To successfully engineermammalian cells for a desired purpose,multiple recombinant genes are required to be coexpressed at a specificand optimal ratio. In this study, we hypothesized that synthetic promotersvarying in transcriptional activity could be used to create singlemultigene expression vectors coexpressing recombinant genes at a predictablerelative stoichiometry. A library of 27 multigene constructs was createdcomprising three discrete fluorescent reporter gene transcriptionalunits in fixed series, each under the control of either a relativelylow, medium, or high transcriptional strength synthetic promoter inevery possible combination. Expression of each reporter gene was determinedby absolute quantitation qRT-PCR in CHO cells. The synthetic promotersdid generally function as designed within a multigene vector context;however, significant divergences from predicted promoter-mediatedtranscriptional activity were observed. First, expression of all threegenes within a multigene vector was repressed at varying levels relativeto coexpression of identical reporter genes on separate single genevectors at equivalent gene copies. Second, gene positional effectswere evident across all constructs where expression of the reportergenes in positions 2 and 3 was generally reduced relative to position1. Finally, after accounting for general repression, synthetic promotertranscriptional activity within a local multigene vector format deviatedfrom that expected. Taken together, our data reveal that mammaliansynthetic promoters can be employed in vectors to mediate expressionof multiple genes at predictable relative stoichiometries. However,empirical validation of functional performance is a necessary prerequisite,as vector and promoter design features can significantly impact performance. While single plasmids encoding multiple transcriptionalunits (TUs) in series can be constructed using Gibson or Golden Gateassembly technology with relative ease,12 control of the relative level at which several individual genesare constitutively expressed to achieve a desired stoichiometry isfar more difficult. Current methods to achieve controlled expressionof recombinant genes in mammalian cells employ multiple single genesynthetic circuits cooperatively functioning using inducible systemsand complex gene switches.15 However, constitutively controllingexpression of multiple genes simultaneously at different stoichiometrieson a single plasmid could be a simpler approach for in vivo systems and engineering.Mammalian cells utilize complex,finely tuned gene networks to maintain essential cellular functions.13 and feedback loops.16 However, this frequently involves the application of synthetic transcriptionfactors, such as transcription activator-like effectors (TALEs),17 zinc fingers,18 chimerictranscription factors,19 or CRISPR transcriptionfactors20 to induce cognate promoters.Alternatively, chemical chaperones,21 aptamers,22 metabolites,19 andother external stimuli15 have all alsobeen employed to induce and regulate synthetic gene circuit expression.These sophisticated biological control systems can be useful, butare also complex and unwieldy, with expression levels determined byligand  concentrationdependent transactivation or repression and the potential of impreciseand leaky expression.23 While complex, programmable geneexpression systems will be required for many applications, “hardwired”components operating at constitutive fixed stoichiometries generallyform the basis of all engineered systems.Recombinant gene expression withinsynthetic circuits can be preciselycontrolled using an assortment of oscillatory, logic gates24 In this case a promoter can be specificallydesigned to utilize the host cell’s existing repertoire oftransactivators to a varying extent in order to achieve a desiredlevel of transcriptional activity.26 As a means to controlmultigene expression stoichiometry, the use of well-defined syntheticpromoters in vector constructs is therefore a potentially attractivesolution.An alternative meansto control recombinant gene expression stoichiometryis the use of synthetic promoters with defined transcriptional activity.In this study, we tested the hypothesis that constitutivesyntheticpromoters varying in transcriptional activity could be used to creatediscrete multigene expression vectors (MGEVs) coexpressing recombinantgenes at a predictable relative stoichiometry in CHO cells. We revealthat mammalian synthetic promoters varying in transcriptional strengthcan be used to achieve variable multigene expression stoichiometry.However, multigene coexpression is inherently context-specific andpromoter and vector design features can significantly affect predictedperformance.24 Syntheticpromoter transcription factor