential VDR binding to gene regulatory regions. Compartment-enriched VDR peaks were also found to differ in their enrichment of secondary transcription factor–binding motifs. This suggests that coordination between other transcription factors and VDR may contribute to differential binding and/or chromatin accessibility. For example, our findings of CDX2 and FOXA1 motifs at or near colon-enriched VDR-binding sites and HNF4a and GATA4 motifs at SI-enriched VDR-binding sites suggest that these transcription factors may promote or stabilize VDR binding at specific sites to mediate compartment-specific gene regulation. A stabilizing role would be similar to the role proposed for ETS1 in the regulation of 1,25(OH)2D3-mediated Cyp24a1 gene expression 2D3-induced crypt transcripts did not have VDR-binding peaks. Also, between 60 to 79% of 1,25(OH)2D3-suppressed transcripts in each of the three compartments lacked a VDR-binding site. Thus, VDR binding does not predict mRNA expression. One hypothesis to explain VDR-independent and compartment-specific regulation of genes is that it is indirectly mediated through other transcription factors. In support of this hypothesis, we found 10 transcription factor genes that contained VDR-binding sites were differentially regulated by 1,25(OH)2 D treatment, and whose downstream targets were differentially regulated by 1,25(OH)2 D despite lacking VDR-binding sites near their genes. In the SI, this includes genes for transcription factors like NFATC3, which regulates intestinal differentiation 2D and their genes included VDR-binding sites bound by VDR. In addition, we found that our list of 1,25(OH)2D3-regulated genes included suppression of HIF1A  and KLF5 target genes. KLF5 has been reported to regulate intestinal epithelial cell proliferation ntiation , GATA4, ntiation , 29, 30,ntiation . Thus, 1feration , particuferation . HIF1A hferation  and 1,25al cells .et al.  on small intestinal gene expression in CYP27B1 KO mice using RNA-seq. Our work confirms some of their findings. For example, of the 72 transcripts we identified as 1,25(OH)2D3-regulated in all three compartments at 10% FDR 2D3 target genes , the manganese exporter Slc30a10 that we 2D3 function in the intestine.Lee et al.  previous 10% FDR , more th that we  and othe that we , 36 havel injury . DHCR24 l injury  that canl injury . SHROOM1l injury . PPAR δ l injury  and may l injury . While tet al.  biosynthesis pathway. This gene was found to be induced by 1,25(OH)2D3 and bound by VDR only in the colon. PGE2 is a bioactive lipid with diverse physiological roles including regulation of inflammatory responses 2D3 may be another mechanism whereby 1,25(OH)2D3 protects mucosal barrier function in the colon.Our work extends the findings of Lee et al.  by demonesponses . Althougesponses . A role uggested , 46. Ind2D3 in the SI but not in the colon are genes encoding drug metabolizing enzymes. Although it was previously suggested that the liver is the major site of xenobiotic metabolism, recent reports show that the intestine also has a major role in xenobiotic detoxification (e.g. cytochrome P450s) and phase II enzymes that catalyze conjugation reactions  , Cyp2b10 (the human homolog is CYP2B6), Gstm1, and Gstm3 mRNAs. Genes involved in xenobiotic metabolism are also induced by the xenobiotic-activated nuclear receptors PXR (pregnane X receptor) and Car (constitutive androstane receptor) fication , 48. Theenzymes) . Phase Ienzymes) . Among ttabolism . Previou/2 class , 53. In eceptor) . Similarn levels . In huma2D3 treatment also regulated 45 solute transporter genes. This includes genes encoding transporters for various amino acids , ion transporters , and organic molecules . Similar to other reports 2D-induced in the SI and with VDR-binding sites associated with the gene. This is a glucose-phosphate transporter  which was induced in all three segments and has VDR-binding sites in its gene. While gene variants of the human SLC39A8 gene have been associated with Crohn’s Disease  and Slc30a5 (encoding ZNT5B), that mediate zinc uptake into cells, and Scl30a1 (encoding ZNT1) that mediates zinc export at the basolateral membrane of enterocytes  reports , we alsonsporter  whose exnsporter . However Disease  its funcerocytes . These terocytes , 59 and e et al. . We prev, Caco-2  but the 2D3 action because it contains stem cells, proliferating