Kluyveromyces lactis contains linear cytoplasmic DNA virus-like elements  that bear genes encoding putative non-canonical two-subunit RNAP. Here, we describe the architecture and identify the evolutionary origin of this transcription machinery. We show that the two RNAP subunits interact in vivo, and this complex interacts with another two VLE-encoded proteins, namely the mRNA capping enzyme and a putative helicase. RNAP, mRNA capping enzyme and the helicase also interact with VLE-specific DNA in vivo. Further, we identify a promoter sequence element that causes 5′ mRNA polyadenylation of VLE-specific transcripts via RNAP slippage at the transcription initiation site, and structural elements that precede the termination sites. As a result, we present a first model of the yeast virus-like element transcription initiation and intrinsic termination. Finally, we demonstrate that VLE RNAP and its promoters display high similarity to poxviral RNAP and promoters of early poxviral genes, respectively, thereby pointing to their evolutionary origin.Extrachromosomal hereditary elements such as organelles, viruses, and plasmids are important for the cell fitness and survival. Their transcription is dependent on host cellular RNA polymerase (RNAP) or intrinsic RNAP encoded by these elements. The yeast  K. lactis VLEs. Using systematic in vivo study, we were able to characterize composition of the previously hypothesized VLE-encoded transcription complex. Further, we identified new DNA and RNA elements that were directly connected with formation of VLE-specific mRNA ends, as demonstrated by mutagenesis of these elements and its effect on VLE transcription in vivo. Finally, our phylogenetic and sequence analysis of VLE-encoded non-canonical RNA polymerase and its promoters, respectively, suggested evolutionary relationship of VLE transcription machinery to much better explored transcription machinery of cytoplasmic double-stranded DNA viruses of the Poxviridae family.Yeast cytoplasmic double-stranded DNA virus-like elements  were widely investigated in the past but the topic was almost entirely abandoned, partly due to an inability to express VLE-encoded proteins using conventional expression systems. In this study, we re-opened investigation of these elements focused on considerably underexplored nucleus-independent transcription of  Kluyveromyces lactis, termed pGKL1 (or K1) and pGKL2 (or K2), have become a model system to study such DNA elements. These VLEs have compact genomes with occasional overlaps of open reading frames (ORFs) and a high AT content of ~74% [K. lactis strains is associated with the extensively studied yeast killer phenotype [Linear double-stranded DNA virus-like elements (VLEs) were found in the cytoplasm of several yeast species. Structural organization of these elements is quite uniform and they are often present as two or three differently sized DNA plasmids in yeast host cells . Charact of ~74% , 6. The Adenoviridae family or Bacillus subtilis bacteriophage φ29 [Functions of protein products for most ORFs encoded by the pGKL plasmids were predicted using bioinformatics approaches and some of these proteins were characterized by biochemical and genetic analyses . Both pGhage φ29 . Consequhage φ29 . NeverthPichia etchellsii pPE1B element acts as a functional promoter when transplanted into the pGKL1 element [cis-acting component of the VLE-specific transcription system and it is essential for transcription initiation. After elongation, transcription then terminates after each gene because only monocistronic transcripts were revealed with Northern blot analyses of transcripts derived from ten pGKL-encoded ORFs [Transcription of VLE-specific genes has been shown to be independent of mitochondrial  and nucl element . Thus, tded ORFs , 19–21. ORF6  and ORF7  of the pGKL2 element should form a non-canonical RNAP. K2ORF6p was found to have a sequence similarity to three conserved regions of the two largest subunits (β and β′ in bacteria) of canonical multisubunit RNAPs [Unique RNAP subunits, and possibly also a putative helicase and the mRNA capping enzyme are the key elements of the VLE cytoplasmic transcription machinery. Protein products of it RNAPs . Sequencit RNAPs , and theit RNAPs .ORF4 sequence of the pGKL2 element shows striking sequence similarity to viral helicases from the superfamily II of DEAD/H family helicases involved in transcription. The K2ORF4 protein product (K2ORF4p) displays similarity with two Vaccinia virus helicases–(i) NPH-I, which is encoded by the D11L gene, and (ii) the small subunit of the heterodimeric Vaccinia virus early transcription factor (VETF) encoded by the D6R gene [The D6R gene , 25. NPHD6R gene . VETF fuD6R gene .ORF3 (K2ORF3p) encoded by the pGKL2 element shows sequence similarity to the Vaccinia virus mRNA capping enzyme encoded by the D1R gene that consists of three domains responsible for the three enzymatic activities necessary to form the 5′ mRNA cap structure [D12L gene [in vitro [The protein product of tructure . The met12L gene , 30. The12L gene . Triphosin vitro .K2ORF3, K2ORF4, K2ORF6 and K2ORF7 genes are indispensable for the maintenance of the pGKL elements in the cell [As reported previously, the the cell , 24, 32.in vivo study focusing on the architecture of the transcription complex of the yeast VLEs. Moreover, we identify a new promoter DNA element which is associated with 5′ mRNA polyadenylation of most pGKL-encoded genes and we uncover a link between RNA stem loop structures and 3′ end formation of VLE-specific mRNAs in vivo. Further, we present an extensive phylogenetic analysis of amino acid sequences of VLE RNAP subunits. Finally, we provide a detailed sequence analysis of pGKL promoters. Collectively, these analyses strongly suggest that the VLE transcription machinery has origin close to poxviruses.Here, we present a systematic in vivo.To start characterizing the transcription machinery of the yeast VLEs we first tested whether the VLE RNAP subunits , the mRNA capping enzyme (K2ORF3p), and the putative helicase (K2ORF4p) form a complex Initially, we tested interactions between K2ORF3p, K2ORF6p and K2ORF7p using a yeast two-hybrid system and its fluorescence variant called bimolecular fluorescence complementation but we failed to detect any interaction with either approach. This was most likely caused by the high AT content of VLE genes that was shown recently to impair their nuclear expression due to RNA fragmentation mediated by the polyadenylation machinery . TherefoWe prepared a strain encoding yeast enhanced green fluorescent protein 3 (yEGFP3)  that wasThen, to verify the interaction between the large RNAP subunit and the mRNA capping enzyme we decided to perform immunoprecipitation using tagged K2ORF3p as the bait. Hence, we prepared a strain encoding yEGFP3 fused to the C-terminus of K2ORF3p (IFO1267_pRKL2-11 strain). Interestingly, selective cultivation of clones after transformation led to a loss of the pGKL1 element . ImmunopTo further validate the results, we prepared several strains containing combinations of these proteins with various tags: (i) strain IFO1267_pRKL2-5 where yEGFP3-K2ORF6p was co-expressed together with K2ORF7p-FLAG, and control strain IFO1267_pRKL2-15 that expressed only one tagged protein—K2ORF7p-FLAG; (ii) strain IFO1267_pRKL2-6 co-expressing yEGFP3-K2ORF6p and K2ORF3p-HA, and control strain IFO1267_pRKL2-14 expressing only K2ORF3p-HA. The results of IP experiments followed with Western blotting clearly showed specific interactions between these proteins .in vivo. For the fourth protein, the putative helicase K2ORF4p, the MS results suggesting it as part of the complex were not convincing due to low protein coverage  yEGFP3-K2ORF4p together with HA-K2ORF6p (IFO1267_pRKL2-9), and (ii) yEGFP3-K2ORF4p together with K2ORF3p-HA (IFO1267_pRKL2-10) were prepared. With the first combination we confirmed that HA-K2ORF6p associated with yEGFP3-K2ORF4p . With thAs an additional control to demonstrate that the observed interactions were specific, we used another pGKL-encoded protein with a function unrelated to transcription. We selected K1ORF4p, a subunit of the toxin responsible for VLE-associated killer yeast phenotype . A straiFinally, we wanted to know whether the association of the putative helicase and mRNA capping enzyme with the large RNAP subunit was dependent on nucleic acids. We prepared lysates from IFO1267_pRKL2-6  and IFO1267_pRKL2-7  strains. The lysates were incubated with GFP-Trap_A beads and, after washing, the beads were split into two parts which were treated or mock-treated with Benzonase Nuclease to digest DNA and RNA. Then, the beads were again extensively washed and the bound proteins were eluted. Subsequent Western blot analysis revealed both K2ORF3p-HA and HA-K2ORF4p to associate with yEGFP3-K2ORF6p regardless of the presence or absence of nucleic acids which was confirmed by PCR .in vivo. This association was independent of nucleic acids. Finally, K2ORF3p, K2ORF6p, and K2ORF7p appeared to form a core transcription complex with relatively strong mutual interactions to which K2ORF4p bound relatively weakly.Taken together, the immunoprecipitation, mass spectrometry, and Western blot results demonstrated the existence of the