MYC expression are regulated. We find that P1 and P2 can be differentially regulated across cell-types and that their selective usage is largely mediated by distal regulatory sequences. Moreover, we show that in colon carcinoma cells, Wnt-responsive enhancers preferentially upregulate transcription from the P1 promoter using reporter assays and in the context of the endogenous Wnt induction. In addition, multiple enhancer deletions using CRISPR/Cas9 corroborate the regulatory specificity of P1. Finally, we show that preferential activation between Wnt-responsive enhancers and the P1 promoter is influenced by the distinct core promoter elements that are present in the MYC promoters. Taken together, our results provide new insight into how enhancers can specifically target alternative promoters and suggest that formation of these selective interactions could allow more precise combinatorial regulation of transcription initiation.In Metazoans, transcription of most genes is driven by the use of multiple alternative promoters. Although the precise regulation of alternative promoters is important for proper gene expression, the mechanisms that mediates their differential utilization remains unclear. Here, we investigate how the two alternative promoters  that drive  The core promoter plays an essential function in integrating multiple transcriptional signals, including tissue-specific enhancer mediated activation to modulate the rate of transcription initiation ,2,3. Thets (DPE) . These dts (DPE) ,6, and tequences ,8,9,10. Numerous reports have shown that in metazoans, most genes are under the control of multiple alternative promoters ,12,13,14MYC is a key transcription factor that has been shown to regulate the expression of thousands of genes involved in a wide-range of key cellular processes including cell growth, proliferation, and metabolism [MYC, it is not surprising then that MYC expression needs to be tightly regulated, especially at the transcription level [MYC in most cells [MYC promoters integrate multiple regulatory signals, including hundreds of tissue-specific enhancers [MYC transcription. In this report, by analyzing CAGE data, we find that the two MYC promoters can be differentially regulated across different human cell samples. Importantly, using synthetic reporter assays, we found that the activity of the MYC promoters embedded in transgenic constructs does not recapitulate the differential promoter usage observed in the context of the endogenous MYC locus, suggesting that promoter usage is likely to be regulated by distal regulatory elements. This led us to test in colon carcinoma cells if distal MYC enhancers that are responsive to Wnt signaling could differentially regulate the endogenous activity of the two promoters. We observed that Wnt-responsive enhancers preferentially activate the P1 promoter upon Wnt induction. In addition, by using CRISPR/Cas9, we confirmed that enhancer deletion preferentially downregulates the activity of the P1 promoter. Finally, we demonstrated that preferential activation of the P1 promoter is mediated by distinct promoter elements associated with P1 that differ from P2. Taken together, these results suggest that alternative promoters can mediate cell-type selective transcription regulation by facilitating selective interactions with distal and proximal Wnt-responsive enhancers.tabolism ,30. Giveon level ,32. Two nhancers ,34,35,36nhancers ,37,38,39MYC locus was obtained from the FANTOM5 database (http://fantom.gsc.riken.jp) [https://bit.ly/2JmEnVR) [MYC P1 and P2 promoters in a 11 bp window around the TSS. A threshold of 0.1 cpm. was set for each promoter before we determined the frequency distribution of the log2 (P2/P1). Similarly, for the time courses, we accessed the Zenbu data viewer human time course (https://bit.ly/2HpU9Pd) [2 (P2/P1) promoter usage ratio at the different time points. The protocols describing the time courses and specific cell lines used are described by Arner et al. [The CAGE profile of the iken.jp) . We acce2JmEnVR)  to obtai2HpU9Pd) , to obtar et al. .2. For HCT-116 cells, Wnt induction was achieved by treating the cells with LiCl solution at different concentrations for 6 h  or 24 h (for luciferase reporter assays).All cell lines used in this study were cultured in accordance with ATCC and Riken Cell bank guidelines in their suggested media supplemented with 10% fetal bovine serum in a humidified incubator at 5% COCells were plated into 96 well E-plates  and grown in a xCELLigence-RCTA SP system . Cells were seeded in quadruplicates for all conditions. In addition, wells with only media were used as a control. The measurement was taken every 10 min for 120 h. We fitted the growth curve using a logistic function with three characteristic parameters: the length of lag phase, the generation time and the maximum cell index.To quantify the P1 and P2 promoter activities, we developed a PCR assay with primers immediately downstream of the P1 promoter (5′-CTTGGCGGGAAAAAGAACGG-3′ and 5′-AGTTAGATAAAGCCCCGAAAACC-3′) and the P2 promoter (5′-AGCGAATAGGGGGCTTCGC-3′ and 5′-TCGTGGATGCGGCAAGGGTT-3′). In addition, we used a 264 bp DNA sequence that could be amplified by both sets of primers to determine the amplification efficiency and the relative transcription from the P1 and the P1 + P2 promoter, and sub sequentially, the transcription from the P2 promoter.MYC promoters P1  or P2  was cloned between XhoI and HindIII sites to generate the promoter alone constructs. Furthermore, to test the effect of the Wnt pathway of the fold activation, a Wnt responsive enhancer consisting of 8 TCF7L2 [Luciferase reporter constructs were generated using pGL3-Basic luciferase reporter vector. A 150 bp stretch of the 8 TCF7L2  or a conhttp://bioinfo.ut.ee/primer3-0.4.0/) [Total RNA was isolated from HCT-116 cells and other cell lines using RNeasy Kit , complementary DNA (cDNA) was generated using Maxima First Strand cDNA Synthesis Kit  and qPCRs reactions were prepared using SYBR FAST qPCR Mix  and ran in a CFX96 Real-Time PCR Detection System . In all cases, manufacturer guidelines were followed to optimize the amplification conditions. Primer sequences used for Real time quantitative PCRs were designed with Primer3 online tool (-0.4.0/)  and are http://crispr.mit.edu/) [To generate enhancer deleted HCT-116 clones, guide RNAs (gRNAs) were designed using the online CRISPR design tool (it.edu/)  and clonMYC promoters might be differentially deployed, we surveyed P1 and P2 promoter usage across 869 human cell types and tissues from the FANTOM database [MYC gene across the different cell types in agreement with previous reports [CAGE = (0.37 to 34.24)). This suggested that multiple regulatory sequences, including cell specific enhancers and proximal promoter elements, may be important to differentially regulate the activity of the tandem MYC promoters.To gain a better understanding of how the two alternative database   and this ratio was maintained across different cell lines ), especially when compared to the endogenous CAGE values. This result suggests that regulation of the two MYC promoters outside of their genomic context does not recapitulate the broad range of P2 to P1 promoter usage ratio observed for endogenous MYC in their native chromatin context. This finding suggests that distal cis-regulatory elements are likely to be, at least in part, responsible for differential activation of MYC promoter P2 versus P1. Based on these preliminary observations, we investigated whether the in vivo promoter usage could be explained by differential promoter activation as a consequence of enhancer bias, derived from the transcriptional network. For this, we measured the independent activity of the P1 and P2 promoters by determining the activity of luciferase constructs harboring the core P2 or P1 promoter sequences across eight cell lines with different P2/P1omosomes C. These MYC expression, since the total levels of MYC decreased in both time courses over time  profiles (MYC promoters, we induced the Wnt pathway by treating the cells with different concentrations of LiCl [MYC mRNA in a LiCl dependent manner in HCT-116 cells (MYC promoter P0 didn’t drive MYC expression in this cell line, no endogenous transcription was detected and the promoter showed extremely low activity by luciferase assays (To investigate if distal regulatory elements could specifically regulate the differential usage of pression . These Wprofiles A. To tes of LiCl , which p16 cells . Importae assays . More ine assays C, whereae assays D. This rMYC promoters, we developed a system to independently quantify the fold activation of the P1 and P2 promoters by the presence or absence of the Wnt-responsive enhancer. We used the previously generated core P1 and P2 promoters to clone a Wnt-responsive enhancer or control sequence. The Wnt-responsive enhancer consists of 8 strong TCF7L2 binding sites, whereas the control sequence has the 8 TCF7L2 binding sites mutated making it unresponsive to Wnt [To further probe how Wnt-responsive enhancers may regulate the e to Wnt . We obsee to Wnt . Taken tMYC promoters. Five enhancers were selected across the topological domain located at approximately 1 Kb, 7 Kb, 335 Kb, 405 Kb and 550 Kb from the MYC gene body based on their strong binding profiles for TCF7L2 and RNA Pol II. Some of these enhancers have been previously shown to form with the MYC gene by capturing Hi-C and regulating promoter activity by luciferase assays [MYC mRNA, we found that in four cases, the deletions caused significant downregulation of total MYC mRNA levels (MYC mRNA levels caused by the single enhancer deletions was accompanied by defects in cell growth (MYC regulation across the cell cycle. When the specific activity of the two alternative MYC promoters was measured, we observed that the enhancer deletions preferentially downregulated transcription initiation from the P1 promoter (MYC.Next, we investigated the effect of deleting Wnt-responsive enhancers on the two alternative e assays ,48,49,50A levels C. Downrel growth , suggestpromoter D, while promoter E. In sumMYC core promoters. Interestingly, we observed that the two alternative MYC core promoter possess rather distinct core promoter element composition and disposition (MYC promoters are known to be highly conserved in mammals [Previous studies have shown that enhancer-promoter specificity can be mediated by the presence or absence of different core promoter elements ,9,10. Toposition A. Import mammals  (SupplemMYC promoters. We first tested the role of the BREu on Wnt-dependent activation, which is only present within the P1 promoter. We observed that mutation of the BREu motif in P1 had little effect on its basal promoter activity (u motif on the P1 promoter was crucial for Wnt-responsive enhancer-mediated activation (u motif into P2 had little effect on its basal activity, but increased its maximum fold activation (u motif suggests that this is a promoter element that potentiates the capacity of P1 to be selectively activated by Wnt-responsive enhancers.To determine the basis for the preferential activation of P1 upon Wnt-induction, we investigated the role of the different core promoter elements that are present within the two activity B. In contivation C. Furthetivation . The funLastly, we tested the role of INR and DPE motif, only present in the P2 promoter, may have on the promoter capacity to be activated by the Wnt-responsive enhancers. We generated P1 promoter containing either a consensus INR or DPE motif. The basal activity of these promoters was increased 