daughter cells, and nonproliferating but undifferentiated cells. Expression of VDR and 1,25(OH)2D3 regulation of target genes in undifferentiated cells typically seen in crypts has been noted using human duodenal enteroids and human colon organoids 2D3, and DNA near the gene was bound by VDR. The Msi1 gene encodes the RNA-binding protein Musushi1, a protein marker of intestinal stem cells and early intestinal cells lineages that is important in crypt regeneration 2D3 in patient-derived colon stem cells rganoids , 61. Herneration . As suchem cells . These f2D3-mediated intestinal gene regulation to date. The novelty of this study is that it is the first to define how 1,25(OH)2D3 action is influenced across critical functional axes within the intestine . As a result, in addition to confirming many 1,25(OH)2D3-regulated target genes from earlier reports 2D3 action on specific enterocyte lineages  nor did we examine any potential age–related effects on intestinal 1,25(OH)2D3 action that might reflect age-associated intestinal resistance to the hormone  by 1,25(OH)2D3 that others reported was present in small intestinal villus but not in crypts 2D3 target genes and to test whether VDR-dependent recruitment of chromatin remodelers influences compartment-specific differences in chromatin accessibility and gene expression.The strength of this study is that it is the most comprehensive examination of 1,25(OH) reports , our fin hormone , 64. Addn crypts . There i2D3 genomic action in different regions of the intestine may account for the compartment-specific, multiple regulatory actions of 1,25(OH)2D3 in the intestine. Further studies related to compartment-specific physiological functions of 1,25(OH)2D3, as well as a more comprehensive understanding of transcription factor networks involved in VDR mediated transcription, will provide new avenues of investigation related to the actions of 1,25(OH)2D3 in the regulation of intestinal physiology.In summary, this study has expanded our understanding of how 1,25(OH)All experiments were approved by the Animal Care and Use Committee at Rutgers University and at Rutgers, New Jersey Medical School. Mice were exposed to a 12h-light, 12h-dark cycle while food and water were given ad libitum.2 D, female mice were fed a vitamin D–deficient diet  for 2 to 3 weeks prior to mating, during pregnancy, and during lactation, and pups from the vitamin D–deficient dams were fed the vitamin D–deficient diet until 12 weeks of age. At the end of the experiment, mice  were injected ip with either 1,25(OH)2D3  or vehicle (9:1 mix of propylene glycol:ethanol) and killed 4 h later. The time, dose of 1,25(OH)2D3, and use of vitamin D–deficient pups were chosen to maximize responsiveness of, and ability to detect, 1,25(OH)2D3-regulated transcripts. Ten to fifteen centimeter of the proximal SI was used to isolate crypts and villi while the entire colon was used for a mucosal scraping (specific sample preparation provided below).C57BL/6J mice were obtained from The Jackson Laboratory. To maximize the transcriptional response to 1,25(OH)2D3 (10 ng/g body weight) and killed 1 h later. The dose and timing of 1,25(OH)2D3 treatment was chosen based on pilot ChIP-PCR studies that showed this treatment protocol enhanced VDR-binding peaks previously reported within enhancers of the Cyp24a1 and Trpv6 genes. Ten to fifteen centimeter of the proximal SI was used to isolate crypts and villi while the entire colon was used for a mucosal scraping (specific sample preparation provided below).C57BL/6J mice were obtained from either the Jackson Laboratory or from breeding colonies maintained at Rutgers University. Mice were fed a standard rodent chow diet . At 10 to 12 weeks old, mice  were treated ip with 1,25(OH)C57BL/6J mice were obtained from either the Jackson Laboratory. Mice were fed a standard rodent chow diet  and small intestinal villus, small intestinal crypts, or colon was harvested at 12 weeks of age. We chose to use a normal chow diet and adult mice to capture the basal, physiologically relevant open chromatin regions in the mouse intestine. SI epithelium was separated into villus and crypt fractions, while the whole, unfractionated epithelium was used from colon .