hypothesized VLE-specific transcription complex because K2ORF3p, K2ORF4p, K2ORF6p, and K2ORF7p were specifically associated in vivo. It is believed that this transcription complex is VLE-specific [in vivo, which was never formally tested. Hence, we performed in vivo chromatin immunoprecipitation. We used the IFO1267_pRKL2-13 strain expressing HA-K2ORF4p, IFO1267_pRKL2-14 strain expressing K2ORF3p-HA, IFO1267_pRKL2-4 strain expressing yEGFP3-K2ORF6p, and the IFO1267 control strain. First, we verified that the mouse monoclonal anti-HA HA-7 agarose efficiently immunoprecipitated HA-K2ORF4p and K2ORF3p-HA (K. lactis actin (ACT) and high-affinity glucose transporter (HGT1) genes as markers of chromosomal DNA, and toxin immunity (K1ORF3), mRNA capping enzyme (K2ORF3), and large RNAP subunit (K2ORF6) genes as markers of pGKL elements. We found, that HA-K2ORF4p, K2ORF3p-HA, and yEGFP3-K2ORF6p were specifically associated with pGKL elements and not with chromosomal DNA  leaders of heterogeneous lengths in mRNAs of 12 pGKL genes except for d K2ORF8 . Interesd K2ORF8 , 38. It in vitro , 40.K. lactis strains with modified pGKL1 elements encoding the G418 resistance marker under the control of the K1UCR2 promoter. We prepared three variants of the K1UCR2 promoter that differed in the INR sequence: (i) TAAAA ; (ii) TAACA (strain IFO1267_pRKL1-2); and (iii) TACCA (strain IFO1267_pRKL1-3). Then, we purified total RNA from the three strains, prepared cDNA, and performed 5′ RACE-PCR to determine the 5′ end sequences. The results showed that the 5′ poly(A) leader was present when the K1UCR2 sequence contained the putative wt INR (TAAAA INR)  . When TAAAA INR) . When TAAAA INR) .K1ORF2 transcripts was the same as for G418R transcripts produced from the K1UCR2 with the wt INR [Therefore, we concluded that slippage of VLE RNAP at the initiation site was the mechanism responsible for 5′ polyadenylation of the transcripts. Moreover, the identified INR sequence constituted an independent DNA element, not influenced by the sequence of the gene because the pattern of the sequenced 5′ RACE-PCR clones for e wt INR .Our previous 3′ RACE-PCR experiments had revealed the absence of 3′ poly(A) tails in mRNAs of all 15 pGKL ORFs . To shedK. lactis strain with a modified pGKL1 element encoding the G418 resistance marker under control of K1UCR2, followed by the 3′ UTR of the K2ORF5 gene  of the K2ORF5 gene. We purified total RNA from the respective K. lactis strains, prepared cDNA, and performed 3′ RACE-PCR experiments. Although the coding sequences of the aforementioned genes differed both in length and AT content, the pattern of their 3′ termini was highly similar  using DaliLite v. 3 showed that all known regions of K2ORF6p and K2ORF7p with sequence similarity to conserved regions of the canonical RNAPs were modelled accordingly. Interestingly, parts of K2ORF6p were modelled by βa1, βa6, βa13, and βa16 conserved regions, which were not detected previously to be present in K2ORF6p. We verified the proper model:template alignment of these regions by manually constructed sequence alignments with other canonical RNAPs and these alignments indeed showed sequence similarities between K2ORF6p and the aforementioned regions  into two polypeptides, similarly as in K2ORF6p and K2ORF7p, is present in RNAPs of some Archaeal species [The overall distribution of the conserved regions within K2ORF6p and K2ORF7p is depicted in  species .S. cerevisiae RNAP II Rpb2 subunit (β subunit homolog) and the K2ORF6p model, respectively. It is clear that almost all conserved β subunit regions are present in K2ORF6p, and only the spacing between them is shorter. S. cerevisiae RNAP II Rpb1 subunit (β′ subunit homolog) and relevant homologous portions of K2ORF6p/K2ORF7p subunits, respectively. Remarkably, more than half of the β′ subunit conserved regions, such as most of the clamp domain (β′a1 - β′a10 regions) and secondary-channel rim helices , are missing in VLE RNAP  were monophyletic, but the ancestral gene was displaced in Asfarvirus and Mimivirus for eukaryotic RNAP I and RNAP II gene, respectively [Asfarviridae and Mimiviridae) was not recovered in our unconstrained tree. Further, we tested alternative evolutionary hypotheses for VLE RNAPs via constrained tree topology approach where we enforced monophyly of VLE RNAPs with other viral and cellular RNAPs, and we compared the likelihood of the original tree with the likelihood of the constrained trees. From these analyses, it was apparent that the likelihood of the unconstrained tree suggesting monophyly of the yeast VLE RNAP with poxviral RNAP was the best, although monophyly of VLE RNAP with RNAP of other NCLDVs could not be rejected by statistical tests  essential for transcription of VLEs was identified a long time ago, no similarities with known promoters that would indicate its origin were reported. We extended the UCSs preceding all pGKL-encoded ORFs both upstream and downstream by ~10 bp, and we created a consensus motif. This consensus was then used to search for similar elements that were associated with transcription by multisubunit RNAPs. We particularly focused on promoters of viral RNAPs because our phylogenetic analysis of VLE RNAPs had suggested a viral origin.th position followed by TGA [Notably, we detected great sequence similarity between the extended UCS  and the d by TGA . This ped by TGA . We annod by TGA .To conclude, it appears that promoters of poxviral early genes and VLE genes are similar both with respect to their sequence and their spacing to the TSSs, implying a common origin.In this study we characterized the considerably underexplored transcription machinery of the yeast cytoplasmic linear double-stranded DNA virus-like elements. We used both experimental and bioinformatic approaches, and determined the composition and interactions of the transcription complex and presented a 3D model of its two main subunits. Further, we defined DNA sequences required for initiation and termination. For a model of the key aspects of transcription of the VLEs see E. coli systems was not possible, most likely due to the different codon usage dictated by the high AT content of VLE genes [E. coli, S. cerevisiae and K. lactis expression systems.Biochemical characterization of proteins encoded by yeast VLEs was shown to be challenging in the past. Expression of genes located on the pGKL elements seemed to be rather weak –17. AlsoLE genes . RecentlLE genes . Accordiin vivo the transcription machinery core complex consisted of the two RNAP subunits  and the mRNA capping enzyme (K2ORF3p). This interaction was independent of the presence of nucleic acids. Subsequently, we showed that the putative helicase (K2ORF4p) associated less tightly with both the VLE large RNAP subunit and the mRNA capping enzyme. This suggested the following molecular model of the VLE transcription components interactions. First, we propose that K2ORF6p and K2ORF7p interact directly because aa residues of β′a15 and β′a16, which are known to participate in intramolecular  or intermolecular (Archaea) bonds in multisubunit RNAPs, are present also in K2ORF6p and K2ORF7p as revealed by our in silico analysis. Second, we suggest that the mRNA capping enzyme interacts directly with the RNAP complex although it is yet to be determined whether it is with K2ORF6p and/or K2ORF7p. Cellular mRNA capping enzymes are known to interact with the C-terminal domain of RNAP II β′ homolog subunit [in vivo, we assume that it possibly acts as a dissociable transcription factor.To overcome this difficulty, we prepared modified and double-modified pGKL elements expressing the transcription components with tags. Using co-immunoprecipitations followed by mass spectrometry and Western blotting we demonstrated that  subunit . However subunit , 51. By  subunit –54. VETF subunit . Howeverin vivo. We propose that the transcription complex interacts directly with pGKL-specific DNA through RNAP subunits, and that the association of the mRNA capping enzyme with pGKL-specific DNA is indirect. Based on Vaccinia virus early transcription system analogy, we believe that the putative helicase also interacts directly with pGKL-specific DNA with the UCS promoter elements being the K2ORF4p binding sites. However, due to compact genomic organisation of pGKL elements precise mapping of the in vivo DNA binding sites of K2ORF4p using ChIP-seq would most likely turn out unsuccessful because resolution of the method would not match close spacing of the UCS elements. Our analysis indeed suggested close spacing of K2ORF4p binding sites in vivo because any pGKL region chosen for PCR amplification showed up to be enriched in anti-HA-K2ORF4p ChIP sample. Importantly, it seems that the transcription machinery of the yeast VLEs is remarkably if not entirely self-sufficient, because we did not find any cellular proteins to be specifically associated with the VLE large RNAP subunit using mass spectrometry analysis.Finally, we found K2ORF4p, K2ORF3p and K2ORF6p to associate with pGKL-specific DNA Our previous 5′ RACE-PCR experiments revealed short poly(A) leaders at the 5′ mRNA ends of most pGKL-encoded genes . These 5in vivo, and it was generated by the same mechanism as shown for Vaccinia virus postreplicative transcripts [G418R