8-fold and 2-fold for the INR and DPE P1 promoter variants, respectively D. IntrigMYC core promoters, further expanding upon the early reports which have focused on MYC proximal promoter elements that regulated transcription factor binding or mRNA stability [MYC promoters, P1 and P2, are differentially deployed across different tissues and cell types, and those distal regulatory elements are necessary for selective regulation of alternative core promoters, in expanding the repertoire of possible mechanisms that could regulate MYC transcription [The usage of alternative promoters as drivers of transcription initiation is a prevalent phenomenon that occurs throughout metazoans ,48. Althtability ,37,38,51cription . In addiu motif potentiates activation of P1 independent of its basal activity. And second, the presence of INR or DPE motifs apparently limits the range of enhancer dependent promoter activation by increasing the basal activity of the core promoter. This means that in HCT-116 cells, the P2 promoter serves as a basal promoter, largely insensitive to Wnt-responsive enhancers, whereas the P1 promoter can be fine-tuned by Wnt-responsive enhancers. The presence of these two differentially regulated promoters could be important for precise regulation of transcription initiation, especially for the genes that are tightly regulated, such as MYC.Previous studies have reported that certain promoter elements present within the core promoter architecture can be important to specify enhancer-promoter communication ,10,52. HMYC gene is exquisitely regulated at the transcriptional level, with more than 270 enhancers annotated in the enhancer atlas to regulate MYC, of which, dozens have been functionally validated [MYC core promoters would significantly expand the network and specificity of possible long distance enhancer/promoter interactions that are necessary to mediate different temporal and spatial transcriptional outcomes.Multiple mechanisms are thought to regulate the specific formation of enhancer-promoter interactions that include chromosome topology, changes in the epigenetic chromatin landscape and biochemical compatibility ,55,56,57alidated ,35,36,59


Heterochromatin, characterized by histone H3 lysine 9 (H3K9) methylation, assembles on repetitive regions including centromeres. Although centromeric heterochromatin is important for correct segregation of chromosomes, its exact role in maintaining centromere integrity remains elusive. Here, we found in fission yeast that heterochromatin suppresses gross chromosomal rearrangements (GCRs) at centromeres. Mutations in Clr4/Suv39 methyltransferase increased the formation of isochromosomes, whose breakpoints were located in centromere repeats. H3K9A and H3K9R mutations also increased GCRs, suggesting that Clr4 suppresses centromeric GCRs via H3K9 methylation. HP1 homologs Swi6 and Chp2 and the RNAi component Chp1 were the chromodomain proteins essential for full suppression of GCRs. Remarkably, mutations in RNA polymerase II (RNAPII) or Tfs1/TFIIS, the transcription factor that facilitates restart of RNAPII after backtracking, specifically bypassed the requirement of Clr4 for suppressing GCRs. These results demonstrate that heterochromatin suppresses GCRs by repressing Tfs1-dependent transcription of centromere repeats. Akiko Okita et al. demonstrate that heterochromatin suppresses gross chromosomal rearrangements by repressing Tfs1/TFIIS-dependent transcription of repetitive sequences. This study underscores the role of heterochromatin for maintaining chromosome stability. The presence of repetitive elements is a threat to genome stability. Recombination events such as crossover and break-induced replication (BIR) between repetitive elements give rise to gross chromosomal rearrangements (GCRs), which cause cell death and genetic diseases including cancer3. Most of the repetitive elements, including centromere repeats, are present in heterochromatin domains and transcriptionally silenced4. Transcriptional de-repression of repetitive elements  has been observed in a variety of cancers6, suggesting a link between GCRs and transcription of repetitive elements.Repetitive DNA elements such as centromere repeats and transposable elements are prevalent in eukaryotic genomes and occupy at least 50% of the human genome7. A clr4 deletion increases RNA polymerase II (RNAPII) localization and de-represses transcription at centromere repeats8, demonstrating that H3K9 methylation causes transcriptional silencing. The H3K9 methylation mark is recognized by chromodomain proteins such as Heterochromatin Protein 1 (HP1)10, which creates phase-separated compartments in the nucleus11. RNA interference (RNAi) that utilizes small RNAs mediates heterochromatin assembly13. In fission yeast, the RNA-induced transcriptional silencing (RITS) complex, which consists of small RNAs, Ago1, Chp1, and Tas3, localizes to the centromeres through the Chp1 chromodomain protein and Ago1 that captures small RNAs18. The RITS complex recruits the Clr4-Rik1-Cul4 (CLRC) complex and facilitates H3K9 methylation at the centromeres. In addition to RNAi, the exosome-dependent RNA degradation also contributes to transcriptional silencing. Cid14 is an essential component of the Trf4/Air2/Mtr4 polyadenylation (TRAMP) complex that promotes exosome-dependent degradation of RNAs including centromere transcripts19. Mlo3 RNA-binding protein, the homolog of budding yeast Yra1 and mammalian Aly/REF, is required for the export of poly(A)+ RNA from the nucleus22. Yra1 directly binds to the C-terminal domain of RNAPII23, facilitating the transcription-coupled loading of RNA export factors. Like RNAPII, Mlo3 localizes to the gene body of the euchromatin, and it binds to centromere repeats in the absence of Clr424. Mlo3 also interacts with Cid14 and facilitates the exosome-dependent RNA degradation24. Loss of either Mlo3 or Cid14 restores H3K9 methylation in ago1∆ cells25, probably via the recruitment of the CLRC complex to non-degraded nascent transcripts at the centromeres.Heterochromatin is marked by histone H3 lysine 9 (H3K9) methylation that is catalyzed by specific methyltransferases such as fission yeast Clr4 and mammalian Suv3927. Robertsonian translocation that occurs around centromeres of acrocentric chromosomes is the most common type of chromosomal abnormality observed in humans 28. The formation of isochromosomes, whose arms are mirror images of each other, is mediated by inverted repeats at the centromeres in Schizosaccharomyces pombe and Candida albicans30. Heterochromatin ensures sister chromatid cohesion at the centromeres31 and prevents incorrect attachment of spindle microtubules to kinetochores32. However, heterochromatin is not always formed at the centromeres: heterochromatin assembly is sometimes lacking at the centromeres that are devoid of repetitive elements35. Heterochromatin may have an important role especially when the centromeres consist of repetitive elements.Centromeres play an essential role in the correct segregation of chromosomes. Centromeres comprise species-specific centromere repeats in many eukaryotes and are one of the fragile sites of the chromosomes. Chromosome breakages frequently occur at centromeres during tumorigenesis, and the centromere sequence and position change rapidly during the process of evolution36. Loss of H3K9 methylation in Caenorhabditis elegans increases instability of repetitive elements probably through the formation of RNA:DNA hybrids37. In fission yeast, heterochromatin appears to prevent replication fork collapse and DNA recombination at the centromeres39. Heterochromatin prevents DNA double-strand break formation at the centromeres in meiosis40. However, how heterochromatin affects GCRs between centromere repeats remains elusive.Heterochromatin plays an important role in the maintenance of genome integrity. Suv39 knockout mice exhibit chromosome aneuploidy and predisposition to develop cancerclr4∆ cells. Tfs1/TFIIS is the transcription factor that facilitates restart of transcription elongation when RNAPII is paused and backtracked on template DNA42. Strikingly, tfs1∆ specifically bypassed the requirement of Clr4 for GCR suppression, without changing chromatin binding levels of RNAPII. These data demonstrate that heterochromatin suppresses GCRs by repressing Tfs1/TFIIS-dependent transcription of repetitive sequences.Here, we found that heterochromatin suppresses GCRs at the centromeres of fission yeast. Deletion of Clr4 increased the formation of isochromosomes, whose breakpoints were located in centromere repeats. Amino acid substitutions in H3K9  also increased GCR rates, suggesting that Clr4 suppresses centromeric GCRs through H3K9 methylation. Mutations in the HP1 homologs, Swi6 and Chp2, and the RNAi component Chp1 synergistically increased the GCR rate, showing that both HP1 and RNAi machinery are required to suppress GCRs. Mutations in the C-terminal domain (CTD) of RNAPII impaired chromatin binding of RNAPII and reduced GCRs in clr4 gene and determined the rate of spontaneous GCRs29. We detected otherwise lethal GCRs in haploid cells, using an extra-chromosome ChL derived from chromosome 3 (chr3)43  were grown in the minimum medium supplemented with uracil and adenine (EMM + UA), and then plated onto YNB + UA and YNB supplemented with 5-fluoroorotic acid and adenine (5FOA + A) to count Leu+ and Leu+ Ura– colonies, respectively. The clr4∆ strain produced slightly fewer Leu+ colonies than wild type . Almost all Leu+ Ura– colonies were Leu+ Ura– Ade–. Using the numbers of Leu+ and Leu+ Ura– Ade– cells (see Methods), we determined the GCR rate by means of a fluctuation analysis44 and found that it was strongly increased by clr4∆ 43 Fig. . Cells hype Fig. , probablype Fig. . Leu+ Ur47. Like clr4∆, rik1∆ increased GCR rate  motif in the SET domain constitutes the binding site of S-adenosyl-L-methionine (SAM), which is essential for the methyl transfer49. It has been shown that single amino acid substitutions in the SET domain impair methyltransferase activity of recombinant Clr4 in vitro, but the mutant strains show residual levels of H3K9 methylation at centromeres in vivo9. To examine if the methyltransferase activity of Clr4 is required to suppress GCRs, we introduced alanine substitutions at the three evolutionally conserved residues, R406, N409, and H410, in the R/HφφNH motif of the SET domain  at the centromeres  nor arginine (mlo3KR) substitution in Mlo3 methylation sites significantly changed GCR rate  , and stained with ethidium bromide (EtBr)  occurs and the right side of cen3 gains an increased number of dg and dh repeats29. Around 6% of GCR products were translocations in this study, where the minimal medium was utilized , most of the GCR products formed in clr4∆ (30 out of 30) and rik1∆ (30 out of 32) strains were isochromosomes . Given the high rates of GCRs in clr4∆ and rik1∆ strains , whereas heterochromatin assembles on the flanking inverted repeats  Fig. 52. Loss ion Fig. 29,53,54.Br) Fig. . In wildBr) Fig. , wt #27.Br) Fig. , suggestBr) Fig. . Short-rBr) Fig. , indicatzed Fig. . In contins Fig. , these dTo see whether breakpoints are located in centromere repeats, GCR products were recovered from agarose gel and analyzed by PCR. In all samples examined, both sides of cnt3–imr3 junctions were amplified Fig. . Togethe57 . A previous study has also shown that swi6∆ does not significantly increase GCRs38. However, the swi6∆ chp2∆ double mutation increased GCR rate, showing that Swi6 and Chp2 redundantly suppress GCRs. Note that GCR rate of clr4∆ is 16-fold higher than