°C.The isolation of small intestinal crypts is a routine procedure in the area of intestinal biology See . For ChI°C.The colon was harvested from the same group of C57BL/6J mice as the crypts and villi isolation at the same time. The entire colon from the terminal cecum to rectum were used for colon samples. After flushing with cold 1XPBS, the colon was opened longitudinally and the epithelial mucosa was removed by scraping until the colon became transparent. For ChIP-seq, the colon epithelial scraping was washed with 1XPBS twice and incubated in 1.5% formaldehyde solution in a rotator for 15 min at 4 °C and 40 min at 25 °C. After fixation, the sample was washed twice with 1XPBS for 3 min at 4 °C and spun down at 300 rcf for 30 s at 4 °C in order to remove any residual PBS. Finally, the samples were frozen on dry ice for 5 min before storage in the −80 °C freezer. For ATAC-seq, samples were prepared as we have previously described . For RNAThree hundred microgram total cell pellets were used per ChIP replicate. The villi, crypts, and colon whole epithelium pellets were thawed on ice and mixed with 3 to 4 times the volume of the lysis buffer ) dissolved in MilliQ water). Lysates were incubated at RT for 10 min, aliquoted at volumes between 300 to 400 μl, and sonicated in cold ultrasonication water bath for intervals of 10 min. After sonication, 5 μl of the lysate was mixed with 100 μl of the reverse cross-linking buffer  and incubated either overnight in 65 °C or for 15 min at 95 °C to reverse crosslinking. The DNA was purified from the lysate using QIAquick PCR Purification Kit   using the bamCoverage (deepTools24). Peak VDR-binding regions were identified in each alignment BAM file using the callpeak utility in MACS28. The resulting peak set files were filtered against the ENCODE blacklist for the mm9 genome assembly29. Pairwise comparisons using DiffBind19  were conducted to identify peaks exhibiting differential VDR-binding affinities between the intestinal compartments. Each set of differentially bound peaks was filtered to only include peaks, which were assigned a confidence value less than 0.001 by DiffBind. Each set of differentially bound peaks was exported to a BED file. A representative sample peak set BED file was generated from each cell type’s composite peak set BED file by randomly selecting 20% of the peaks in each composite peak set for use in HOMER motif calling analysis to save computational time. Several iterations of subsampling were compared and found to not majorly impact the results.Sequencing adapters were removed from the read FASTQ files using NGmergehttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE161038, token= klufacscdfurnyv).Raw and processed data files have been deposited into GEO as entry GSE161038. .Samples in Trizol were thawed on ice and RNA was isolated using the RNeasy Plus Universal Kit with RiboZol RNA extraction reagent (Amresco), according to manufacturer’s instructions. All nucleic acid extracts were treated with gDNA Eliminator Solution for 15 s at 37 °C in order to remove contaminating chromosomal DNA. The resulting RNA was analyzed for quantity and quality with a NanoDrop spectrophotometer ND-1000 (Isogen Life Science) 31 was utilized to quantify the transcript abundances of the RNA-Seq samples with a RefSeq mm9 transcriptome build index. The tximport (v1.8.0)32 package was run in R (version 3.6.2) to create gene-level count matrices for use with DESeq2 (v1.2.0)33 by importing quantification data obtained from Kallisto. DESeq2 was then used to generate FPKM values per kilobase of gene length per million mapped fragments in each tissue sample with comparison of vitamin D–deficient replicates with 3 x D treated replicates. Two kinds of data were outputted from DESeq2: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE133949, token=kvaduyksbvindeb).Raw and processed data files have been deposited into GEO as entry GSE133949.  