was used for selection of clones containing recombinant VLEs. To assess possible role of INR alterations on VLE gene expression, we prepared a strain (IFO1267_pRKL1-9) where we introduced TACCC mutations to the TAAAC INR of toxin subunit gene K1ORF4, and this strain showed reduced killer toxin production (It has been shown that 5′ untranslated regions composed of 5′ poly(A) leader sequences prior to start codon have a regulatory role in translation initiation , 58. Usinscripts . In Vaccnscripts , 59. Thioduction .Mycoplasma hyopneumoniae , an organism with a similarly high AT content [in vivo, suggesting an intrinsic transcription termination model for the yeast VLEs, similar to that in bacteria , these values were comparable to the genome-wide predicted intrinsic terminators in  content . Further content –63. Inteiewed in ). Even tin vitro [in vivo. However, we were not able to locate a specific termination signal (UUUUUNU) of Vaccinia virus early mRNAs that is required for RNAP pausing induced by viral mRNA capping enzyme [Even though Vaccinia virus RNAP, presumably related to VLE-specific RNAP, terminates transcription of early genes in a factor-dependent manner it was recently shown that RNA stem loops can influence both efficiency and location of transcription termination in vitro . Therefoin vitro , 31. Duein vitro , 28, resg enzyme  in VLE mBioinformatic analysis of VLE-specific RNAP proved to be challenging due to its unique reduced architecture and great evolutionary distance from other multisubunit RNAPs. From the 3D model it is evident that VLE RNAP significantly differs from canonical RNAPs in several aspects:(i) Almost the entire clamp structure element is absent. Only a basal portion of the clamp formed by βa15, βa16 and β′a20 conserved regions is maintained. The clamp is a mobile RNAP element and its closure is important for high stability and processivity of the enzyme. The clamp conformation is regulated by interaction of universally conserved elongation factors NusG and Spt4/5 with the clamp coiled-coil motif –an elemein vitro [in vivo. The rudder element interacts with the upstream edge of the DNA-RNA hybrid [(ii) The lid and rudder elements are likely missing. The lid acts as a wedge to facilitate dislocation of RNA from the DNA-RNA hybrid molecule, and thereby maintains a constant size of the DNA-RNA hybrid between 7 to 10 base pairs . Howeverin vitro . TherefoA hybrid . ExperimA hybrid . This ma(iii) The secondary-channel rim helices are missing. These helices are the binding sites for some transcription factors of multisubunit RNAPs, such as the transcription elongation factor TFIIS . TherefoA viral origin of the VLE genes encoding the mRNA capping enzyme and the putative helicase was suggested previously . HoweverMimiviridae family, although its location prior to the TSSs seems to differ [Our promoter analysis suggests that not just the VLE RNAP, but also the VLE promoters are related to nucleo-cytoplasmic large DNA viruses. We noticed sequence similarity between the UCE motif of Vaccinia virus early genes and the extended UCS motif of pGKL elements, as well as their similar location prior to the TSSs. Invariant G residue and several A residues in the minor groove were proposed to be the UCE nucleotides contacted by Vaccinia virus VETF helicase . Presenco differ .A recent evolutionary hypothesis suggests that both yeast VLEs and nucleo-cytoplasmic large DNA viruses branched from a Polintovirus—eukaryotic dsDNA virus that had acquired transcription machinery genes required for escape from nucleus to cytoplasm . Yeast VIn conclusion, the transcription apparatus of the yeast VLEs has most likely an origin close to poxviruses and uses transcription initiation mechanisms similar to those used by poxviral genes. Unlike poxviruses, however, VLEs are beneficial for the cell, and this exemplifies the ability of the cell to domesticate potentially pathogenic elements.Escherichia coli cells were grown at 37°C in 2xTY medium which was supplemented with kanamycin (50 μg/ml) or ampicilin (100 μg/ml) for selection of transformants. Transformations of E. coli cells were performed by electroporation using Gene Pulser Xcell (Bio-Rad). K. lactis cells were grown at 28°C in YPD medium which was supplemented with G418 (250 μg/ml) and/or hygromycin B (200 μg/ml) for selection of transformants. Transformations of K. lactis cells were performed using the one-step LiCl method [All of the strains used in this study are listed in l method  and follThe nucleotide sequences of the primers used for construction, verification and sequencing of recombinant pGKL elements, and RACE-PCR amplification are listed in K. lactis IFO1267 strain was transformed with PCR-generated fragment consisting of 5′ and 3′ ends homologous to the part of the pGKL element to be modified and non-homologous part that introduced purification and/or detection tag  into VLE-specific ORF together with a gene encoding resistance marker (G418 or hygromycin B) whose expression is driven by pGKL1-derived upstream control region . This type of construct was prepared by PCR or fusion PCR methodology  and used for transformation. After transformation, yeast cells were plated onto selective media and analyzed using agarose electrophoresis for the presence of the modified pGKL element. Usually, it was possible to detect both, modified and wild-type target element directly after transformation. Colonies containing both (modified and wild-type variants of the target pGKL element) were selected and cultivated under selective conditions for approximately 60 generations and analyzed again using agarose electrophoresis. For subsequent analysis and preparation of double-modified pGKL elements, colonies containing only modified variant of the target pGKL element were used. Absence of unmodified variant of the respective pGKL element was also verified by PCR and subsequent electrophoresis.Modified protocol based on  was used600 = 4–8) were used. The cells were harvested with centrifugation, washed with distilled water and lysed in 2–3 ml of ice-cold GFP-wash buffer  supplemented with 1 mM PMSF, cOmplete Mini protease inhibitors (Roche) and 0.45 mm glass beads using Mixer Mill MM 301 (Retsch) at a frequency of 30/s for 5 min. The glass beads and cell debris were pelleted with centrifugation at 8 000 g for 5 min at 4°C. The lysates were clarified with centrifugation at 20 000 g for 20 min at 4°C. Co-immunoprecipitations were performed using 25 μl of GFP-Trap_A (Chromotek) beads with gentle mixing overnight at 4°C. Mock immunoprecipitations were performed using 25 μl of empty agarose beads (Chromotek) with gentle mixing overnight at 4°C. Bound proteins were washed three times in 1 ml of ice-cold GFP-wash buffer with gentle mixing for 5 min at 4°C. The immunoprecipitated complexes were dissolved in 60 μl of 2X sample loading buffer , incubated for 5 min at 95°C and subjected to SDS-PAGE. The gel was stained with Coomassie Brilliant Blue G-250 or silver. Gel lanes or bands of interest were excised from the gel, digested with trypsin [For the co-immunoprecipitation experiments, 100 ml of the yeast cells from the late exponential growth phase  were used. The cells were cross-linked with formaldehyde  added to the growing culture for 40 min at 28°C. The cells were harvested with centrifugation, washed with distilled water and lysed in 2 ml of ice-cold non-denaturing lysis buffer  supplemented with 1 mM PMSF, cOmplete Mini protease inhibitors (Roche) and 0.45 mm glass beads using Mixer Mill MM 301 (Retsch) at a frequency of 30/s for 5 min. The lysate was sonicated  sixty times with 10 sec pulses. The glass beads and cell debris were pelleted with centrifugation at 8 000 g for 5 min at 4°C. The lysates were clarified with centrifugation at 20 000 g for 20 min at 4°C and 50 μl aliquot of the clarified lysates representing chromatin before immunoprecipitation (Input) was taken. The DNA fragments were immunoprecipitated using 30 μl of mouse monoclonal anti-HA HA-7 agarose (Sigma Aldrich) beads or 20 μl of GFP-Trap_A (Chromotek) beads with gentle mixing overnight at 4°C. The beads were then washed once in 1 ml of ice-cold Wash buffer  supplemented with single stranded salmon sperm DNA , twice in 1 ml of ice-cold Wash buffer, and once in 1 ml of ice-cold Final wash buffer (Wash buffer containing 500 mM NaCl), each time with gentle mixing for 5 min at 4°C. Immunocomplexes were then eluted from the beads in 120 μl of Elution buffer  for 30 min at 37°C (anti-HA HA-7 beads) or for 30 min at 65°C (GFP-Trap_A beads). Eluted immunocomplexes and 50 μl of the clarified lysates representing chromatin before immunoprecipitation (Input) were mixed with 400 μl of TBS  supplemented with 5 μl of proteinase K , and the cross-linking was reversed by incubation for 5 hr at 65°C. The immunoprecipitated and input DNA was isolated by phenol-chloroform extraction followed by ethanol precipitation supplemented with 1 μl of linear polyacrylamide , and then used for PCR amplification for 25–30 cycles followed by electrophoresis. PCR amplifications were carried out on 1/30 of the chromatin immunoprecipitation (ChIP) and 1/1200 of the chromatin before immunoprecipitation (Input) using primers listed in For the chromatin immunoprecipitation