that of chp2∆ swi6∆ (P < 0.0001), indicating that H3K9 methylation suppresses GCRs only partly through HP1 homologs. Deletion of Chp1, the chromodomain subunit of the RITS complex, increased GCR rate, suggesting that RNAi machinery is required for GCR suppression. swi6∆ chp2∆ and chp1∆ synergistically increased GCR rate to the level similar to that of clr4∆. Collectively, these results demonstrate that both HP1 homologs and RNAi component Chp1 are the chromodomain proteins that are essential for full suppression of GCRs.Clr4, Swi6, Chp2, and Chp1 bind to H3K9me2 and H3K9me3 through the chromodomain59. Dcr1 cleaves double-stranded RNAs to produce small RNAs. Loading of small RNAs onto Ago1 occurs in the Argonaute small interfering RNA chaperon (ARC) complex that contains Ago1, Arb1, and Arb260. Then, Ago1 bound to small RNAs forms the RITS complex with Chp1 and Tas3, and localizes to the centromeres through Chp1 and through base pairing between small RNAs and nascent transcripts at the centromeres16 , suggesting that Ago1 suppresses GCRs partly through the formation of ARC and RITS complexes. GCR rate of ago1∆ was also higher than those of rdp1∆ and dcr1∆ (P < 0.0001), probably due to a Dcr1-independent pathway of small RNA production that uses the exosome61. Analysis of GCR products formed in ago1∆ cells showed that most of them (15 out of 16) were isochromosomes whose breakpoints were located in centromere repeats . Most of the GCR products formed in cid14∆ ago1∆ cells were isochromosomes whose breakpoints were located in centromere repeats (14 out of 16 samples) . In contrast to cid14∆, mlo3∆ reduced GCR rate in ago1∆ cells  and at a non-centromere region (adl1). In wild type, H3K9me2 was specifically detected at dg, dh, and imr3, but not at adl1 , suggesting that intrinsic transcriptional activity of imr3 is low64. cid14∆ did not significantly change RNAPII levels in ago1∆ cells (P ≥ 0.24), suggesting that Ago1 acts downstream of H3K9me2 to reduce RNAPII localization at the centromeres. In contrast to cid14∆, mlo3∆ reduced RNAPII levels at dg and dh, consistent with previous reports25. Epe1 is a putative H3K9 demethylase that antagonizes heterochromatin assembly65. Loss of Epe1 restored H3K9me2 and reduced RNAPII chromatin occupancy and GCR rate in ago1∆ cells , suggesting that Mlo3 directly affects chromatin binding of RNAPII. These results suggest that the CLRC complex suppresses centromeric GCRs by repressing RNAPII.To examine whether repression of RNAPII suppresses GCRs even in the absence of H3K9 methylation, we deleted lls Fig. , showingtes Fig. . As expells Fig. . Repress69. Ser7 of the CTD is required for transcription of noncoding small nuclear RNAs in human cells70. Changing all the Ser7 to Ala, rpb1-S7A, reduces chromatin-bound RNAs and H3K9me2 levels at fission yeast centromeres72. To obtain the direct evidence that RNAPII is involved in centromeric GCRs, we created the rpb1-S7A strain that harbored ChL. Consistent with low levels of H3K9me272  imr3 RNAs , indicating that RNAPII chromatin binding per se does not cause GCR events. However, tfs1∆ slightly reduced the levels of centromeric noncoding RNAs in clr4∆ cells CRs Fig. .58. Like clr4∆, ago1∆ reduced H3K9 methylation and increased RNAPII localization, resulting in high incidence of isochromosome formation. However, GCR rate of ago1∆ was higher than that of clr4∆, showing that Ago1 has a Clr4-independent function in GCR suppression. A discrepancy between RNAi mutants  and clr4∆ phenotypes has been observed in the formation of uniparental disomy (UPD), a pair of homologous chromosomes originating from only one parent79. However, in contrast to the case of UPD, Ago1 may have a unique function to suppress GCRs, which is independent of other RNAi factors including Dcr1, Rdp1, Chp1, Tas3, Arb1, and Arb2. Human Ago1 directly binds to RNAPII80, and Drosophila Ago2 interacts with the negative elongation factor NELF and represses heat-shock genes under normal conditions81. Thus, Ago1 might directly affect transcription and suppress GCRs. This study provides the evidence that transcriptional repression is important to suppress GCRs between centromere repeats. Mlo3 as well as RNAPII localizes to the centromeres in clr4∆ cells24, and Yra1/Mlo3 directly binds to the CTD of Rpb1 in budding yeast23. mlo3∆ reduced chromatin binding of RNAPII and bypassed the requirement of Clr4 methyltransferase to suppress GCRs at the centromeres. In addition, mutations in the largest subunit of RNAPII, rpb1-S7A, also reduced RNAPII chromatin binding and bypassed the requirement of Clr4 for GCR suppression. Transcriptional repression by heterochromatin seems to be important to suppress homology-mediated GCRs not only at the centromeres but also at other chromosomal loci, e.g., subtelomeres82.The RITS complex recruits the CLRC complex and facilitates H3K9 methylation at the centromeres. Loss of Clr4 eliminates H3K9 di-methylation and tri-methylation and increases RNAPII localization at centromere repeats84. After backtracking, Tfs1/TFIIS cleaves nascent RNAs by enhancing the intrinsic nuclease activity of RNAPII and facilitates the restart of RNAPII42. Here, we found that tfs1∆ greatly reduced GCRs in clr4∆ cells. In contrast to GCRs, tfs1∆ did not significantly change the level of RNAPII chromatin binding and only slightly reduced the amount of centromeric noncoding RNAs, showing that neither RNAPII binding nor transcription per se induces GCRs. Interestingly, the effect of tfs1∆ on transcription levels was most prominent at imr3 repeats where the intrinsic transcription activity is low. Tfs1 facilitates passing through the transcription termination site and produces readthrough transcripts of the adl1 gene. We propose a model in which, with the aid of Tfs1, RNAPII competes with DNA-binding proteins that block transcription elongation  between centromere inverted repeats results in isochromosome formation88. Rad51 suppresses isochromosome formation by promoting non-crossover recombination at the centromeres53. In the absence of Rad51, GCRs occur in a manner-dependent on the crossover-specific endonuclease Mus8153, demonstrating that crossover is the mechanism of GCRs in rad51∆ cells. Tfs1-dependent transcription passing through pausing or termination sites may extend the length of RNA:DNA hybrids and produce R-loops  of clr4∆ cells , 5 g L−1 of ammonium sulfate , and 2% glucose. YNB medium was supplemented with 1 g L−1 of 5-fluoroorotic acid  and 56 mg L−1 of uracil to make 5FOA medium. Solid media contained 1.5% agarose . Yeast transformation was performed by the lithium acetate method. The transformants that contain the kanamycin, hygromycin, or nourseothricin resistance gene were selected on the media supplemented with G418 , hygromycin B , or clonNAT  at a final concentration of 100 µg mL−1. clr4-R406A,N409A,H410A (clr4-set), mlo3K165A,K167A (mlo3KA), and mlo3K165R,K167R (mlo3KR) mutant strains were created by the pop-in/pop-out gene replacement95: pTN1220 plasmid containing the wild-type ura4+ and mutant clr4-set genes was digested with NgoMIV and introduced into ura4-D18 mutant cells. pTN1179 containing ura4+ and mlo3KA and pTN1178 containing ura4+ and mlo3KR were digested with HpaI and introduced into ura4-D18 cells. Ura+ transformants were selected on EMM plates, and then, Ura– progenies resulting from ura4+ pop-out were selected on 5FOA plates. We performed PCR and DNA sequencing to confirm correct integration of the clr4 and mlo3 mutations.Fission yeast strains used in this study are listed in Supplementary Table ura4+ gene was introduced between HindIII–EcoRV sites of pBluescript II KS+ (Stratagene) to make pTN782. clr4-set, mlo3KA, and mlo3KR mutant genes were constructed by a two-step PCR method. From yeast genomic DNA, a 0.7 kb PCR fragment was produced using clr4-1 and clr4-NHR-F primers, and a 1.0 kb fragment using clr4-NHR-R and clr4-2 primers, independently. These partially overlapping PCR fragments were mixed and used for the 2nd PCR in the presence of clr4-1 and clr4-2 primers. A 1.4 kb SpeI–PvuII restriction fragment prepared from the 2nd PCR product was introduced between SpeI–NaeI sites of pTN782 to make pTN1220. A 2.0 kb genomic region that contains the mlo3+ gene was amplified using mlo3-1 and mlo3-5, and digested with XbaI at one site. A 1.9 kb restriction fragment with XbaI–blunt ends was introduced between XbaI–NaeI sites of pTN782 to make pTN1169. From yeast genomic DNA, a 1.0 kb PCR fragment was produced using mlo3-1 and mlo3-KA-R primers, and a 0.7 kb fragment using mlo3-KA-F and mlo3-4 primers. These partially overlapping PCR fragments were mixed and used for the 2nd PCR in the presence of mlo3-1 and mlo3-4 primers. A 1.0 kb SacI–XbaI restriction fragment of the 2nd PCR product that contains the mlo3KA mutation was introduced between SacI–XbaI sites of pTN1169 to make pTN1179. mlo3-KR-R and mlo3-KR-F primers were used in place of mlo3-KA-R and mlo3-KA-F to make pTN1178 that contains the mlo3KR mutation. A 9.6 kb XbaI–EcoRI fragment containing cen1 sequence from pRS14096 was introduced between XbaI–EcoRI sites of pUC19 to make pTN834. From yeast genomic DNA, a 1.7 kb region that contains a portion of dh was amplified using dh-1 and dh-2 primers. A 1.5 kb NheI–ClaI restriction fragment of the PCR product was introduced between SpeI–ClaI sites of pBluescript II KS+ to make pTN770. A 2.3 kb region that contains a portion of imr3 was amplified using otr3-2 and imr3-XhoI-R primers. A 1.7 kb PvuII–MfeI restriction fragment of the PCR product was introduced between HincII–EcoRI sites of pBluescript II KS+ to make pTN1226. A 0.9 kb region that contains a portion of the adl1 gene was amplified using adl1-F and adl1-R primers. A 0.9 kb XbaI–ApaI restriction fragment of the PCR product was introduced between XbaI–ApaI sites of pBluescript II KS+ to make pTN1227. A 2.1 kb region that contains a portion of the act1 gene was amplified using act1-F and act1-R primers. A 1.9 kb XhoI–EcoRV restriction fragment of the PCR product was introduced between XhoI–EcoRV sites of pBluescript II KS+ to make pTN1225. DNA sequencing confirmed that no mutations were introduced during PCR amplification.A 1.8 kb HindIII–SspI fragment containing the + and that of Leu+ Ura– cells, respectively. Leu+ Ura– colonies formed on 5FOA + A plates were incubated on EMM + UA plates and then replicated onto EMM + A and EMM + U plates to confirm Ura– and to inspect Ade+/–, respectively. The number of Leu+ Ura– Ade– cells indicative of GCR was obtained by subtracting the number of Leu+ Ura– Ade+ cells from that of Leu+ Ura– cells. Using the number of Leu+ cells and that of Leu+ Ura– Ade– cells in 10 mL of EMM + UA culture, we determined GCR rate by means of the fluctuation analysis44.Rates of spontaneous GCR were determined by the fluctuation analysis. Yeast cells were incubated for 6–9 days on EMM + UA plates, and 10 mL of EMM + UA medium was inoculated with a single colony formed on the EMM + UA plates. After 2-days incubation, cells were plated onto YNB + UA and 5FOA + A media. After incubation for 6–12 days, the number of colonies formed on YNB + UA and 5FOA + A plates were counted to determine the number of Leu+ Ura– Ade–) clones obtained from biologically independent experiments, chromosomal DNAs were prepared in 1.6% low melting agarose plugs  as described previously54. Chromosomal DNAs were separated in 0.55% Certified Megabase agarose gel  using CHEF-DRII system (Bio-Rad) under the following conditions. Broad-range PFGE: 1500 s pulse time at 2 V cm−1 for 42 h and then, 180 s pulse time at 2.4 V cm−1 for 4 h, at 4 °C in 1× TAE buffer . Short-range PFGE: from 40 to 70 s pulse time at 4.2 V cm−1 for 24 h, at 4 °C in 0.5× TBE buffer . After electrophoresis, DNAs were stained with 0.2 µg mL−1 of EtBr  and detected using a Typhoon FLA9000 .From parental and GCR , and Random primer labeling kit ver. 2  were used to prepare radioactive probe A. Radioactive signals were detected using BAS2500 phosphorimager (Fuji Film).After EtBr staining, agarose gel was irradiated with 300 mJ ultraviolet (UV) light using GS Gene Linker (Bio-Rad) for DNA fragmentation, and then soaked into 800 mL of alkaline buffer  for 40 min with gentle shaking to denature DNA. DNA was transferred to ClearTrans nylon membrane 0.45 µm  by capillary action in 25 mM sodium phosphate buffer (pH 6.5) and covalently attached to the membrane by 150 mJ UV irradiation. A 0.6 kb EcoRI–EcoRI fragment prepared from pTN755−1 of EtBr, and detected using a Typhoon FLA9000. PCR primers used in this assay are listed in Supplementary Table After separation of chromosomal DNA by PFGE, GCR products were recovered from agarose gel using a FastGene Gel/PCR Extraction kit . KOD FX Neo polymerase  was utilized to amplify cnt3–imr3 junctions, whereas Q5 polymerase  was used to amplify irc3. PCR products were separated by 1.7% Seakem GTG agarose gel  electrophoresis in 1× TBE buffer, stained with 0.2 µg mL54. 