regulatory element (TFRE) compositionis shown in 24Three heterotypicmammalian synthetic promoters previously designed to provide a relativelylow, medium, and high level of gene expression were selected froma library developed by Brown et al.Figure S1). These were cotransfected in three groups, each group consistingof three SGVs encoding each fluorescent protein under the controlof the same transcriptionally active promoter, either low (group 1),medium (group 2), or high (group 3), at a total plasmid DNA load rangingfrom 100 to 800 ng for subsequent comparison with gene expression from MGEVs, each syntheticpromoter was inserted upstream of three spectrally discrete fluorescentreporter proteins, eGFP, mCherry, and tagBFP to create a library ofnine SGVs  were measured by qRT-PCR.For each experiment an equal mass of eGFP, mCherry, and tagBFP SGVsutilizing either low, medium, or high strength promoters were mixedprior to transfection such that all reporters were cotransfected aseither low, medium of high strength synthetic promoter groups as previouslydescribed . To enable directcomparison of transcriptional activities for each reporter mRNA measurement,mRNA copies measured at each DNA load and varying promoter strengthwere arithmetically combined and normalized with respect to the lowstrength promoter data sets, incorporating the assumption that whilereporter-specific mRNA dynamics likely vary post-transcription 31 the transcriptional rate mediated by a given promoter was constantfor each reporter gene. This enabled reporter-specific external calibrationcurves to be created, relating measured mRNA copies to a cross-reportercomparable relative transcriptional activity (RTA). In each case,a third-order polynomial regression provided the line of best fit, potentially indicative of reducedself-inhibition  with increasedpromoter complexity at higher DNA loads.However, as the sensitivity of flow cytometric detectionof fluorescentproteins at low expression levels was limited, and in order to quantitativelycompare transcriptional activity more directly, we measured recombinantcellular reporter mRNA content using absolute quantitation qRT-PCR.To externally calibrate measured mRNA copies to variation in transcriptionalactivity for each reporter, recombinant mRNA copies derived from arange of transfected SGV total plasmid DNA loads .We tested the hypothesis that synthetic promoterscould be used to predictably control the relative level of expressionof recombinant genes arranged in series in MGEVs. We constructed alibrary of 27 MGEVs encoding eGFP, mCherry, and tagBFP in a fixedseries utilizing all possible combinations of synthetic promoters in the different positions within the series,while keeping the core (hCMV-MIE core), and the 5′ (AZ’sproprietary UTR) and 3′ (SV40 late polyA) UTRs constant, asshown in 6 cells. Under these conditions, the number of fluorescent gene copiestransfected (29113 ± 212 copies of each fluorescent reportergene per cell) was approximately equivalent to the number of genecopies transfected using 600 ng of combined SGV vector plasmid DNA and withinthe linear range . Therefore,derived from SGV expression data at the same plasmid DNA load employed,the predicted RTAs for low, medium, and high strength synthetic promoters,respectively, were 552 (±20), 2915 (±284), and 4384 (±874),respectively, a ratio of 1:5.3:7.9. For each MGEV, reporter mRNA copieswere measured by qRT-PCR and RTAs derived using the SGV external calibration within the vector backbone of both the MGEVs and SGVswere identical, hence potential bacterial sequence related transgenessilencing41 causing differences in transcriptionalrepression in a MGEV context compared to SGVs is unlikely. Furthermore,other studies have reported that transgene silencing may not be bacterialsequence specific.41The most obvious general trend observed was a substantialoverallrepression of reporter gene transcription relative to that observedusing SGVs .This repression is quantified per synthetic promoter in 42 Alternatively, the RNA pol IIelongation complex can also dislodge transcription factors bound tothe enhancer region of a downstream promoter resulting in repressedtranscription.42 Other dual promoter systemsin tandem arrangement within a standard or lentiviral vector havealso exhibited unpredictable gene expression both caused by transcriptionalinterference.44 Similarly, a triple gene cassetteconstructing a synthetic pathway in Saccharomyces cerevisiae also exhibited substantial discrepancies from predicted expressionattributed to transcriptional interference.45Gene positionaleffect within the library of vectors was quantifiedsimply by summation of all RTAs deriving from positions 1, 2, and3 for the detected RTA in position 2 and 3 of