experiments, 50 ml of the yeast cells from the late exponential growth phase (ODSDS-PAGE gels were electroblotted onto Immun-Blot PVDF Membrane (Bio-Rad). The membranes were blocked in 5% non-fat dry milk (Hero) in a TBS-Tween buffer  and incubated with a primary antibody overnight at 4°C. After washing in TBS-Tween buffer and blocking with 5% non-fat milk, the membranes were incubated with a goat anti-mouse HRP-conjugated antibody . Finally, after washing in TBS-Tween buffer, the membranes were immersed in a luminol detection solution and the signal was detected using ImageQuant LAS 4000 . To confirm the expression of the target protein and the successful immunoprecipitation, a mouse monoclonal anti-FLAG M2 antibody , mouse monoclonal anti-HA 6E2 antibody , and mouse monoclonal anti-GFP B-2 antibody  was used.600 = 0.5–1) were quickly pelleted and frozen. Total yeast RNA was isolated by the hot acidic phenol procedure followed by ethanol precipitation [free Kit (Ambion). The quality of RNA was assessed by electrophoresis and UV spectrophotometry [25 ml of the yeast cells from the exponential growth phase  were used for cDNA synthesis using 100U of SuperScript III Reverse Transcriptase (Invitrogen). The cDNA was purified using High Pure PCR Product Purification Kit (Roche) and used for cDNA tailing using 800U of rTdT (Fermentas) and 0.5 mM dGTP (Roche) in 50 μl reaction for 30 min at 37°C with subsequent heat inactivation of rTdT for 10 min at 70°C. For PCR amplification of cDNA ends, 2.5 μl of the reaction mixture was used with olig2(dC)anchor primer and appropriate gene-specific primer for 35 cycles.In the case of 3′ RACE, 1 μg of total yeast RNA was polycytidinylated using Poly(A) Tailing Kit (Applied Biosystems) and 2 mM CTP (Thermo Scientific) for 90 min at 37°C. Following reverse transcription was performed using 100U of SuperScript III Reverse Transcriptase (Invitrogen) and 1 μg of oligo(dG)anch2 primer. The cDNA was purified using High Pure PCR Product Purification Kit (Roche) and 2.5 μl of the purified cDNA was used for PCR amplification of cDNA ends with anch2 primer and appropriate gene-specific primer for 35 cycles.E. coli XL-1 Blue cells (Stratagene), isolated using GenBond Plasmid FlexSpin Kit (Renogen Biolab) and sequenced using universal T7 promoter primer or T3 primer.In both RACE experiments, after PCR amplification and electrophoresis, obtained fragments were verified using restriction digestion and fragments exhibiting correct digestion pattern were gel-purified using FavorPrep GEL/PCR Purification Kit (Favorgen) and cloned to the pCR4-TOPO vector (Invitrogen). Vectors were transformed into Modelling of the tertiary structure of the non-canonical RNA polymerase of the pGKL elements was carried out on the Robetta server . SelectiSchizosaccharomyces pombe RNAP II (PDB ID: 3H0G.B), residues 754–882 were modelled by β′ subunit of Thermus thermophilus RNAP (PDB ID: 2A6H.D), and residues 894–974 were modelled by β′ subunit homolog of S. pombe RNAP II (PDB ID: 3H0G.A). Amino acid residues 1–52 of K2ORF7p were modelled by β′ subunit homolog of S. pombe RNAP II (PDB ID: 3H0G.A), and residues 103–132 were modelled by β′ subunit homolog of Saccharomyces cerevisiae RNAP II (PDB ID: 1TWF.A).Amino acid residues 1–693 of K2ORF6p were modelled by β subunit homolog of S. cerevisiae RNA polymerase II elongation complex (PDB ID: 2NVQ) using DaliLite v. 3 in pairwise option and visualized together using PyMOL 1.3 software . The template modeling score (TM-score) value [http://zhanglab.ccmb.med.umich.edu/TM-score/). Methionine 9 residue of the K2ORF6p sequence was considered to be the methionine 1 residue because it is thought that the second ATG within the ORF is the true start codon [Models of each part of K2ORF6p and K2ORF7p with the best Z-scores from previous comparison using DaliLite v. 3 were individually superimposed to e) value , which mKluyveromyces lactis (P05472.1 and P05473.1), Saccharomyces kluyveri (CAA38625.1 and CAA38626.1), Pichia acaciae (CAJ57280.1 and CAJ57281.1) and Pichia etchellsii (CAC08226.1 and CAC08227.1). The joined sequences of RNAP subunits were aligned using default settings of Clustal Omega [Lausannevirus (YP_004346983.1 and YP_004346982.1), Marseillevirus marseillevirus (YP_003406803.1 and YP_003406800.1), Pandoravirus dulcis (YP_008318947.1), and Pandoravirus salinus (YP_008436862.1) RNAP β′ subunits were aligned using default settings of Clustal Omega, and their β′ conserved regions were then manually added to the final alignment. PhyML 3.0 with Smart Model Selection [Phylogenetic analysis was performed using β′ subunit conserved sequence regions of multisubunit RNAPs that are present in all canonical RNAPs together with corresponding regions that were found in yeast VLE RNAPs. A single amino acid (aa) sequence was created by joining the aa sequences of K2ORF6p and K2ORF7p for each of the following organisms: al Omega . β subunal Omega , 24 idenal Omega  of β′ suelection  was usedelection  was usedhttp://weblogo.berkeley.edu/logo.cgi) was used to create a consensus motif. The literature was searched for elements with similar motives known to be associated with promoter activity of multisubunit RNAPs.The known UCSs preceding all 15 pGKL-encoded genes  were useK2ORF2, K2ORF3 and K2ORF8 transcripts displayed a single peak TSS pattern, where more than 60% clones had the same initiator nucleotide and other initiator nucleotides were not represented in more than 25% of the clones. Sequencing of cDNA clones corresponding to transcripts of other pGKL-encoded ORFs displayed a multiple peaks TSS pattern, due to presence of a non-templated 5′ poly(A) leader of a heterogeneous length. For those ORFs the first adenosine residue of the clones that could be aligned to the promoter template sequence was considered to be the initiator nucleotide.Transcription start sites (TSSs) were annotated according to our previous 5′ RACE-PCR experiments covering all pGKL genes . BrieflyS. cerevisiae S6/1 as a sensitive strain. Approximately 2x105 of sensitive yeast cells were plated onto YPD plates  for testing of pGKL1 killer toxin activity. Wells were made with an 8 mm diameter cork borer and 100 μl of filter sterilized culture medium or 100 μl of serial dilution of the filter sterilized culture medium was pipetted into well.Filter sterilized culture medium was tested for the presence of the killer toxin activity by the agar well diffusion assay using S1 FigK. lactis strain with wild-type pGKL elements.For each electrophoretogram DNA mass markers are indicated on the left, and native or recombinant pGKL elements are indicated on the right side, respectively. Recombinant pGKL elements are marked in red. M1, DNA molecular mass marker (Lambda DNA/Eco130I (StyI) Marker, Fermentas). M2, DNA molecular mass marker . IFO1267—(TIF)Click here for additional data file.S2 Fig(A) IFO1267_pRKL2-4 (containing yEGFP3-K2ORF6p) and IFO1267_pRKL2-11 (K2ORF3p-yEGFP3) cells were grown to late exponential phase. The cells were lysed, yEGFP3-K2ORF6p and K2ORF3p-yEGFP3 were affinity-purified using GFP-Trap agarose beads. Bound proteins were digested with trypsin, and then analyzed by mass spectrometry. Also, an aliquot of the beads was taken and bound proteins were eluted and resolved by SDS-PAGE, and the gel was silver-stained. Proteins identified by mass spectrometry are indicated with arrows on the right side. Proteins identified by mass spectrometry in previous experiments described in (TIF)Click here for additional data file.S3 Fig(A) yEGFP3-K2ORF6p was purified with GFP-Trap agarose beads from lysates of the IFO1267_pRKL2-6 strain cells. After washing the bound immunoprecipitated proteins (α-GFP IP), the beads were split into two parts which were mock-treated (Benz-) and treated (Benz+) with Benzonase Nuclease to digest DNA and RNA. Then, the beads were extensively washed again and the bound proteins were eluted and analysed by Western blotting using anti-GFP (α-GFP) and anti-HA (α-HA) antibodies. Also, an aliquot of the beads was taken and bound nucleic acids were eluted and analyzed for presence of pRKL2-6 VLE DNA by PCR amplification for 25 cycles and visualized by electrophoresis. M, DNA molecular mass marker ; respective molecular mass values of two DNA fragments (500 bp and 1 000 bp) are indicated. (B) yEGFP3-K2ORF6p was purified with GFP-Trap agarose beads from lysates of IFO1267_pRKL2-7 strain cells. After washing the bound immunoprecipitated proteins (α-GFP IP), the beads were split into two parts that were mock-treated (Benz-) and treated (Benz+) with Benzonase Nuclease to digest DNA and RNA. Then, the beads were extensively washed again and the bound proteins were eluted and analyzed by Western blotting using anti-GFP (α-GFP) and anti-HA (α-HA) antibodies. Also, an aliquot of the beads was taken and bound nucleic acids were eluted and analyzed for presence of pRKL2-7 VLE DNA by PCR amplification for 22 cycles and electrophoresis. M, DNA molecular mass marker ; respective molecular mass values of two DNA fragments (500 bp and 1 000 bp) are indicated.