1.5 × 108 cells from log-phase cultures in YE media supplemented with leucine, uracil, adenine, and histidine (YE4S) were collected by centrifugation, and suspended in 60 mL of EMM. After the addition of formaldehyde  to a final concentration of 1%, the cell suspension was vigorously mixed for 15 min at room temperature. The cell suspension was further mixed for 5 min, after the addition of 3 mL of 2.5 M glycine to neutralize the crosslinker. Mouse antibodies against H3K9me297, H3K9me397, H3K9ac98, H3K14ac98, Rpb1 , and FLAG , and rabbit antibodies against histone H3  were used. Mouse and rabbit antibodies were attached to Dynabeads M-280 sheep anti-Mouse IgG  and Dynabeads M-280 sheep anti-Rabbit IgG , respectively. DNAs in whole-cell extracts and immunoprecipitates were quantified by real-time PCR using SYBR FAST  in a StepOnePlus real-time PCR system (Applied Biosystems). The primers used in ChIP are listed in Supplementary Table ChIP was performed as described previously53. From 1 × 109 log-phase cells grown in YE4S media, RNA was extracted by heating and freezing cells in the presence of phenol and SDS. 10 µg of total RNAs was suspended in 8.5 µL of MOPS buffer  supplemented with 8% formaldehyde, 50% deionized formamide, and 10 µg mL−1 EtBr, and heat denatured by incubation at 55 °C for 60 min. RNAs were separated by 1.0% PrimeGel agarose LE  gel/2.2 M formaldehyde electrophoresis in MOPS buffer. RNAs stained with EtBr were detected using a Typhoon FLA9000. After soaking the gel in 50 mM NaOH  for 20 min, RNAs were transferred to a ClearTrans nylon membrane 0.45 µm by capillary action in alkaline transfer buffer  and covalently attached to the membrane by 150 mJ UV irradiation. A 2.0 kb KpnI–KpnI fragment prepared from pTN834, a 0.9 kb NsiI–XbaI fragment from pTN770, a 1.7 kb PstI–XhoI fragment from pTN1226, a 0.9 kb XbaI–ApaI fragment from pTN1227, and a 1.9 kb XhoI–EcoRV fragment from pTN1225, were used to prepare radioactive DNA probes for the detection of dg, dh, imr3, adl1, and act1 RNAs, respectively. Radioactive signals were detected using a BAS2500 phosphorimager.Northern blotting was carried out as described previouslyt-test was performed using Excel (Microsoft).The two-tailed Mann–Whitney test and the two-tailed Fisher’s exact test were performed using GraphPad Prism version 6.0 g for Mac (GraphPad Software). The two-tailed Student’s Supplementary Information


Arabidopsis thaliana CELLULOSE SYNTHASE A4/7/8 (CESA4/7/8) are three non-redundant subunits of the secondary cell wall cellulose synthase complex. Transcript abundance of these genes can vary among genotypes and expression quantitative trait loci (eQTL) were identified in a recombinant population of the accessions Bay-0 and Shahdara. Genetic mapping and analysis of the transcript levels of CESAs between two distinct near isogenic lines (NILs) confirmed a change in CESA4 expression that segregates within that interval. We sequenced the promoters and identified 16 polymorphisms differentiating ShaCESA4 and BayCESA4. In order to determine which of these SNPs could be responsible for this eQTL, we screened for transcription factor protein affinity with promoter fragments of Bay, CESA4ShaCESA4, and the reference genome ColCESA4. The wall thickening activator proteins NAC SECONDARY WALL THICKENING PROMOTING FACTOR2 (NST2) and NST3 exhibited a decrease in binding with the ShaCESA4 promoter with a tracheary element-regulating cis-element (TERE) polymorphism. While NILs harboring the TERE polymorphisms exhibited significantly different CESA4 expression, cellulose crystallinity and cell wall thickness were indistinguishable. These results suggest that the TERE polymorphism resulted in differential transcription factor binding and CESA4 expression; yet A. thaliana is able to tolerate this transcriptional variability without compromising the structural elements of the plant, providing insight into the elasticity of gene regulation as it pertains to cell wall biosynthesis and regulation. We also explored available DNA affinity purification sequencing data to resolve a core binding site, C(G/T)TNNNNNNNA(A/C)G, for secondary wall NACs referred to as the VNS element. While a primary cell wall surrounds all plant cells, a secondary cell wall is also found in xylem cells responsible for water transportation, structural fibers, and cells that serve as an outside barrier to the external environment. These thick and relatively inflexible walls are composed of a complex of cellulose, hemicelluloses, and the polyphenolic polymer lignin. Cellulose is the most abundant fraction in the majority of tissues and exists as long unbranched β-1,4-linked glucan chains. Cellulose chains coalesce in parallel to form a single microfibril via hydrogen bonding and van der Waals forces. Depending on their density and nature of the commingling polymers and linkages, microfibrils can contribute to a matrix that ranges from fairly elastic to extremely rigid. Aside from the obvious functional virtues to plants, secondary cell wall rich in cellulose are a valuable feedstock for the pulp and paper and the biofuel industries . UnderstA. thaliana CESA family, while direct polymerization activity remains to be documented, all but CESA10 have been shown to be associated with cellulose biosynthesis (Cellulose is synthesized at the plasma membrane by multiple Cellulose Synthase A (CESA) proteins organized into rosette shaped complexes . The rosynthesis . Mutatioynthesis . All memynthesis . Additioynthesis . These aynthesis . Across ynthesis .A. thaliana first revealed the non-redundant nature of the secondary CESAs, as mutants harboring null mutations in any of the three secondary CESAs exhibited irregular cell walls and weaker stems  of CESA genes in Nicotiana benthamiana led to dwarfed phenotypes and reduced cellulose content  and Shakdara (Sha) recombinant inbred line (RIL) population , proCESA4Bay (539 bp), and proCESA4Sha (540 bp) were cloned and recombined with reporter genes. Promoter sequences and primers used are described in Supplementary Table Col were independently cloned according to Yeast one-hybrid protein–DNA interaction assays were conducted as previously described . The traE. coli strain BL21-AI (Invitrogen) transformed with pDEST15-GST:NST2 were grown in liquid media to an OD600 of 0.4, treated with 0.2% L-arabinose to induce expression overnight and harvested by centrifugation the following day. Cells were treated with 1 mg/mL lysozyme on ice for 30 min in minimal volume of 1X PBS buffer and lysed by sonication. Cell lysates were clarified by centrifugation and incubated with 100 μL of glutathione sepharose beads  for 30 min at 4°C with rotation. The beads were transferred to a column, washed with 10 volumes of 1X PBS. Protein was eluted in 100 mM Tris-HCl pH8.0, 100 mM NaCl and 3 mg/mL glutathione buffer and purified protein was re-suspended in 50% glycerol and stored at -80°C.To express recombinant NST2 or SND1 protein, coding sequences were cloned and fused to glutathione S-transferase tag in the pDONR211 vector and then transferred into pDEST15 (Invitrogen). Bay-C, CESA4Bay-D, CESA4Bay-E, CESA4Sha-C, CESA4Sha-D, CESA4Sha-ECESA4 promoter fragments using the same oligonucleotides described in Supplementary Table Three overlapping probes were generated for CESA4 locus in the F6 generation were identified  was quantified in 5 cm stems. Stem tissue was frozen in liquid nitrogen and pulverized with metal beads in a Retsch  Mixer Mill MM400. RNA extraction and cDNA synthesis was conducted as described above. Primers for CESA4 real-time PCR are described in Supplementary Table To develop NILs, RILs maintaining heterozygosity at the entified . One plaSpectraMax M5 and corrected to a known glucose standard to calculate the percent cell wall composition of cellulose.To quantify and compare levels of crystalline cellulose, the Updegraff assay as adapted and described by Kumar and Turner was used . BrieflyLeica Biosystems VT1000S Vibrating-blade microtome. Sections were incubated on the bench top for 2 min in a 2% w/v phloroglucinol/ethanol solution , mounted in a 1:1 concentrated hydrochloric acid: water solution, and immediately imaged using a Nikon Eclipse E200 microscope and a PixeLink scope camera. For each stem, three sections were imaged, and five cells from three different portions of each stem were measured to get an average of 45 measurements per biological sample.To investigate the histological effects of expression variation on the cell wall, 100 μm cross sections were sliced using a To qualitatively analyze cellulose crystallinity under polarized light, internode segments were first cut on a vibratome into 100 μm sections, fixed in 2% glutaraldehyde and then embedded in Epon/Araldite  before slicing into 0.5 μm sections on an Reichert-Jung, Ultracut E microtome . Polarized light microscopy takes advantage of variation in the passage of light through crystalline structures to uncover discrete differences in crystallinity configurations. Specifically, the LC-PolScope  employed for this assay uses polarized light to measure birefringent retardance and the intensity of the images generated directly correlates to the retardance value, thus qualitative differences in crystallinity can be observed. Several stem samples from each HIF were imaged and analyzed.A. thaliana TAIR 10 sequence assembly and for each DAP-seq set of binding sites. Sum of the counts determined the total number of VNS motif sites and percentages were calculated as the proportion of each motif variant relative to the sum and multiplied by 100. The process was repeated for the DAP-seq binding site data using the reference genome file as a guide to extract DAP-seq peak fasta sequences. ThenarrowPeak DAP-seq peak data files were then searched iteratively through each of the peak binding sites. The percentage shown for DAP-seq data represents the mean percentage across all binding site files.In order to determine a consensus VND, NST, and SND (VNS) protein binding motif, the MEME.txt motif files from CESA genes pairwise using replicate gene expression data for Bay-0 and Sha parental lines and RILs using the rcorr function from the Hmisc library in R 3.2.5. Two tailed Student’s t-tests were carried out