and compared with gene expression data obtained from an RNA-seq assay of various intestinal regions in juvenile and adult mice.Enriched motifs were identified within VDR ChIP-seq peaks using HOMER findMotifsGenome.pl2D3-mediated differential expression at 10% FDR were used for analysis so that subanalyses for upregulated or downregulated transcripts would contain > 200 differentially expressed genes. Analyses were conducted for pathway, GO processes, and protein class enrichment as well as transcription factor interactome analysis using a 5% FDR cut-off for significance. A list of all genes determined to be “present” in the intestinal compartments was used as the background gene set for analyses. The top 50 pathways or processes from each analysis were downloaded and lists for the three compartments were integrated for interpretation.Functional analysis of differentially expressed gene lists was conducted with the MetaCore analysis tool (Clarivate). Gene lists from the various compartments with 1,25(OH)GSE133949 (RNA-seq), GSE161038 (VDR ChiP-Seq), and GSE134579 (ATAC-seq). In addition, summarized lists of differentially expressed genes and tables of the functional analysis the genomics data are presented in the supplementary materials as tables.Raw and processed data files for the genomics experiments have been deposited into GEO as This article contains The authors declare that they have no conflicts of interest with the contents of this article.


The expression of SLIM1-dependent genes in sulfur deficiency appeared to be additionally induced by a high concentration of either mannitol or glucose, but also by sucrose, which is not only the source of glucose but another signaling molecule. Additionally, SLIM1 affects PAP1 expression during sulfur deficiency by directly binding to its promoter. The lack of PAP1 induction in such conditions leads to much lower anthocyanin production. Taken together, our results indicate that SLIM1 is involved in the glucose response by modulating sulfur metabolism and directly controlling PAP1 expression in Arabidopsis during sulfur deficiency stress.The homeostasis of major macronutrient metabolism needs to be tightly regulated, especially when the availability of one or more nutrients fluctuates in the environment. Both sulfur metabolism and glucose signaling are important processes throughout plant growth and development, as well as during stress responses. Still, very little is known about how these processes affect each other, although they are positively connected. Here, we showed in Arabidopsis that the crucial transcription factor of sulfur metabolism, SLIM1, is involved in glucose signaling during shortage of sulfur. The germination rate of the  PAP1/MYB75expression to induce sucrose-dependent anthocyanin biosynthesis, and hexokinase1 participates in signaling glucose levels through SLIM1 to transcriptional machinery.During sulfur deficiency, the SLIM1 transcription factor directly and positively regulates  Plants are continuously challenged with environmental variables and therefore need to balance the use of resources between growth processes and adaptation to adverse conditions. One of the most essential compounds in this process is sugar, which has both trophic and signaling activities. Sugar metabolism must be carefully coordinated with environmental cues and different developmental stages . Most imsir1-1 mutant with reduced sulfite reductase activity, sulfide availability is diminished, TOR activity is abolished, and glucose content is significantly lower than that in wild-type Arabidopsis  kinase activity . In the gin2-1) mutant, which is defective in HXK1 function, together with a substantial reduction of glucosinolate level, indicates an interaction between HXK1 and MYB factors in the regulation of glucosinolate biosynthesis. Glucose-specific induction of glucosinolate accumulation is also evident from the fact that fructose and mannose are ineffective in the stimulation of glucosinolate synthesis  and PHOSPHATIDYLINOSITOL MONOPHOSPHATE 5-KINASE 9 (PIP5K9), repressing and enhancing their expression, respectively. In turn, PAP1 binds directly to the promoter of CINV1 and induces its expression, while PIP5K9 interacts with and inhibits CINV1. CINV1 hydrolyses sucrose, releasing the glucose signal, which negatively regulates the stability of EIN3 via HXK1, thereby allowing further accumulation of CINV1. This regulatory mechanism participates in the synchronization of sucrose catabolism with growth and development requirements.The rising awareness of the importance of proper plant sulfur nutrition has resulted in substantial growth in our understanding of sulfur metabolic pathways and their regulatory elements. However, there are many issues to clarify how sulfur metabolism is regulated in response to the changing supply and the internal demand given the plant developmental program and environmental conditions. SULFUR LIMITATION1 (SLIM1), an ethylene insensitive 3-like (EIL) family transcription factor, plays a central role in inducing transcriptional responses whenever more sulfate is needed . EIN3, tS-transferase mutants to accumulate anthocyanin in vacuoles . Deletion in the SLIM1 gene was generated using the CRISPR/Cas9 method (SLIM1 locus in Arabidopsis (SLIMsgRNA1: CTATGTCCGTAGCAGACATC and SLIMsgRNA2: GGGGTAATAACGCTGACAGG), and were assembled in a modified pEn-Chimera. The sgRNAs were then combined with Cas9 nickase by Gateway® cloning into the pDe-Cas9 binary plasmid. The construct was transformed into Arabidopsis by Agrobacterium tumefaciens-mediated transformation  and Bensheim, respectively, were a kind gift of Prof. Leszek Kleczkowski.Arabidopsis Columbia ecotype (Col-0) was used as the parental line for the ormation . The conp method . gin2/hxArabidopsis seeds were surface-sterilized using a vapor-phase seed sterilization method . Seedling for 2 min and the supernatant was used for photometric measurements. To quantify anthocyanin in the samples the following equation was used: (A530 – 0.25A657)/M (g) = relative units of anthocyanin (where A is absorption and M is plant fresh weight)  HCl) was added and the samples were incubated overnight with moderate rotation at 4 °C in the dark. Next, 400 µl of deionized water was added, followed by chlorophyll extraction with 400 µl chloroform. The homogenate was cleared by centrifugation at 13 400  weight) .PAP1 open reading frame was ligated into the SacI and MluI sites of the pHIS2.1 vector , upstream of the HIS3 minimal promoter. Two short DNA sequences present in the PAP1 promoter and matching cis binding sites for EIN3 or SLIM1  assay, a 958 bp DNA fragment harboring sequence upstream or SLIM1  flankingTUBULIN 3 gene  was selected as an internal control to normalize the quantity of total RNA present in each sample. Stable expression of TUA3 under all experimental conditions was confirmed by its steady Cq values in qRT-PCR   (TIP41 (At4g34270)  and melting curve analysis following qRT-PCR. The reaction mixture (6 µl) contained 3 µl of DyNAmo Color Flash SYBR Green Master Mix (Thermo Fisher Scientific), 0.6 µM each of forward and reverse primers, and 1 µl of cDNA template. qRT-PCR was carried out in triplicate  of three to four biological repeats to ensure the reproducibility of the results. Relative gene expression levels were calculated using the ΔΔCt method  according to the manufacturer’s protocol . For qua, n=180)  but also4g34270) . All prit method .O-acetylserine (OAS) levels were determined after derivatization using an amino acid analysis protocol  have been introduced by PCR with mutated primers. To ensure an equal physiological state, all leaves were infiltrated with a control  and the tested interaction (right side of the leaf). Three to four discs of 10 mm diameter were cut from the infiltrated leaf and used directly for staining or frozen and later used for a quantitative assay. All oligonucleotides and plasmids used in the assay are described in Qualitative and quantitative assays of β-glucuronidase (GUS) activity were performed as described (slim1_KO versus Col-0) was tested by a two-tailed, unpaired Student’s t-test in Microsoft Excel. The statistical significance of group differences was assessed with Fisher’s least significant difference (LSD) test of two-way ANOVA using Statistica 12 software . Differences were considered significant at a P-value <0.05. Asterisks indicate significant differences between values of two genotypes within one treatment . All experiments were independently repeated at least three times.The statistical difference between the two genotypes  (SALK_089129) was recently compared with those original slim1 mutants and displayed similar defects in the transcriptional response to sulfur deficiency, although its phenotype was not described (SLIM1 deletion mutant that was generated by applying CRISPR/Cas9 technology. The genomic region covering the deletion was amplified and sequenced. The deletion starts from the 10th residue of the SLIM1 protein with the open reading frame reconstituted from residue 310 up to 567 (stop codon) (SLIM1 coding sequence from slim1_KO to check its ability to activate reporter gene expression in a Y1H assay using a construct containing the SLIM1 binding site (UPE-box) .Several p codon) . We presve media . HoweverO mutant . Additioslim1_KO mutant displays a clear phenotype