(TIF)Click here for additional data file.S4 FigK. lactis pGKL1 (X00762.1) and pGKL2 (X07776.1) sequences. Sequences situated below represent individual sequenced cDNA clones . Cytosine residues at the 3′ end of cDNA corresponding to the RNA tail are omitted in this representation for clarity. RNA secondary structures close to 3′ ends of VLE-specific mRNAs were predicted using default settings of RNAstructure Server (http://rna.urmc.rochester.edu/RNAstructureWeb/) [K1ORF1, (B) K1ORF2, (C) K1ORF3, (D) K1ORF4, (E) K2ORF1, (F) K2ORF2, (G) K2ORF3, (H) K2ORF4, (I) K2ORF5, (J) K2ORF6, (K) K2ORF7, (L) K2ORF8, (M) K2ORF9, (N) K2ORF10, (O) K2ORF11.This figure represents results of 3′ RACE-PCR analysis of individual mRNAs corresponding to all genes encoded by the pGKL elements. Total RNA was isolated from wild-type IFO1267 strain, DNase treated and 3′ polycytidinylated. Reverse transcription was carried out using oligo(dG)anch2 primer. Purified cDNA was used for 3′ RACE-PCR using anch2 primer and gene-specific primers listed in ureWeb/) . Predict(PDF)Click here for additional data file.S5 FigK2ORF5 gene in the same transcriptional orientation. (B) Total RNA was isolated from IFO1267_pRKL2-3 and IFO1267_pRKL2-12 strains, DNase treated and 3′ polycytidinylated. Reverse transcription was carried out in the presence (+RT) and absence (-RT) of reverse transcriptase using oligo(dG)anch2 primer. Purified cDNA was used for 3′ RACE-PCR using anch2 primer and gene-specific primers listed in This figure represents results of 3′ RACE-PCR analysis of individual mRNAs corresponding to the G418 and hygromycin B resistance genes expressed under control of K1UCR2 and K1UCR3, respectively. (A) Schematic representation of recombinant pGKL2 elements where the resistance marker genes are inserted prior to (TIF)Click here for additional data file.S6 FigThermus aquaticus β (CAB65465.2), Escherichia coli β (AAC76961.1) Methanocaldococcus jannaschii B′′ (Q58444.1), Methanocaldococcus jannaschii B′ (Q60181.1), Saccharomyces cerevisiae Rpa135 (CAA95050.1), Saccharomyces cerevisiae Rpb2 (NP_014794.1), Sascharomyces cerevisiae Rpc128 (CAA99422.1), Vaccinia virus Rpo132 (AAQ93241.1), Kluyveromyces lactis ORF6-pGKL2 (P05472.1), Saccharomyces kluyveri ORF6-pSKL (CAA38625.1), Pichia acaciae ORF6-pPac-1 (CAJ57280.1), Pichia etchellsii ORF6-pPE1B (CAC08226.1).Conserved regions are named according to ref. . In the (DOCX)Click here for additional data file.S7 FigThe trees are ranked from best to worst based on their likelihood. c-ELW, Expected Likelihood Weight. p-AU, p-value of approximately unbiased (AU) test. logL, log-likelihood. Plus signs denote the 95% confidence sets. Minus signs denote significant exclusion. All tests were performed with 10 000 resamplings using the RELL method.(XLSX)Click here for additional data file.S8 FigS. cerevisiae S6/1 sensitive strain cells grown on YPD plates at 24°C for 2 days. Result from post-cultivation medium taken at 12 hours is shown.This figure represents results of killer toxin activity assay. IFO1267_pRKL1-9 (TACCC INR) and IFO1267 (control) cells were cultivated in YPD medium at 24°C. Aliquots were taken at 0, 3, 6 and 12 hours, and the culture medium was filter-sterilized, diluted, and assayed for the presence of the killer toxin activity by an agar well diffusion test using a lawn of (TIF)Click here for additional data file.S9 Fighttp://www.biossc.de/seqool/dwnload.html) and plotted. The occurrence of the T residue in the 8 3′ terminal nucleotides of cDNA ends (including TSS) is ≥ 50%.This figure represents results of sequence analysis of all cDNA clones whose sequences were detected in at least two independent clones obtained from the 3′ RACE-PCR analysis of mRNAs corresponding to all genes encoded by the pGKL elements. Those cDNA sequences (58 non-redundant sequences) were manually aligned according to their transcription termination site (TSS), and frequencies of individual nucleotides were calculated for each position within the 50 nt upstream of the TSS using Seqool 3.1 ((DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Telomeres, the nucleoprotein structures at the ends of eukaryotic chromosomes, play an integral role in protecting linear DNA from degradation. Dysregulation of telomeres can result in genomic instability and has been implicated in increased rates of cellular senescence and many diseases, including cancer. The integrity of telomeres is maintained by a coordinated network of proteins and RNAs, such as the telomerase holoenzyme and protective proteins that prevent the recognition of the telomere ends as a DNA double-strand breaks. The structure of chromatin at telomeres and within adjacent subtelomeres has been implicated in telomere maintenance pathways in model systems and humans. Specific post-translational modifications of histones, including methylation, acetylation, and ubiquitination, have been shown to be necessary for maintaining a chromatin environment that promotes telomere integrity. Here we review the current knowledge regarding the role of histone modifications in maintaining telomeric and subtelomeric chromatin, discuss the implications of histone modification marks as they relate to human disease, and highlight key areas for future research. Saccharomyces cerevisiae and Schizosacchromyces pombe have long served as models for understanding telomere function. We focus on key findings described in yeast, and highlight findings connecting chromatin structure, telomere function, and disease in mammalian systems.Due to the linear nature of eukaryotic chromosomes, their ends must be protected in order to prevent the progressive loss of sequence with each round of cell division, which can eventually lead to genomic instability, disease, and cell death ,2. TelomThe basic unit of chromatin, the nucleosome, consists of 146 base pairs of DNA wrapped around an octamer of histone proteins . One of the main mechanisms for the regulation of chromatin structure is through the post-translational modification (PTM) of histones, which most commonly occur on the amino acid residues lysine, arginine, serine, tyrosine, and threonine. The addition or removal of these chemical groups to or from specific residues on histones can both alter histone–DNA association and serve as a signal to be recognized by chromatin-modifying or remodeling proteins, which ultimately alter transcriptional activity. Post-translational histone modifications can include phosphorylation, methylation, acetylation, sumoylation, and ubiquitylation. Crosstalk between these different modifications results in context-dependent mechanisms through which gene expression and other genomic functions are regulated ,10,11,12The enzymes that facilitate the addition or removal of these marks have been termed “writers” and “erasers,” respectively. Examples of writers include histone acetyltransferases (HATs), arginine and lysine methyltransferases , ubiquitin ligases, and kinases. Conversely, the respective erasers include histone deacetylases (HDACs), arginine and lysine demethylases , deubiquitinases, and phosphatases. Proteins that recognize and bind to PTMs, or to an unmodified region of a histone, are classified as “readers”. These proteins typically rely on specialized domains, a few of which include the methyl–lysine or acetyl–lysine chromodomains, PHD fingers, or bromodomains ,14,15. T1–3) repeats with 3’ overhangs of the G-rich strand, and the adjacent subtelomeres contain repeat sequences known as X elements, which are found on all chromosome ends, and Y’ elements, which are found in variable numbers in a subset of subtelomeres [A requirement for proper telomere function is the maintenance of the adjacent chromatin landscape. Disruptions to chromatin structure through the mutation or altered expression of chromatin-modifying factors have been linked to telomere dysfunction and genomic instability ,16,17,18elomeres  Figure –3 repeatelomeres . The t-lelomeres .In yeast, the telomeric repeats do not appear to be associated with nucleosomes; however, subtelomeric DNA is associated with nucleosomes and is thought to be predominantly composed of transcriptionally silent chromatin  Figure A. The suThe nature of chromatin at mammalian telomeres is subject to more debate. While there is significant evidence indicating that telomeres are likely to be largely heterochromatic, the existence of heterochromatin at telomeres may vary in different cell types or under different conditions , and thehTERT gene in humans, which encodes the telomerase reverse transcriptase [hTERT transcription. However, as telomeres shorten, the silencing loop structure is no longer in close proximity to hTERT, leading to the transcriptional activation of hTERT, and therefore to increased telomerase activity, which counteracts the telomere shortening. These findings suggest that TPE may function to both regulate gene expression and maintain telomere length as cells senesce.In diverse systems, genes adjacent to telomeric regions are often lowly expressed and subject to transcriptional silencing, an observation known as the telomere position effect, or TPE . TPE is criptase ,38. When+-dependent deacetylase [Acetylation of lysines on histones lessens their overall positive charge, weakening histone–DNA interactions and providing a permissible chromatin landscape for transcriptional activation. Silent regions of the genome, such as within yeast subtelomeres and at mammalian heterochromatin, often