in Excel to assess gene expression, cell thickness, and cellulose content data sets. BoxPlots were generated with the BoxPlotR Web Tool , CESA2 (At4g39350), CESA3 (At5g05170), CESA4 (At5g44030), CESA5 (At5g09870), CESA6 (At5g64740), CESA7 (At5g17420), CESA8 (At4g18780), CESA9 (At2g21770), CESA10 (At2g25540), NST1 (At2g46770), NST2 (At3g61910), NST3/SND1 (At1g32770), PP2AA3 (At1g13320), TIP41 (At4g34270).CESA1  or the Sha CESA4 promoter allele (ShaCESA4)  Figure . No diffa Figure .cis-eQTL include functional polymorphisms in the promoter of the gene in question, and we hypothesized that this change may disrupt interaction of SND1 or NST2, which we previously identified to interact with the CESA4 promoter using yeast one-hybrid  of BayCESA4 and ShaCESA4 promoters by yeast one-hybrid with ColCESA4 promoter interacting proteins  but only a single ShaCESA4 fragment (E). One SNP in this region disrupts the second position of a perfect TERE motif in ShaCESA4 fragment C  and HIF350, segregating for the CESA4 promoter interval that demonstrated differential binding. Transcript abundance in developing stems containing BayCESA4 was twofold greater than that of ShaCESA4 in both pairs of NILs  caused by constitutive expression using the CaMV35S promoter was only sometimes (25% of the time) associated with a compromised cell wall phenotype (CESA, CESA4, 7, and 8, resulted in lower transcript levels for all three endogenous secondary cell wall CESAs. Moreover, transcript levels of all three CESAs tended to correlate, regardless of which CESA cDNA was driven by the CaMV35S promoter, suggesting that regulatory mechanisms limited by synthase complex stoichiometry are at play (The presence of loss-of-function variance . The reshenotype . Transge at play .Co-regulation of the CSC subunits at the transcript level was not reported in HIFs for this study, but posttranscriptional regulation poses another possible mechanism to explain the lack of phenotypic effect. While transcript analysis is a powerful tool and can provide invaluable data on genome regulation, stress response, and more, mRNA accumulation alone is not enough to draw definitive conclusions about protein expression. Indeed, the global correlation between the transcriptome and proteome in both prokaryotes and eukaryotes has been found to be weak, at best . PosttraA number of studies have identified cases where protein expression of complex subunits is posttranscriptionally controlled and seemingly limited by complex stoichiometry; generally, mRNA levels of individual subunits have not been found to correlate with complex expression . For exaJO, LL, and SH conceived and designed the study. JO, LL, and GT acquired the data. JO, LL, SL, KM, and SH analyzed and interpreted the data. JO, LL, and SH drafted the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


COBLL1 are achieved by at least two distinct mechanisms. Furthermore, we discovered that EBNA3C interacts with the lysine demethylase KDM2B and that this interaction is important for its transcriptional repressive function. The findings in this study provide new insights into the mechanism used by the oncoprotein EBNA3C to repress cellular target genes.EBV is a virus associated with human cancers and is well known for its ability to transform B lymphocytes into continuously proliferating lymphoblastoid cell lines. EBNA3C is considered an oncoprotein and has been shown to be essential for B cell transformation by EBV. EBNA3C is well characterized as a viral transcription factor, but very little is known about its mechanisms of action. In the present study, we demonstrate that removal of the activating histone mark H3K4me3 and deposition of the repressive mark H3K27me3 by EBNA3C on  COBLL1, ADAM28, and ADAMDEC1, we investigated the mechanism of EBNA3C-mediated transcriptional repression. Chromatin immunoprecipitation (ChIP) analysis indicated that EBNA3C HDmut is still able to recruit Polycomb proteins BMI1 and SUZ12 to COBLL1 as efficiently as EBNA3C WT does, leading to the full deposition of the repressive histone mark H3K27me3. However, we found that the activation-associated chromatin mark H3K4me3 is highly enriched at EBNA3C target genes in LCLs expressing EBNA3C HDmut. We show here that EBNA3C interacts with the histone lysine demethylase KDM2B and that this interaction is important for H3K4me3 removal and for the EBNA3C-mediated repression of COBLL1 and the ADAM28-ADAMDEC1 locus.Epstein-Barr virus nuclear antigen 3C (EBNA3C) is a well-defined repressor of host gene expression in B cells transformed by Epstein-Barr virus (EBV) that cooperates with various cellular factors. It is established that EBNA3C interacts with the cellular factor RBPJ (RBP-Jκ or CBF1) through two distinct motifs: the TFGC motif, also called the homology domain (HD) motif, and the VWTP motif. In this study, we investigated the role of each motif in EBNA3C transcriptional repression activity by using two novel recombinant viruses with single RBPJ interaction motifs mutated (EBNA3C HDmut and EBNA3C W227S). Infection of primary B cells with either of these recombinant EBVs led to the successful establishment of lymphoblastoid cell lines (LCLs). Gene expression analysis showed that full repression of EBNA3C target genes is not achieved by EBNA3C HDmut compared to that with EBNA3C W227S or the EBNA3C wild type (WT). Focusing on the well-characterized EBNA3C-repressed genes IMPORTANCE EBV is a virus associated with human cancers and is well known for its ability to transform B lymphocytes into continuously proliferating lymphoblastoid cell lines. EBNA3C is considered an oncoprotein and has been shown to be essential for B cell transformation by EBV. EBNA3C is well characterized as a viral transcription factor, but very little is known about its mechanisms of action. In the present study, we demonstrate that removal of the activating histone mark H3K4me3 and deposition of the repressive mark H3K27me3 by EBNA3C on COBLL1 are achieved by at least two distinct mechanisms. Furthermore, we discovered that EBNA3C interacts with the lysine demethylase KDM2B and that this interaction is important for its transcriptional repressive function. The findings in this study provide new insights into the mechanism used by the oncoprotein EBNA3C to repress cellular target genes. In vivo, infection of mature B cells by EBV initially leads to their activation and differentiation into proliferating B blasts. These activated B blasts then migrate through germinal centers, where they further differentiate, resulting in resting memory B cells that carry the EBV genome as extrachromosomal episomes, forming long-lived reservoirs of EBV infection  resembling activated B blasts. LCLs express all EBV latency-associated genes, producing six EBV nuclear antigens , three latent membrane proteins , two small noncoding RNAs (EBER1 and -2), and microRNA transcripts from the BHRF1 and BamHI A (BART) regions, which act in concert to induce and maintain continuous proliferation (Epstein-Barr virus (EBV) is a large DNA virus that belongs to the gammaherpesvirus subfamily and persistently infects >90% of the human population. Despite being a ubiquitous virus, EBV is also one of the most transforming viruses identified. It is etiologically associated with a variety of B cell malignancies in humans, including Burkitt lymphoma (BL), Hodgkin's lymphoma (HL), and diffuse large B cell lymphoma (DLBCL) , 2. EBV nfection , 5. In vferation , 7.CDKN2A (coding for p16INK4A)  , 9. EBNA16INK4A) –13. Inte6INK4A) –, 14, 15,K4A) –16–. FurtherK4A) –16–.209TFGC212) within the homology domain of EBNA3C were identified as being important for the interaction with RBPJ. The TFGC motif is not a known RBPJ interaction motif. However, mutation of these residues to 209AAAA212 (HDmut) in EBNA3C destabilized its interaction with RBPJ as determined by coimmunoprecipitation (co-IP) , is conserved across species and is core to Notch binding of RBPJ  was required for an effective loss of RBPJ interaction , and on the ADAM28-ADAMDEC1 locus, which is regulated by both EBNA3A and EBNA3C. We demonstrated that EBNA3C-mediated repression of transcription involved a two-step mechanism—a rapid loss of activation-associated histone marks that led to repression of mRNA expression and then recruitment of Polycomb group (PcG) proteins and increased repressive histone H3K27me3 marks  –39. EBNA–poptosis , 42, 43.e3 marks .COBLL1 and ADAM28-ADAMDEC1, whereas the PRC2 subunit SUZ12 was found only at the TSS of COBLL1 (PcG proteins form two multiprotein Polycomb repressive complexes (PRC1 and PRC2). PRC1 and PRC2 are known to catalyze lysine 119 monoubiquitination of histone H2A (H2AK119ub1) and H3K27me3, respectively , 45. PRCf COBLL1 . Other Ef COBLL1 , 43, 48.Recent studies have also shown that the composition of these PcG complexes is considerably more diverse than previously thought, particularly for PRC1. Noncanonical PRC1 complexes have been discovered that are capable of binding to chromatin and that function in the absence of PRC2 , 50. One53–COBLL1, ADAM28, and ADAMDEC1 as models, we found that EBNA3C HDmut was unable to fully remove the activation-associated chromatin mark H3K4me3 from the transcription start sites of its target genes. Further characterization revealed that the interaction between EBNA3C and KDM2B was important for the rapid loss of H3K4me3 and subsequent repression of EBNA3C target genes.In the present study, following the construction and use of novel EBNA3C recombinant EBVs, we demonstrated that EBNA3C uses its TFGC motif (HD motif) to epigenetically repress gene expression. Using the well-characterized EBNA3C target genes 209AAAA212 and W227S mutations, giving a construct named EBNA3C RBPJ BM [21209TFGC212 mutated to 209AAAA212  36 days after primary B cell infection, showed that around 50% of EBNA3C W227S cells were synthesizing DNA, which was the same as the level for cells infected with either the wild-type (WT) or EBNA3C revertant  virus  virus . Interestant LCL . The EBNtant LCL . The levFinally, we investigated whether the newly generated EBNA3C mutants were impaired in the ability to interact with RBPJ. To do this, we performed co-IP assays with extracts of the WT LCL as well as LCLs 3C HDmut, 3C W227S, and 3C RBPJ BM . The co-+ B cells with different recombinant EBVs. Samples of cells were harvested at the time of infection and at 5-day intervals, and mRNA was extracted for analysis by reverse transcription-quantitative PCR (RT-qPCR). As expected, the EBNA3C knockout (3C KO) and EBNA3C RBPJ BM (3C RBPJ BM) viruses failed to regulate the COBLL1 mRNA level, whereas EBNA3C-competent viruses (WT and 3C Rev) resulted in a rapid reduction of COBLL1 gene expression over a period of 30 days after infection  . Furthermore, EBNA3C HDmut and EBNA3C W227S recruited RBPJ to the EBNA3C peak on both the COBLL1  assays were performed on LCLs WT, 3C HDmut, 3C W227S, and 3C RBPJ BM, followed by qPCR to determine the levels of EBNA3C protein bound to the ectively . We founectively , was stiBNA3C WT  and B. Ikground)  that hade COBLL1  and ADAML1  and its transcription start site (COBLL1 TSS), respectively. It is unclear whether BMI1 and SUZ12 are recruited by direct interaction with EBNA3C or if this represents a default mechanism of COBLL1 repression. The EBNA3C-mediated regulation of the ADAM28-ADAMDEC1 locus is different, as BMI1 recruitment increases at the ADAM peak in the first few weeks of repression and then quickly decreases, reaching the same start level as that found when EBNA3C is inactive  to target genes, leading to deposition