when grown in sulfur-deficient conditions, which is even more severe than that observed in SALK_089129 and slim1-2 (SLIM1 coding sequence under the control of its native promoter to the slim1_KO mutant (slim1_KO mutant when exposed to short 2-day sulfur starvation. We followed the expression of the ‘OAS cluster’ genes and β-GLUCUSIDASE 28 (BGLU28), which are known to be mostly dependent on SLIM1 transcriptional activity  and sulfur-deficient (dS) conditions  when plated on medium with 5% glucose and no sulfate. The slim1_KO mutant also showed sensitivity to osmotic stress, as it had a lower germination rate in the presence of 5% mannitol with no sulfate limitation  and RESPONSE TO LOW SULFUR 1 (LSU1)  diurnal fluctuations, but they are also on the list of genes that are regulated by SLIM1 ), (ii) a HXK1 enzymatic activity-dependent pathway (PATHOGENESIS-RELATED GENE 1 (PR1), induced by unknown metabolite(s) downstream in the glycolytic pathway), and (iii) a HXK1-independent pathway (gene encoding the large subunit of ADP-Glucose Pyrophosphorylase (APL3) involved in starch biosynthesis)  sulfur starvation on the expression of genes in 7-day-old seedlings grown in the presence of high glucose (3%) . PAP1 waand LSU1 .HXK1 expression in the slim1_KO mutant, the role of HXK1 in this process was assayed. The changes induced by glucose can be eliminated or exaggerated by decreasing or increasing the amount of the glucose sensor HXK in transgenic Arabidopsis plants  sulfur deficiency on gene expression in 7-day-old seedlings grown in the presence of high glucose (3%) , similarPAP1 is induced by sulfur deficiency, and this effect seems to be dependent on SLIM1. Because the PAP1 gene was previously described as sucrose dependent in the process of anthocyanin biosynthesis induction in Arabidopsis  within the promoter of PAP1, which is only 21 nucleotides downstream of the published EIN3 binding site   and check the GUS expression. Co-expression with SLIM1 showed GUS activity at the level of control, i.e. unmutated promoterPAP1::GUS reporter gene expressed with no effector (promPAP1m2). As expected, no visible differences in GUS activity were noticed between promPAP1m2 co-expressed with EIN3 and control, i.e. unmutated promoterPAP1::GUS reporter gene expressed with no effector (Our findings suggest that SLIM1 directly and positively regulates t codon) , indicateffector . Alongsieffector .PAP1 promoter (958 bp upstream of the start codon) appeared to be able to autoactivate the reporter gene, probably due to the interaction with yeast transcription factor(s)  . Therefoing site . This unSLIM1 has been described in Arabidopsis as a crucial transcription factor governing the expression of dozens of genes during sulfur deficiency. Moreover, its role in cadmium and arsenic resistance and signaling has recently been underlined  of targeted genes, while the SALK_089129 T-DNA insertion in the 5ʹ-UTR of the SLIM1 coding sequence could reduce the SLIM1 protein levels. In contrast, the truncated SLIM1 protein produced in the slim1_KO mutant contains only the C-terminal part and is unable to bind to the DNA   . Surprisslim1 mutants is the inhibition of germination by glucose during dS conditions. Environmental factors, such as light, temperature, water, and nutrients, primarily regulate seed germination via the metabolism and signaling pathways of gibberellin, and abscisic acid (ABA), with ABA promoting and maintaining dormancy and gibberellin promoting germination  cannot produce enough H2S to cope with osmotic stress, hence the observed sensitivity to mannitol.It is noticeable that the 5% mannitol that served in our experiments as an osmotic control to glucose treatment exerted a comparable effect on ‘OAS cluster’ gene expression. Similarly, ynthesis   and the 19S regulatory particle of proteasome subunit (RPT5B), and directly controls the transcription of specific target genes independent of glucose metabolism .Fig. S5. Phenotypes of PAP1 expression in slim1-2 and SALK_089129 lines under sucrose treatment.Fig. S6. Phenotypes, anthocyanin content, and Table S1. List of oligonucleotide primers and constructs used in this study.Table S2. Gene-specific primers used for qRT-PCR.slim1_KO mutant.Table S3. Metabolite contents in the shoots of the wild-type and the erac371_suppl_Supplementary_Figures_S1-S6_Tables_S1-S3Click here for additional data file.