contain hypo-acetylated histones. Histone acetylation marks that are typically associated with transcriptionally active areas of the genome include those on H2AK7, H2BK11 and K16, H3 at lysines 9, 14, 18, 27, and 56, and on H4 at lysines 5, 8, 12, and 16 ,40. In ycetylase . The abscetylase , suggestcetylase ,48,49,50cetylase ,52,53 thcetylase ,54. SIRTcetylase , indicatWhile the majority of lysines on histones are hypoacetylated near telomeres, it has been shown in budding yeast that H4K12ac, established by the NuA4 acetyltransferase complex, is found in telomeric regions . This maS. pombe. The proper distribution of histone methylation marks at telomeric and subtelomeric regions is established through multiple mechanisms, some of which are not completely understood; however, a recent study identified a link between the non-coding RNA TERRA and the establishment of heterochromatic marks at telomere repeats in human cell lines [In contrast to histone acetylation, which is predominantly associated with gene activation, the addition of methyl groups to histone lysine  or arginll lines . This woDot1L alters the distribution of marks within telomeres, including decreasing the repressive mark H4K20me3 and increasing the mark of euchromatin H3K9ac [The H3K79 methyltransferase Dot1, or disruptor of telomeric silencing, was originally described for its role in promoting the silencing of subtelomeric genes in yeast and its loss leads to defects in telomere length . It has n H3K9ac . Addition H3K9ac . TogetheGene silencing within subtelomeric regions and the regulation of telomere length are also known to depend on H3K4 methylation, particularly in yeast and likely in mammalian systems ,80,81,82In budding yeast, a proposed explanation for defective telomere silencing in the absence of Set1 involves the titration of the SIR deacetylase complex away from the euchromatin–heterochromatin boundary, leading to increased histone acetylation ,96. HoweThe ubiquitination of H2BK123 in budding yeast, mediated by Rad6 and Bre1, is often a prerequisite for the establishment of other histone PTMs, including H3K4me and H3K79me ,108,109.DKC1, as well as telomere protection factors, including shelterin components like POT1 [Telomere syndromes, or telomeropathies, are genetic disorders that have generally been linked to germline mutations in genes encoding telomere maintenance factors, including telomerase subunits and the proteins required for its function, such as ike POT1 . These sike POT1 ,118. Whiike POT1 ,7, thereThe pleiotropic nature of mutations in or the dysregulation of chromatin-modifying enzymes make it challenging to attribute cellular abnormalities to defects in the localized function of a chromatin modifier, such as at telomeres. However, some modifiers, such as SIRT6, distinctly manifest phenotypes associated with telomere dysfunction and have molecular roles at telomeres ,54,55, iDUX4 in a subset of patients, particularly in the presence of short telomeres [SORBS2 and TMEM8C, each important for muscle differentiation, were also identified as being subject to regulation via TPE in a manner dependent on telomere length and as possibly contributing to the development of FSHD [Intriguingly, there are key examples indicating that the regulation of gene expression via TPE is critical for preventing diseases associated with telomere maintenance defects. Facioscapulohumeral muscular dystrophy, or FSHD, is a myopathy characterized by facial, scapular, and arm muscle atrophy and shows a late onset associated with telomere attrition . The etielomeres . In anot of FSHD . TogetheThe examples of chromatin regulation discussed here suggest that the composition and distribution of histone modifications within telomeric regions is important to telomere function, as aberrant patterns of modifications are linked to telomeric instability and altered gene expression patterns in subtelomeres. Studies from model systems, in particular budding and fission yeast, have provided great insight into the role of histone modification marks and chromatin structure in telomere maintenance pathways. Much of the foundational knowledge gained from these systems is also applicable to humans; however, there are still substantial gaps in our understanding of the interplay between chromatin structure, gene repression, and telomere integrity in human cells. Detailed mechanistic studies are required to further elucidate the specific contributions of histone modifications to chromatin regulation near telomeres and their roles in telomere maintenance in mammalian systems. For example, the findings in yeast implicating the conserved marks H2BK123ub and H3K4me in telomere maintenance suggest that the exploration of the roles of these marks in human cells at telomeres may uncover new mechanisms by which telomeric and subtelomeric chromatin are regulated. Furthermore, while studies of some chromatin modifiers, such as SIRT6, and the characterization of some telomere-linked diseases, such as FSHD, indicate a key role for chromatin organization and histone modifications in preventing the accumulation of telomere-linked defects, there are relatively few defined links between chromatin regulation and pathologies associated with abnormal telomere function. Determining the precise contribution of chromatin-modifying proteins to genetic telomeropathies or the epigenetic mechanisms of disease associated with telomere dysfunction, including pathologies associated with aging, will shed light on both basic cellular mechanisms of telomere regulation and genome stability, and open new avenues for alleviating diseases linked to dysfunctional telomeres.


The chromosomal loci known as centromeres (CEN) mediate the equal distribution of the duplicated genome between both daughter cells. Specifically, centromeres recruit a protein complex named the kinetochore, that bi-orients the replicated chromosome pairs to the mitotic or meiotic spindle structure. The paired chromosomes are then separated, and the individual chromosomes segregate in opposite direction along the regressing spindle into each daughter cell. Erroneous kinetochore assembly or activity produces aneuploid cells that contain an abnormal number of chromosomes. Aneuploidy may incite cell death, developmental defects (including genetic syndromes), and cancer . While kinetochores and their activities have been preserved through evolution, the CEN DNA sequences have not. Hence, to be recognized as sites for kinetochore assembly, CEN display conserved structural themes. In addition, CEN nucleosomes enclose a CEN-exclusive variant of histone H3, named CENP-A, and carry distinct epigenetic labels on CENP-A and the other CEN histone proteins. Through the cell cycle, CEN are transcribed into non-coding RNAs. After subsequent processing, they become key components of the CEN chromatin by marking the CEN locus and by stably anchoring the CEN-binding kinetochore proteins. CEN transcription is tightly regulated, of low intensity, and essential for differentiation and development. Under- or overexpression of CEN transcripts, as documented for myriad cancers, provoke chromosome missegregation and aneuploidy. CEN are genetically stable and fully competent only when they are insulated from the surrounding, pericentromeric chromatin, which must be silenced. We will review CEN transcription and its contribution to faithful kinetochore function. We will further discuss how pericentromeric chromatin is silenced by RNA processing and transcriptionally repressive chromatin marks. We will report on the transcriptional misregulation of (peri)centromeres during stress, natural aging, and disease and reflect on whether their transcripts can serve as future diagnostic tools and anti-cancer targets in the clinic. During cell division, the replicated chromatids that are associated by cohesin rings bind to the microtubules of the metaphase spindle, which extend from two opposite spindle poles Figure . This biSaccharomyces cerevisiae chromosome 3, and all three CEN of the fission yeast Schizosaccharomyces pombe were the first CEN loci to be characterized  are made up of 10-bp repeats followed by 11/12-bp tandem repeats n followed by doceda tandem repeats (CCCGTACT[C]GGT) that show an asymmetric distribution of G and C residues. In vitro, the C-rich dodeca satellite single strand produces an “i-motif”; a cubic structure that is formed by the head-to-tail association of two parallel strands combined in antiparallel fashion  and Lys36  Figure . These mS. pombe), is required for CEN transcription , and at neocentromeres  was removed, indicating a fast turn over of these transcripts. This approach revealed a 1.2-kb CEN3 RNA species, revealing that RNAPII proceeds into the pericentromere  tails in a portion of the beetle CEN transcripts indicates RNAPII-dependent transcription. Indeed, treatment of larvae with alpha-amanitin at concentrations that selectively inhibit RNAPII activity reduced the amount of PRAT transcripts. These transcripts derived from one, two, or three monomers, and were produced from both strands  (The PRAT CEN satellite monomer in the beetle  strand) . Within  strand) . In huma strand) , was ide strand) .per se is not enough for CEN formation since trypanosomes