of the repressive mark H3K27me3. Notably, it has clearly been shown that the PRC1 family member BMI1 and the PRC2 component SUZ12 are recruited to the inactive . FurtherH3K27me3 .COBLL1, as ChIP-qPCR experiments showed that BMI1 was efficiently recruited to the COBLL1 peak in the EBNA3C HDmut LCL , it was essential to verify whether this repressive mark was found around the 3C HDmut . It is iNever HT . Interes compare  and B.COBLL1 expression is repressed) revealed a loss of H3K4me3 at the COBLL1 TSS compared to the high H3K4me3 enrichment in the positive-control LCLs RBPJ BM and 3CHT Never HT, in which COBLL1 is expressed  or cDNA encoding EBNA3C WT, EBNA3C HDmut, or EBNA3C W227S were produced and used to infect LCL 3CHT Never HT. Analysis of protein levels by Western blotting showed that the levels of EBNA3C WT and EBNA3C mutants were similar to the level of EBNA3C found in LCL WT (COBLL1 mRNA (compared to that in mCherry control cells), whereas expression of both EBNA3C WT and EBNA3C W227S induced a decrease in COBLL1 expression , LCL EBNA3C HDmut, and LCL EBNA3C W227S were measured by Western blotting, and KDM2B was expressed at the same level across the different LCLs . An antiCOBLL1, ADAM28, and ADAMDEC1 is rapid, and it was previously shown that activation of EBNA3C in the conditional LCL 3CHT resulted in a reduction of COBLL1, ADAM28, and ADAMDEC1 mRNAs (with the COBLL1 reduction being more drastic than those of ADAM28 and ADAMDEC1) after only 5 days of HT addition to the culture medium  treated with HT.Finally, to assess the effect of KDM2B on the H3K4me3 level in LCLs, we performed a ChIP analysis using the LCLs 3CHT shNT, shKDM2B-1, and shKDM2B-2 left without HT (Never HT) or treated with HT for 5 days . As expeCOBLL1, ADAM28, and ADAMDEC1 TSS and that KDM2B is needed for efficient repression of COBLL1, ADAM28, and ADAMDEC1 by EBNA3C.Taken together, these results show, for the first time, that KDM2B is important for the removal of H3K4me3 from the 212 to AAAA212) was still able to recruit PcG proteins, as EBNA3C WT can, this mutant was found to be impaired in the capacity to efficiently remove the activation-associated chromatin mark H3K4me3 from its target genes.Although it is now well established that the Epstein-Barr virus protein EBNA3C is essential for transformation of primary B cells and for repression of host tumor suppressor genes, the precise molecular mechanisms by which EBNA3C silences cellular target genes are still poorly understood. In the present study, by using novel EBNA3C recombinant EBVs encoding EBNA3C with mutations in two previously described motifs important for RBPJ interaction, we explored the molecular mechanisms by which EBNA3C represses cellular gene expression. This revealed that the TFGC motif, located in the N-terminal homology domain of EBNA3C, is important for transcriptional repression of EBNA3C's cellular target genes. While EBNA3C HDmut  at its TSS followed by the deposition of a high level of the repressive mark H3K27me3 (histone 3 lysine 27 trimethylation), catalyzed by PRC2. Because EBNA3C-mediated gene repression at COBLL1 is known to involve the Polycomb protein complexes PRC1 and PRC2, we first decided to investigate whether EBNA3C HDmut was deficient in the recruitment of both complexes to COBLL1. EBNA3C HDmut still recruited BMI1 and SUZ12 to the COBLL1 peak and TSS, respectively. However, we found that whereas EBNA3C could interact with BMI1, EBNA3C could not be found in complex with the PRC2 component SUZ12. This result strongly suggests that the PRC2 complex is recruited indirectly to COBLL1 by EBNA3C, most likely via interaction with the PRC1 complex. It is known that EBNA3C mediates regulation of transcription by long-distance chromatin interactions (chromosome “looping”) between promoter and enhancer elements. Furthermore, Polycomb complexes have been shown to mediate the formation of higher-order chromosome structures  in LCL EBNA3C HDmut might explain why the level of H3K27me3 is impaired in this cell line. Indeed, both chromatin modifications (acetylation and trimethylation) take place at the same lysine residue on histone H3. It has been shown that H3K27Ac and H3K27me3 are mutually exclusive and that H3K27Ac prevents trimethylation of H3K27 at sites in Polycomb target genes . EBNA3C HDmut expression failed to fully repress COBLL1 as well as ADAM28 and ADAMDEC1 and did not completely remove the activation-associated H3K4me3 mark on their transcription start sites. Interestingly, we next found that KDM2B, a protein implicated in the demethylation of H3K4me3, interacts with EBNA3C. This interaction was greatly impaired when the TFGC motif of EBNA3C was mutated (HDmut). Finally, to determine whether KDM2B was important for EBNA3C-mediated repression of its target genes, we made use of the conditional LCL 3CHT Never HT combined with lentiviruses carrying shRNA to knock down the expression of KDM2B. We showed that KDM2B was important for the loss of the H3K4me3 mark as well as for the repression of EBNA3C target genes. We speculate that EBNA3C may recruit KDM2B to target genes to remove the activation mark H3K4me3. So far, we have not found an antibody against KDM2B that works in a ChIP assay with LCLs to test this point directly. Published ChIP studies of the KDM2B protein have mainly used tagged versions of KDM2B or antibodies which are not available commercially , CXCL9 and CXCL10 are coregulated by both EBNA3A and EBNA3C, suggesting that different mechanisms of repression are used depending on the target genes and the cofactors involved.Further studies will be needed to explore whether this mechanism of repression is conserved throughout the EBNA3 family of proteins and, more importantly, whether the same mechanism is used at other EBNA3C target genes. It is highly likely that different mechanisms of repression are involved, depending on the chromatin state of the target genes as well as on the presence of other EBV transcription factors. For instance, the EBNA3A protein has been shown to cooperate extensively with EBNA3C in the repression of thousands of cellular genes , and a tlleagues  describey EBNA3A . InteresAICDA . Further work is needed to investigate and reveal the molecular mechanisms behind EBNA3C-mediated gene activation. It was, however, not surprising to find that EBNA3C HDmut also fails to activate gene transcription, as EBNA3C binding partners, such as HDAC1 and KDM2B, have been demonstrated to have transcriptional activation functions  supplemented with 10% fetal calf serum (FCS), penicillin, and streptomycin. Puromycin was added at 1 μg/ml when selection was required. The activating ligand 4-hydroxytamoxifen (HT) was added to 400 nM where indicated.Cells were cultured at 10% COWT and RBPJ binding mutant (BM) EBNA3C recombinant viruses were already constructed and used in a previous study . The creOpen reading frames (ORFs) for EBNA3C WT, EBNA3C HDmut, EBNA3C W227S, and mCherry were cloned into a Gateway recombinational cloning system as previously described . BrieflyFor viral packaging, lentivirus-based vectors pLKO.1 and pLenti CMV Puro DEST were cotransfected with helper plasmids psPAX2 and pMD2.G into 293T cells by the calcium phosphate precipitation method. Media containing viruses were collected 48 h after transfection.g for 1.5 h at 22°C. The infected cells were then resuspended in RPMI medium and transferred to flasks.Primary B cells used in this study were isolated from buffy coat residues (UK Blood Transfusion Service) and infected as previously described . For lenGNB2L1, 5′-GCTTGCAGTTAGCCAGGTTC-3′ and 5′-GAGTGTGGCCTTCTCCTCTG-3′; for ALAS1, 5′-TCCACTGCAGCAGTACACTACCA-3′ and 5′-ACGGAAGCTGTGTGCCATCT-3′; for COBLL1, 5′-CTGTTCAGCTGACAACAGATCG-3′ and 5′-ACGTTGAACTCTCAGTGGTCCT-3′; for ADAM28, 5′-GTTGCAGGGACAATGGCACA-3′ and 5′-TGAGACGGCTGCAGGAACTG-3′; and for ADAMDEC1, 5′-CCTTGGTATGCCTGATGTTCCA-3′ and 5′-CAGCAGGCACTTTGGTTTCTGA-3′. The comparative threshold cycle (ΔΔTC) method was used to calculate relative mRNA expression, with the housekeeping gene GNB2L1 used as an endogenous control. Error bars in graphs show standard deviations for three triplicate qPCR replicates for each mRNA sample.RNA was isolated from cells by use of an RNeasy minikit (Qiagen) with DNase digestion per the manufacturer's instructions. Reverse transcription of RNA into cDNA was performed using Superscript III first-strand synthesis supermix for qRT-PCR (Invitrogen). Ten nanograms of cDNA was run per qPCR, using a Platinum SYBR green qPCR supermix UDG kit (Invitrogen) and a QuantStudio 7 Flex real-time PCR machine. Primers used in the study were as follows: for 7 LCL cells were harvested and lysed in immunoprecipitation (IP) buffer plus protease inhibitors . Cell extracts were then precleared with 55 μl of protein G-Sepharose beads  at 4°C for 1 h. Next, the protein of interest was immunoprecipitated from 200 μg of protein lysate overnight at 4°C. The antibodies used for IP were as follows: antibodies against RBPJ , BMI1 , SUZ12 , and KDM2B . Thirty microliters of protein G-Sepharose beads was added, incubated with rotation for 1 h at 4°C, and washed four times in IP buffer, and the immunopurified proteins were resolved by SDS-PAGE and detected by Western blotting. All IPs shown are representative examples of at least two independent experiments.IPs were performed essentially as described previously . BrieflyCells were washed twice in phosphate-buffered saline (PBS) and lysed in RIPA buffer for 30 min on ice. After centrifugation at 4°C for 10 min, the supernatant was removed, and the protein concentration was estimated calorimetrically using a Bio-Rad detergent-compatible assay. Protein samples (30 μg) were loaded onto SDS-polyacrylamide gels at a percentage appropriate for electrophoretic separation. Antibodies used for Western blotting were as follows: antibodies against EBNA3A , EBNA3B , EBNA3C , EBNA1 , EBNA2 , EBNA-LP (JF-186) , LMP1 , H3K4me3 , H3K27Ac , EBNA3C , RBPJ , BMI1 , and SUZ12 . Input DNA was 5% of the DNA used for immunoprecipitations and was diluted to 2.5% prior to PCR quantification. Enrichment relative to the input level was calculated using four 5-fold-dilution series, and error bars were calculated as standard deviations for triplicate PCRs for both input and IP samples. All ChIPs shown are representative of at least two independent experiments, each performed on LCLs established by two independent primary B cell infections.ChIP assay and qPCR analysis were performed as described previously . PrimersCell proliferation assays were performed as described previously , by meas+ primary B cells were purchased from the UK Blood Transfusion Service. As these were derived from anonymous volunteer blood donors, no ethical approval is required.The buffy coat residues used in this study for the isolation of CD19