each MGEV. These positionaleffect normalized RTAs from MGEV expression were compared againstexpected RTAs  yielding a percentage of transcriptional repression foreach position. The distribution of transcriptional repression irrespectiveof position showed that the majority (90.1%) of expression was repressedby >50% and the median transcriptional repression was 68.6% 4A.Positional or promoter specific gene repression trends were highlightedin 48 When referring to the TFRE composition of the low, medium, and highstrength synthetic promoter, all six transcription factors and theircognate TFREs are shared between the promoter variants  with respect to genedosage, it is unlikely that competition for transcription factorswas caused by saturation of transcriptional activity from recombinantgenes. This would suggest the repressed state of the medium strengthpromoter is potentially caused by squelching. The enhanced activityof the low strength synthetic promoter neighboring a higher strengthsynthetic promoter variant could be caused by interaction betweentranscription factors. Literature has shown cofunctionality betweenderivatives of the C/EBP and NFκB transcription factors to initiatetranscription in immune and cancer cells.50 We speculate that the abundance of NFκB and its potency ontranscription rate,51 and interaction with C/EBP transcriptionfactors, may be enhancing transcription of the low strength syntheticpromoter within the local MGEV environment.We rationalize that the deviation of promoter activity  is context-specific to the localized environmentwithin a MGEV where promoter squelching may be impacting transcription.Promoter squelching refers to competition of transcription factorsand associated cofactors involved in regulating transcription betweenpromoter variants resulting in bias gene expression activity.variants 1A. The mde novo design ofMGEVs for future applications. The major factor impacting nonpredictablerecombinant gene expression performance was general repression ofcolocated transcriptional units utilizing synthetic promoters. A solutionto this could derive from a consideration of two related design criteria.Our data can be used to aid 52 where broad access to the hostcell’s transcriptional landscape is clearly a functional advantage in vivo. A variation of this design criterion would be theuse of synthetic promoters composed of different TFREs to avoid competitiveinterference.First, synthetic promoter design could be deliberately more complex,effectively increasing the diversity of TFREs and thus the availabletranscription factor repertoire that can drive expression of recombinantgenes, thus minimizing promoter–promoter competitive interference.More complex, larger viral promoters such as CMV exhibit lower self-repressionwithin a MGEV context,42 While other solutionsthat employ different means to simultaneously deliver multiple transcriptionunits could be utilized , in the currentcontext (delivery of plasmid DNA), a simpler practical solution couldbe the inclusion of efficient transcription terminator and insulatorelements within the MGEV56 to avoid, for example,transcriptional run-through.57 For example,a β-globin cotranscriptional cleavage (CoTC) terminator elementhas been shown to improve transcription termination efficiency andpotentially reduce transcriptional interference,58 whereas a chicken hypersensitivity site 4 (cHS4) insulatorwould avoid distal promoter-mediated transcription by functioningas a enhancer-blocker.61 However, both of these elements are large (rangingfrom 800 to 1200 bp)63 resulting in increased plasmidsize—risking cellular toxicity and poor transfectability.65 More recently, as part of their development, shorter insulatorswhich include the core CCCTC-enriched elements (250 bp) derived fromthe cHS4 element have maintained insulator functionality and couldbe a viable option to alleviate positional effects.62 We believe future studies exploring different transcription unitorientations such as a divergent conformation and inclusion of efficienttranscription terminators and insulators within a MGEV context wouldreveal transcriptional interference mechanisms contributing to unpredictableexpression. Moreover, we emphasize that while we show that syntheticpromoter technology can be used to control relative expression ofindividual recombinant genes encoded in a MGEV, additional testingof MGEV performance in a particular context is essential , and specific to the application.Second, spatial organization and isolation oftranscriptional unitsshould be improved to minimize positional and repressive effects.Co-located assemblies of synthetic DNA elements (an underpinning concept)may be inherently susceptible to corepression through steric limitationson transcription