and insects with holocentric chromosomes lack a CENP-A ortholog . Kinetochores then reassemble to attach the still-replicating chromatids to the interphase spindle   . Studiesn (FACT) . Of noten (FACT) . In fungn (FACT) . In Drosed state .Alpha-satellite arrays amplified from human CEN and cloned into a BAC plasmid form a functional HAC that recruits kinetochores and stably propagates in HT1080 fibrosarcoma cells . In HACsDrosophila CENP-A is phosphorylated at Ser75 and Ser77, which could be the analogs of Ser16 and Ser18 in human CENP-A  is recruited to early mitotic kinetochores by the Bub1-phosphoryated centromeric histone H2A . Next, Sgo1 binds to RNAPII and travels along with it to the inner CEN (region between the two sister CEN) where it binds to the cohesin rings to protect them from precocious cleavage by the protease separase . TranscrIn contrast to human alpha-satellite transcripts, murine MinSat transcripts are absent in G0/G1. They appear in S-phase, peak at G2/M, and become undetectable after mitosis, when cells re-enter the cell cycle . This dySchizosaccharomyces pombe CEN are transcribed during DNA replication, which may generate transcription–replication conflicts. Encounters between RNAPII and the replisome may cause RNAPII to halt and produce immature transcripts  but predominantly contain 40 and 75-nt species . Althougdicer-1 (DCRΔ/Δ) are defective in global RNAi activity but retained ES cell characteristics. Although viable, they proliferated more slowly (Δ/+) produced 150-nt MinSat and MajSat species, as well as 21–30 nt long specimen, suggesting the contribution of Dicer (Mouse embryonic stem (ES) cells knocked out in e slowly . No aberof Dicer . Furtherof Dicer .In tammar wallaby cells, 34–42 nt double-stranded (ds) RNAs with homology to the CEN retroelement kLTR  were ideDolichopoda cave crickets , their binding to a specific satellite monomer box (CENP-B), their recruitment by the CEN H2A T120P modification (Sgo1), and their recruitment by Thr3-phosphorylated CEN histone H3 and CEN H2A T120P (CPC).In silico predictions of potential RNA-binding sites indicated that 286 out of the 748 HJURP residues, and 79 out of the 140 CENP-A residues, have RNA-binding capacity. However, the vast majority of these CENP-A residues may well be buried inside the nucleosome and/or be bound by CENP-C and CENP-N. The predicted residues lie in the N-terminal half of CENP-A, the protein’s most rapidly evolving part , CEN DNA binding is stabilized by long ssRNA in vitro. The RNAs that stabilize this contact correspond to the ssCEN RNAs present in kinetochores  to reduce local transcription by promoting the methylation of CEN DNA and histone H3. Consequently, CENP-C depletion caused increased CEN transcription . CENP-C The binding of CENP-A and CENP-C to CEN DNA and alpha-satellite RNA promotes kinetochore assembly, including the recruitment of the 4-protein CPC , which regulates chromosome-spindle attachment and activates the SAC upon chromosome misalignment . The CPCXenopus egg extracts  to form ribonucleoprotein complexes, as observed in anti-Aurora B immunoprecipitation experiments with interphase and mitotic cells, followed by RNA-sequencing. Over 600 RNAs were identified, 465 of which were specific for mitosis, suggesting a cell cycle-regulated binding of target RNA. Identified RNAs encode proteins of the cytoskeleton, centrosome, transcription factors, and RNAs that are enriched on spindle microtubules . They further contain DNA transposons (1 kb), long terminal repeat (LTR)-endogenous retroviral elements (10 kb), non-LTR autonomous retrotransposons (transposons that are formed after reverse transcription of an intermediate RNAPIII-generated transcript) including long interspersed elements  and short interspersed elements SINE (100–300 bp) Figure . PericenS. pombe, repressive H3K9 methylation occurs at the outermost dg and dh pericentromere repeats and ends at the tRNA clusters inside the innermost repeats that surround the CEN’s central domain. Their presence prevents the pericentromeric heterochromatin from expanding into the CENP-A chromatin  produced after the processing of longer transcripts are required for the propagation and maintenance of the heterochromatic identity of pericentromers  and the SHREK-associated protein Chp2, as well as histone deacetylase Clr3 (HDAC1), which removes the local permissive H3K14ac marks. The SHREK complex inhibits RNAPII activity, resulting in silent heterochromatin  caused chromosome missegregation due to defective silencing of the pericentromeric heterochromatin. S. pombe strains mutated in RNAPII subunits Rpb2 and Rpb7 also suffered from increased chromosomal instability, impaired transcriptional silencing, and a reduced association of H3K9me and Swi6 at dg/dh 3-9 and HP1 recruitment (Drosophila has been hypothesized since a nuclear pool of transposable element-derived siRNAs (21 nt) was shown to promote heterochromatin formation in somatic cells of Drosophila. Components of the RNAi pathway contributed to heterochromatin maintenance 3-9. RNAi activity seems to help establish heterochromatin in the early embryo, which can then be maintained in the absence of RNAi in somatic tissues . Comparable ectopic overexpression of MinSat DNA led to decondensed CEN and mitotic defects such as multiple spindle attachments, loss of sister chromatid cohesion, aneuploidy, and cell death .per se does not trigger the DNA damage response . Possibly, the transcripts could regulate local RNA splicing during the stress response by sequestering splicing factors. Via a position-effect mechanism, they might counteract the repressive nature of heterochromatin and activate nearby genes in cis or trans (How do (peri)centromere transcription/transcripts contribute to a stress response and recovery from it? The transcripts processed via an RNAi-dependent or -independent pathway could mediate heterochromatin reformation, as in or trans .Heterochromatin structure and expression change during aging. An up-regulation of MajSat expression in senescent cardiac muscle cells of aging mice but not in their brain or kidneys may be linked to mitochondria-induced oxidative stress . TranscrPericentromeric heterochromatin was show to lose H3K9me3 and HP1 proteins in older flies and human cells, leading to an anomalous expression of satellite sequences . Loss ofex vivo, high expression levels similar to those observed in tumors in vivo were measured in immortalized PDAC tumor cells treated with 5-aza-2′-deoxycytidine, suggesting that transcription is regulated by DNA methylation, which might be re-established together with other epigenetic silencing mechanisms ex vivo. Furthermore, SatII expression showed a median 21-fold increase in human PDAC samples in comparison with “healthy” tissue samples centromeric DNA at cytosines in CpG dinucleotides, is recruited by CENP-C. Impairment of this interaction causes an overproduction of CEN and pericentromere transcripts . BesidesThe histone demethylase JMJD2B acts as an oncogene in certain breast cancers . When ovSatII and SatIII transcripts were markedly overexpressed in human osteosarcoma cells depleted in tumor suppressor SIRT6, which deacetylates histone H3K16ac in pericentric heterochromatin. Its inactivation led to H3K18 hyperacetylation likely by the histone acetyltransferase GCN5, reversal of heterochromatin silencing, mitotic defects, genomic instability, and senescence. Importantly, depletion of the transcripts through RNAi rescued the phenotypes .Mutations in the hereditary ovarian and breast cancer susceptibility gene BRCA1, which acts as a tumor suppressor, led to genomic instability. While BRCA1 acts in DNA replication and damage repair, control of the cell cycle, and many other regulatory functions, the protein was recently shown to also determine the epigenetic states of centromeric and pericentric chromatin . ThroughThe tumor-suppressing transcription factor Prep1 is associated with DNA damage control and the management of histone methylation levels . Indeed,Tumor-suppressing transcription factor p53 cooperates with DNA methylation activity to silence a large part of the mouse genome. A massive transcription of major classes of retroelements, near-CEN tandem repeat satellite DNAs, and numerous species of non-coding RNAs was observed in p53-deficient mouse fibroblasts treated with 5-aza-2′-deoxycytidine (not observed in treated p53 wild-type cells). The levels of these transcripts exceeded those of β-actin mRNA by more than 150-fold. Accumulation of these transcripts, which are capable of forming dsRNAs, was complemented by a potent apoptosis-inducing type I interferon response. The authors suggested a model in which the downregulation of these repeat sequences is controlled by p53-driven transcriptional silencing, DNA methylation-based suppression of transcription, and the suicidal type I interferon response, which eliminates the cells that escaped the first two lines of control .Rb1 mutant mice, which are defective in recruiting EZH2 to repetitive sequences, a transcriptional derepression of satellite DNA was observed, which induced susceptibility to lymphoma ((Peri)centromere silencing is also regulated by the Polycomb repressive complexes PRC1 and PRC2, which are commonly misregulated in cancer . PRC2 lylymphoma .in situ hybridization analysis of SatII expression in biopsies proved a better diagnostic for pancreatic cancer than standard histopathological analysis  that impinge on centromeric and pericentromere domains via ever more sensitive mass spectrometry approaches will take our understanding of CEN, kinetochore, and pericentromere biology to the next level. In addition, transcription factors that drive (peri)centromere transcription in healthy and diseased cells must be identified as their biology and influences on the spatiotemporal regulation of the CEN and pericentric regions remains largely unknown. The same is true for regulators that act upon the RNAPII complex to orchestrate its activity  at (peri)centromeres. Kinases and phosphatase may be prime candidates. A better understanding of RNAi pathway involvement in mammalian biology would be welcomed as well. For sure, exciting (peri)centromere biology will continue to be “written” in laboratories worldwide, and hopefully at some point in cancer clinics as well.KS and PDW wrote the paper and made the figures. Both authors have approved of 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.