Little is known about the molecular dynamics governing this transition. We have previously shown that overexpression of the transcriptional regulator TBX3 in DCIS‐like cells increases survival, growth, and invasiveness. To explore this mechanism further and assess direct transcriptional targets of TBX3 in a high‐resolution, isoform‐specific context, we conducted genome‐wide chromatin‐immunoprecipitation (ChIP) arrays coupled with transcriptomic analysis. We show that TBX3 regulates several epithelial–mesenchymal transition (EMT)‐related genes, including SLUG and TWIST1. Importantly, we demonstrate that TBX3 is a direct regulator of SLUG expression, and SLUG expression is required for TBX3‐induced migration and invasion. Assessing TBX3 by immunohistochemistry in early‐stage (stage 0 and stage I) breast cancers revealed high expression in low‐grade lesions. Within a second independent early‐stage non‐high‐grade cohort, we observed an association between TBX3 level in the DCIS and size of the invasive focus. Additionally, there was a positive correlation between TBX3 and SLUG, and TBX3 and TWIST1 in the invasive carcinoma. Pathway analysis revealed altered expression of several proteases and their inhibitors, consistent with the ability to degrade basement membrane in vivo. These findings strongly suggest the involvement of TBX3 in the promotion of invasiveness and progression of early‐stage pre‐invasive breast cancer to invasive carcinoma through the low‐grade molecular pathway. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.The acquisition of cellular invasiveness by breast epithelial cells and subsequent transition from ductal carcinoma  Our findings suggest that TBX3 facilitates the process of early invasion in DCIS by promoting the induction of EMT and tumor progression through the low‐grade pathway, as described by Bombonati and Sgroi In this study, we explored the role of TBX3 in breast cancer progression pathways, focusing specifically on its involvement in the induction of EMT and the transition from DCIS to invasive carcinoma. Immunohistochemical analysis revealed that TBX3 levels are elevated in low‐grade, pre‐invasive DCIS with an associated invasive component, and significantly correlated with the size of the invasive focus. Using genome‐wide bioinformatic approaches along with more conventional m l‐glutamine, 1 μg/ml insulin, 12.5 ng/ml EGF, 2.8 μm hydrocortisone, 10 mm HEPES, 1 mm sodium pyruvate, 0.1 mm non‐essential amino acids, 50 μg/ml gentamycin sulfate, and 10% FBS (αHE10F). All reagents were obtained from Wisent Inc . Stable 21NT transfectants previously generated m hygromycin (SLUG knockdown lines) as a selection marker. Details related to the generation of cell lines are located in the supplementary material, Supplementary materials and methods.The 21T series cell lines (21NT and 21MT‐1) were obtained as a gift from Dr Vimla Band Cell lines representing other molecular breast cancer subtypes were purchased from ATCC . Cell lines were maintained in their respective media: T47D (RPMI), SKBR3 (McCoy's 5A), and MDA‐MB‐468 (αMEM), each supplemented with 10% FBS.in vitro gelatin degradation assays Detailed protocols related to western blots, immunofluorescence staining, migration assays, Transwell invasion assays, chick choriallantoic membrane (CAM) assays, gelatin zymography, cell–cell adhesion assay, and Detailed protocols related to qRT‐PCR, chromatin immunoprecipitation (ChIP), RNA‐Seq (GEO Accession Number: GSE126153), ChIP promoter arrays (GEO Accession Number: GSE126154), and the bioinformatics analyses Patients with early‐stage breast cancer were identified from the London Breast Cancer Database on the basis of having either DCIS only (stage 0) or DCIS with an associated invasive component . Cohort 1 consisted of 186 patients with low‐, intermediate‐ or high‐grade DCIS with no invasion or early invasion (stage 0 or stage I). Cohort 2 consisted of 118 patients with non‐high‐grade DCIS with the aforementioned characteristics, with either no invasion or early invasion (stage 0 or stage I). Clinicopathological variables for cohort 1 and cohort 2 patients entered into this study are listed in Table in vitro . In the presence of calcium ions (+ CaCl2) or after adding increasing doses of EDTA to chelate free calcium, there were reduced levels of cell‐to‐cell adhesion, implying that several cell‐adhesion molecules may be involved (not solely Ca2+‐dependent cadherins). Loss of E‐cadherin has been associated with the switch to an EMT phenotype To examine the role of TBX3 in breast cancer, non‐invasive 21NT cells (which endogenously express low levels of TBX3) were stably transfected with either an empty vector, TBX3iso1 or TBX3iso2. Structural differences between TBX3 isoforms are shown in the supplementary material, Figure S1. Isoform specific up‐regulation was confirmed at the mRNA level using isoform‐specific primers, in addition to up‐regulation of total TBX3 protein levels  model. Fluorescently labeled cells were injected into the vasculature of chicks at embryonic day 12 and cell extravasation was assessed as previously described TBX3 in invasive 21MT‐1 cells, which natively express TBX3 at high levels . To confirm that the cells are better able to exit the vasculature (and are not simply better able to survive at T = 24 h), we performed live functional invadopodia assays within the CAM using confocal microscopy. Six hours after injecting fluorescently labeled cells into the CAM vasculature, a binary quantification of invadopodia formation was conducted , SKBR3 , and MDA‐MB‐468  breast cancer cell lines, representing different molecular subtypes . Collectively, this suggests that these findings have broad applicability and are not cell type‐specific.More invasive phenotypes were also observed using transient transfections of TBX3iso1 or TBX3iso2 into T47D , as well as EMT‐associated transcription factors  with TBX3 overexpression . Several other protein classes related to EMT were also altered, including extracellular matrix proteins, metalloproteases, and serine protease inhibitors. Importantly, within this list of 194 genes directly induced by TBX3 was SNAI2 (which encodes SLUG), a transcriptional regulator and potent inducer of EMT, which may provide a mechanism for the previously described EMT and invasion‐associated functional changes in TBX3‐transfected cells.To determine whether the TBX3‐dependent changes in gene expression are direct, chromatin immunoprecipitation (ChIP) experiments were conducted using either TBX3 antibody or rabbit IgG antibody as a non‐specific control, and immunoprecipitated DNA was hybridized to Affymetrix Promoter 1.0R arrays. Using this approach, we identified 5652 and 5512 specific binding sites for TBX3iso1 and TBX3iso2, respectively. Comparison of TBX3 binding sites with our expression analysis identified 194 genes that were directly regulated by both TBX3 isoforms  . We conducted ChIP qPCR validation using primers spanning this identified TBE. Published primer sequences were used for the transcription start site (CDH1 TSS) and coding region (CDH1 coding) of E‐cadherin SNAI2. Interestingly, this binding region overlaps with a DNase I hypersensitive region and RNA polymerase II binding region, which is suggestive of open and transcriptionally active chromatin  of our list of EMT‐ and invasion‐associated genes previously assessed in this study were still significantly altered with high TBX3 isoform expression in cells with SLUG knockdown   across two patient cohorts Table A. Earlie) Figure E.p < 0.05) and TWIST1 (p < 0.001) expression in the invasive component  in our second patient cohort. TWIST1 was exclusively nuclear localized, and SLUG was predominantly nuclear with occasional cytoplasmic staining . By further profiling transcriptomic data from the TCGA BRCA and the Farmer Breast study TBX3 levels in luminal breast cancers, and ER‐ and PR‐positive cancers  between patients with high and low TBX3 relative to the median  and Desmedt , consistent with our immunohistochemical data. The Desmedt dataset also showed reduced TWIST1 levels in high‐grade breast cancers. There was a high degree of overlap between TBX3 and SLUG in terms of patient characteristics and lesion types identified in our immunohistochemical staining and within the transcriptomics datasets. Additionally, upon examination of the TCGA TARGET GTEx dataset, we observed co‐expression of SNAI2 and TBX3 and tight association of MMP14 with high levels of both transcription factors  and the high‐grade (ER−/PR−) pathway The prevailing breast cancer progression model supported by numerous genomic and transcriptomic studies includes two divergent molecular pathways of progression – the low‐grade , is the main driver in the transition from an In conclusion, we propose a unique pathway in which TBX3 promotes progression through advancement of low‐grade DCIS to invasive carcinoma Figure . Our proin vivo invadopodia formation assay. KW conducted in vitro invadopodia formation assays. HSL provided us with CAM models. MB identified patients for immunohistochemical studies and gathered clinical information. ABT and AFC supervised the work. MK drafted the manuscript with support from ABT, BK, and JT. All the authors provided critical feedback and therefore shaped the research content and manuscript.MK, ABT, and AFC conceived the ideas, associated experiments, and contributed to interpretation of the results. MK performed the experiments and data analysis. MK and BK conducted bioinformatics analyses. MJC conducted pathological examination for immunohistochemical studies. COP conducted immunohistochemical staining for TBX3, SLUG, and TWIST1. HMH conducted a portion of the western blot experiments in Figure Supplementary materials and methodsFigure S1. TBX3 isoform protein structure and functional domainsFigure S2. TBX3 expression in 21NT transfectant and 21MT‐1 transductant cell linesFigure S3. Functional assessment of TBX3‐overexpressing cell linesFigure S4. Representative images of cell extravasation and invadopodia formation in vivo in the chick chorioallantoic membrane (CAM)Figure S5. TBX3‐mediated invadopodia formationFigure S6. Effect of TBX3 overexpression on invasiveness in cell lines representing other breast cancer molecular subtypesFigure S7. Expression of EMT markers with modulation of TBX3 levelsFigure S8. Protein class analysis of direct transcriptional targets of TBX3Figure S9. Analysis of T‐box binding elements (TBEs) in TBX3‐bound genes identified by ChIP‐arrayFigure S10. Subcellular localization of TBX3Figure S11. H‐scores for TBX3, SLUG, and TWIST1 immunostains in various cell compartmentsTable S1. Integrated ChIP‐array and RNA‐Seq data for assessment of direct and overlapping binding sites of TBX3iso1 and TBX3iso2Table S2. Primer sequences utilized for qRT‐PCR in mRNA studies Table S3. Primer sequences utilized for ChIP‐qPCR validation studies Table S4. Publicly available datasets utilized for analysis Supplementary materials and methodsClick here for additional data file.Figure S1. TBX3 isoform protein structure and functional domains.Figure S2. TBX3 expression in 21NT transfectant and 21MT‐1 transductant cell lines.Figure S3. Functional assessment of TBX3 overexpressing cell lines.Figure S4. Representative images of cell extravasation and invadopodia formation in vivo in the chick chorioallantoic membrane (CAM).Figure S5. TBX3‐mediated invadopodia formation.Figure S6. Effect of TBX3 overexpression on invasiveness in cell lines representing other breast cancer molecular subtypes.Figure S7. Expression of EMT markers with modulation of TBX3 levels.Figure S8. Protein class analysis of direct transcriptional targets of TBX3.Figure S9. Analysis of T‐box binding elements (TBEs) in TBX3‐bound genes identified by ChIP‐array.Figure S10. Subcellular localization of TBX3.Figure S11. H‐scores for TBX3, SLUG, and TWIST1 immunostains in various cell compartments.Click here for additional data file.Table S1. Integrated ChIP‐array and RNA‐Seq data for assessment of direct and overlapping binding sites of TBX3iso1 and TBX3iso2Click here for additional data file.Table S2. Primer sequences utilized for RT‐qPCR in mRNA studies Table S3. Primer sequences utilized for ChIP‐qPCR validation studies Table S4. Publicly available datasets utilized for analysis Click here for additional data file.


AMI) is mainly caused by atherosclerosis, an inflammatory and metabolic disease. Autophagy has been demonstrated to play critical roles in lipid metabolism and inflammation. Altered autophagic activity has been reported in AMI patients. However, molecular basis for dysfunctional autophagy in AMI remains unexplained.Coronary artery disease including acute myocardial infarction  and ethnic‐matched healthy controls (n = 363). Related molecular mechanisms were also explored.In this study, the promoter of the DNA sequence variants (DSVs) including single‐nucleotide polymorphisms (SNPs) were found in the ATG7 gene promoter. Two novel DSVs and five SNPs were only identified in AMI patients group. These DSVs and SNPs, except one SNP, significantly altered the transcriptional activity of the ATG7 gene promoter in both HEK‐293 and H9c2 cells (p < 0.05). Further electrophoretic mobility shift assay revealed that the DSVs and SNPs evidently affected the binding of transcription factors.A total of 19 ATG7 gene DSVs and SNPs identified in AMI patients may alter the transcriptional activity of the ATG7 gene promoter and change ATG7 level, contributing to the AMI development as a rare risk factor. Dysregulated lipid metabolism and inflammation have been shown to play critical roles in the initiation and progression of atherosclerosis and its complications, particularly plaque rupture and subsequent AMI  mainly degrades long‐lived macromolecules and damaged organelles and has been extensively studied. Autophagy is involved in many physiological processes, including lipid metabolism and inflammation. Altered autophagic activity has been implicated in a wide range of human diseases, including cardiovascular diseases  proteins have been identified in the formation of autophagosome  were recruited during the period April 2014 to April 2016, from Cardiac Care Unit, Division of Cardiology, Affiliated Hospital of Jining Medical University, Jining, Shandong, China. AMI patients were diagnosed with medical records, physical examination, electrocardiogram, and three‐dimensional echocardiography. Ethnic‐matched healthy controls  were recruited from the Physical Examination Center in the same hospital. The controls with CAD family history were excluded from this study. The study was approved by the Human Ethic Committee of Affiliated Hospital of Jining Medical University and conducted according to the principles of the Declaration of Helsinki. Informed consent was obtained from all individual participants included in the study.Acute myocardial infarction patients (2.2ATG7 gene promoter region (from −1145 bp to +112 bp to the transcription start site) was directly sequenced. Two overlapped DNA fragments, 698 bp (−1145 bp ~ −448 bp) and 622 bp (−510 bp~+112 bp), were generated by PCR. PCR primers were designed based on genomic sequence of the human ATG7 gene  to construct expression vectors. These expression vectors were then transfected into cultured cells, and dual‐luciferase activities were examined. Briefly, DNA fragments of wild‐type and variant ATG7 gene promoters  were generated by PCR and inserted into the KpnI and Hind III sites of pGL3‐basic to generate expression vectors. The PCR primers are shown in Table ATG7 gene promoters were represented as ratios of firefly luciferase activities over renilla luciferase activities. Transcriptional activity of the wild‐type ATG7 gene promoter was designed as 100%. All the experiments were repeated three times independently, in triplicate.Wild‐type and variant 2.4® Nuclear and Cytoplasmic Extraction Reagents . Protein concentrations were determined with Bio‐Rad protein assay. EMSA examining DNA–protein interactions was carried out using LightShift® Chemiluminescent EMSA kit . DNA‐protein binding reaction was carried out with nuclear extracts (3.0 μg) and double‐stranded biotinylated oligonucleotides (30 bp) containing wild‐type and variant DNA sequence of the ATG7 gene promoter. After 20 min at room temperature, the reaction mixtures were loaded onto a native 6% polyacrylamide gel in 0.5× TBE and run at 100 V for 1 hr, which were subsequently transferred onto a nylon membrane  at 100V for 30 min. The oligonucleotides were cross‐linked to the membrane using the UV Stratalinker 1800 . The biotin‐labeled oligonucleotides were then detected by chemiluminescence.Nuclear extracts were prepared from HEK‐293 and H9c2 cells using NE‐PER2.5SEM and compared by a standard Student's t test. DSV frequencies in AMI patients and controls were analyzed and compared with SPSS v13.0. p < 0.05 was considered statistically significant.The quantitative data were represented as mean ± 33.1rs2594975), g.11271763G>A (rs76708041), g.11272004C>G (rs550744886), g.11272071G>A (rs544484285), and g.11272355A>G (rs2606729), were found in nine AMI patients. In the 1000 Genome Project, frequencies of C allele in g.11271281T>C (rs2594975) and G allele in g.11272355A>G (rs2606729) were 100% in Han Chinese people. These DSVs and SNPs were not found in controls. Sequencing chromatograms of these DSVs and SNPs are shown in Figure p > 0.05).A total of 19 DNA sequence variants (DSVs), including 12 single‐nucleotide polymorphisms (SNPs), were identified in this study population. Locations and frequencies of the DSVs are depicted in Figure 3.2ATG7 gene promoter was analyzed with JASPAR program (http://jaspar.genereg.net/). The DSVs identified in AMI patients may abolish, create, and modify the putative binding sites for transcription factors, which are summarized in Table rs2594975) may abolish a binding site for zinc finger protein 354C (ZF354C), a ubiquitously expressed transcriptional repressor. The DSV g.11271467C>T may abolish a binding site for THAP domain containing 1 (THAP1), a C2CH THAP‐type zinc finger factor with roles in proliferation, apoptosis, cell cycle, chromosome segregation, chromatin modification, and transcriptional regulation. The SNP g.11271763G>A (rs76708041) may modify the binding site for GATA binding protein 2 (GATA2), a transcriptional activator involved in the development and proliferation of hematopoietic and endocrine cell lineages. The SNP g.11272004C>G (rs550744886) may create the binding sites for Rhox homeobox family member 1 (RHOXF1), a transcription factor that may play a role in human reproduction, and NF‐kappaB‐related factors. The SNP g.11272071G>A (rs544484285) may abolish the binding site for transcription factor 7‐like 2 (TCF7L2), which is highly expressed in fat. The DSV g.11272338G>A may modify the binding site for nuclear respiratory factor 1 (NRF1), which regulates cellular growth and nuclear genes required for respiration, heme biosynthesis, and mitochondrial DNA transcription and replication. The SNP g.11272355A>G (rs2606729) may modify the binding site for nuclear factor 1 X‐type (NFIX), a SMAD/NF‐1 DNA‐binding domain transcriptional activator.To determine whether the DSVs and SNPs identified in AMI patients affected putative binding sites for transcription factors, the 3.3ATG7 gene promoters were subcloned into luciferase reporter vector (pGL3‐basic) to generate expression vectors, including empty pGL3‐basic (negative control), pGL3‐WT (wild‐type ATG7 gene promoter), pGL3‐11271281T, pGL3‐11271431T, pGL3‐11271467T, pGL3‐11271576C, pGL3‐11271763A, pGL3‐11272004G, pGL3‐11272071A, pGL3‐11272171T, pGL3‐11272227G>T, pGL3‐11272338A, and pGL3‐11272355A. The expression vectors were transfected into HEK‐293 and H9c2 cells, and dual‐luciferase activities were assayed.Wild‐type and variant rs76708041), g.11272004C>G (rs550744886), g.11272071G>A (rs544484285), and g.11272355A>G (rs2606729)] identified in AMI patients significantly decreased the transcriptional activity of the ATG7 gene promoter (p < 0.01). In contrast, the DSV (g.11272338G>A) and the SNP [g.11271281T>C (rs2594975)] identified in AMI patients significantly increased the transcriptional activity of the ATG7 gene promoter (p < 0.01). As expected, the DSVs  identified in controls and the SNP [g.11272171G>T (rs140788185)] found in both AMI patients and controls did not affect the activity of the ATG7 gene promoter  and the SNPs [g.11271763G>A ] did not significantly affect the transcriptional activity of ATG7 gene promoter in H9c2 cells, indicating its tissue‐specific effect (p > 0.05). Therefore, the DSVs and SNPs identified in AMI patients significantly altered the activity of the ATG7 gene promoter in both HEK‐293 and H9c2 cells.To further determine the tissue‐specific effects of the DSVs and SNPs in cardiomyocytes, the activity of wild‐type and variant 3.4rs550744886)] reduced the binding of a transcription factor, which was weakly expressed in H9c2 cells. The predicted creation of RHOXF1 binding site may not exist or beyond the detection of EMSA. The DSV g.11272338G>A enhanced the binding of a transcription factor. The SNP [g.11272355A>G (rs2606729)] enhanced the binding of a transcription factor in HEK‐293 cells ] did significantly decrease the transcriptional activity of the ATG7 gene promoter in HEK‐293 cells, but had no effect in H9c2 cells, indicating its tissue‐specific effects   is significantly associated with breast cancer‐specific survival  in AMI patients. Therefore, these DSVs and SNPs may change ATG7 levels, contributing to the AMI development as a low‐frequency risk factor.Recently, low‐frequency and rare genetic variants have been suggested to account for the missing heritability as a major cause for human diseases Lettre, . PreviouATG7 gene has been mapped to chromosome 3p25.3 and is ubiquitously expressed in human adult and fetal tissues , a master regulator of heat shock responses, directly binds to the ATG7 gene promoter and transcriptionally upregulating its expression , which bind to the 3′ UTR of ATG7 gene mRNA   gene, which is independent of its E1‐like enzymatic activity (Lee et al., ATG7 gene promoter was genetically and functionally analyzed in AMI patients and healthy controls in this study. The DSVs and SNPs were identified in AMI patients, which significantly altered the transcriptional activity of the ATG7 gene promoter. These DSV and two SNPs evidently affected the binding of transcription factors. Therefore, these DSVs and SNPs may change ATG7 level, contributing to AMI development through its autophagic and non‐autophagic functions as a low‐frequency risk factor.In conclusion, the The authors declare that they have no conflict of interest.