CXXC5 is a member of the CXXC‐type zinc‐finger protein family. Proteins in this family play a pivotal role in epigenetic regulation by binding to unmethylated CpG islands in gene promoters through their characteristic CXXC domain. CXXC5 is a short protein (322 amino acids in length) that does not have any catalytic domain, but is able to bind to DNA and act as a transcription factor and epigenetic factor through protein‐protein interactions. Intriguingly, increasing evidence indicates that expression of the CXXC5 gene is controlled by multiple signaling pathways and a variety of transcription factors, positioning CXXC5 as an important signal integrator. In addition, CXXC5 is capable of regulating various signal transduction processes, including the TGF‐β, Wnt and ATM‐p53 pathways, thereby acting as a novel and crucial signaling coordinator. CXXC5 plays an important role in embryonic development and adult tissue homeostasis by regulating cell proliferation, differentiation and apoptosis. In keeping with these functions, aberrant expression or altered activity of CXXC5 has been shown to be involved in several human diseases including tumourigenesis. This review summarizes the current understanding of CXXC5 as a transcription factor and signaling regulator and coordinator. Members of this family are characterized by a specialized CXXC domain and include CXXC1/CFP1, CXXC2/KDM2B, TET1/3 and DNMT1, among others.The CXXC5 protein, though much less understood, has been shown to act as a transcriptional regulator by binding directly to DNA.2The combined pattern of DNA and histone modifications confers an epigenetic code that can profoundly affect both chromatin structure and gene expression.4‐5CxxCxxC and CxxRxC, wherein x indicates residues other than cysteine), with three cysteines from the first motif and one from the latter together coordinating one Zn2+ ion, therefore forming two zinc‐finger structures.Twelve CXXC zinc‐finger protein family members have been identified in mammalians Table .1, 2, 3,3Although CXXC4 and CXXC5 do not have any catalytic domains, it is interesting that both have been found to interact with and recruit TET2, which functions in a similar way as TET1/3 to promote DNA demethylation but is unable to bind DNA directly due to the absence of the CXXC domain.Several other reports have provided additional evidence that CXXC5 could act as a transcription factor and/or epigenetic factor by binding to DNA via its CXXC domain Table . In neurIn other contexts, CXXC5 may repress gene expression. For instance, in CD8+ Th cells, CXXC5 could associate with the histone‐lysine N‐methyltransferase SUV39H1, inducing histone H3K9 methylation in the Cd40L gene promoter region and inhibiting CD40 ligand expression.44.1Transforming growth factor‐β is a multi‐functional peptide that plays a critical role in regulating various cellular functions and exerts a diverse array of physiological functions. Deregulation of TGF‐β signaling has been connected with several major human diseases, including developmental defects, tumourigenesis, cardiovascular disorders and metabolic syndromes.One major cellular response to cytokine stimulation is transcriptional alteration. Target gene identification and their functional analyses are crucial for understanding the context‐dependent biological roles of specific cytokines, including TGF‐β.Another important point to understand the pleiotropic nature of TGF‐β is the spatiotemporal regulatory mechanisms of the signal transduction process.4.2The Wnt family of cytokines plays a critical role in embryonic development and adult tissue homeostasis in multicellular animals.Not surprisingly, the Wnt/β‐catenin pathway is also subject to intense regulation.4.3The p53 tumour suppressor acts as a major defence against cancer by inducing DNA repair, cell cycle arrest, senescence or apoptosis in response to diverse cellular stresses.1α,25‐dihydroxyvitamin D3  is the active hormonal metabolite of vitamin D. It incites a variety of biological functions by binding the vitamin D receptor (VDR), thus stimulating its transcriptional activity.5The CXXC5 gene is evolutionarily conserved from Xenopus and zebrafish to mammals. It is located on chromosome 5q31.3 in human, spans 35.5 kb and contains 4 exons, with an mRNA product of approximately 1.4 kb.As mentioned above, the expression of the CXXC5 gene is transcriptionally induced by TGF‐β, BMP4 and Wnt3a, which rely on Smad proteins and β‐catenin respectively.Several transcription factors have been shown to bind to the CXXC5 promoter or enhancer, thereby regulating its transcription Table . The zinTogether, different cytokines and transcription factors converge on the CXXC5 gene to regulate its expression, suggesting that CXXC5 may act as an important downstream integrator and mediator of various signal inputs. Taking into account its ability to fine‐tune various signal transduction processes, CXXC5 is likely to function as a crucial coordinator of multiple cellular signaling pathways Figure .6Considering its roles in regulating gene expression and coordinating cellular signaling, it is not surprising that CXXC5 regulates a diverse array of cellular events including cell proliferation, differentiation and apoptosis in developmental processes, adult tissue maintenance and pathological circumstances.With respect to cell differentiation, retinoid‐induced CXXC5 has been shown to play a role in normal primary leukaemia cell differentiation and tumoral myelopoiesis.CXXC5 is involved in regulating cell proliferation and death in various cell types. In primary leukaemia cells, CXXC5 not only promotes differentiation, as mentioned above, but it also contributes to retinoid‐induced cell cycle arrest and cell death.Aberrant expression or altered activity of CXXC5 is closely linked to multiple human diseases, especially tumourigenesis Figure . The CXXCXXC5 has also been observed to play a role in some other pathological processes. Aberrant Wnt signaling has been found to be associated with hair loss  and anabolic osteoporosis. A previous study demonstrated that expression of CXXC5 is upregulated and is inversely correlated with that of β‐catenin in both haired and bald individuals.7CXXC5 shares a conserved CXXC domain with other CXXC‐type zinc‐finger protein family members, which utilize this domain to recognize unmethylated CpG dinucleotides, especially those within CpG islands in gene promoters. Therefore, the majority of the proteins in this family, including CXXC5, act as transcription factors and epigenetic regulators.First, although CXXC5 is capable of associating with unmethylated CpG islands via its CXXC domain and regulating the expression of certain genes, the specific genomic sites at which CXXC5 binds to in a given cell type or context remain to be determined.Second, CXXC5 is regulated by various cytokines and transcription factors and in turn regulates the intensity and duration of several cellular pathways. However, it is unknown whether and how the different signals converge on CXXC5 expression and how CXXC5 coordinates various signaling pathways in a certain circumstance.Third, the CXXC domain plays a crucial role in CXXC5‐mediated regulation of gene expression or signal transduction. However, as this domain is conserved in other CXXC‐type zinc‐finger proteins, it remains unclear how the functional specificity of CXXC5 is achieved and what functions the amino acids out of the CXXC domain may exert.Fourth, targeting of CXXC5, for example through inhibition of its interaction with the Dvl protein using competitive peptides or small molecules, is emerging as a promising means of therapeutic intervention in animal models. Given that aberrant CXXC5 expression is involved in various human diseases, especially cancer, it is reasonable to believe that targeting CXXC5 could be an effective clinical approach to treat these diseases.8The authors declare that they have no conflicts of interest. This is a review of previous studies and it does not contain any original human or animal studies performed by any of the authors.