initiation.67 For example,adeno-assisted viruses (AAVs) achieve a precise expression stoichiometryof multiple genes (7) within a compact genome (∼5 kb).68 This is achieved using a combination of internal (within open-readingframe) promoters, differential mRNA splicing, overlapping open readingframes (ORFs), varying translation initiation rates (by varying startcodons) and feedback loops (using transactivators or repressors).69 Combined aspects of these control systems could be employed in syntheticDNA assemblies to achieve predictable coexpression performance; however,this would be entirely dependent on the desired application. Mostsynthetic systems would require coexpression of multiple distinctopen reading frames encoding different, nonoverlapping functions.The most advanced natural MGEV systems are arguably viruses—naturalbiological systems specifically designed for compact encoding of complexmultimeric assemblies and functions.In conclusion, our study highlights the importance of genetic engineeringplatform design systems. While it is entirely possible to rapidlysynthesize and assemble discrete DNA elements, whole synthetic assemblyfunctionality requires platform engineering control systems and partsthat enable essential system performance parameters such as relativeexpression level and stoichiometry to be predictably embedded.de novo synthesized by GeneArt  comprisinga human cytomegalovirus major-intermediate early (hCMV-MIE) core promoter,a fluorescent protein coding DNA sequence (CDS) for eGFP, mCherryand tagBFP, optimized for expression in Cricetulus griseus (Chinese hamster), and a simian virus 40 (SV40) late polyadenylationsequence . The proximal regionof the synthetic promoters developed by Brown et al.24 exhibiting a relatively low, medium, and high level oftranscriptional activity within CHO cells were PCR modified to facilitatesubcloning into the TUs by restriction digestion–ligation cloningupstream of the hCMV-MIE core element. The MGEV library was assembledby cloning the TUs into a recipient expression vector called pExp-Vec-GG(which was de novo synthesized by GeneArt comprisinga glutamine synthetase (GS) expression cassette regulated by an SV40promoter, a mammalian episomal origin of replication element, anda β-lactamase gene for ampicillin resistance selection; Figure S1). This was performed by using the GoldenGate assembly kit  according tothe manufacturer’s protocol. Successful SGV and MGEV constructionswere identified by a restriction digest of purified DNA.The transcription units (TUs) were designed in-house and l-glutamine (Thermo Fisher Scientific), hereby describedas CD CHO culture medium, for 72 h. Transfection was performed byelectroporation using the Amaxa Nucleofector system  coupled with the Nucleofector 96-well Shuttle system(Lonza) for high throughput (HT) transfection according to manufacturer’sprotocol. Each reaction comprised either 600 or 800 ng DNA resuspended into 2.5 μL of nuclease-free d.H2O (Qiagen)combined with 7.5 μL of nucleofection solution (prepared accordingto Amaxa SG Cell Line IV 96-well electroporation kit instructions(Lonza)). A total of 1.86 × 106 cells were preparedby resuspending into 10 μL of nucleofection solution. The DNAand cells were combined and 20 μL of the DNA-cell mix was transferredinto a 96-well Nucleocuvette plate (Lonza) and electroporated usingthe program FF-158. The transfected cells per well were recoveredby the addition of 80 μL of prewarmed CD CHO culture medium.The transfected cells were cultured by seeding 20 μL of transfectantsinto 180 μL of prewarmed CD CHO culture medium per well (10-folddilution) into a 96-well culture plate (Thermo Fisher Scientific).The culture plate was incubated at 37 °C, 5% (v/v) CO2 with humidity for 24 h prior to quantification by qRT-PCR or flowcytometry.Transient expressionwas performed in a CHO-K1 host cell line adaptedto growth in suspension in chemically defined medium, hereby referredto as CHO cells. Three biological replicates were performed in thisstudy where each biological replicate comprised three technical replicates.In each case, the cells were cultured prior to transfection in CDCHO medium  supplemented with6 mM g for 5 min and resuspended in 200 μL of RNAlater stabilizationreagent (Qiagen) prior to extraction. The total RNA from the transfectedcells was extracted using a RNeasy mini kit (Qiagen) according tothe supplier’s protocol, and purity was determined by the Nanodropspectrophotometer 2000 (Thermo Fisher Scientific). The extracted RNA(800 ng) was reverse transcribed using the QuantiTect reverse transcriptionkit according to the manufacturer’s protocol . The complementary DNA (cDNA) was diluted10-fold with nuclease-free d.H2O to a final volume of 200μL prior to qRT-PCR analysis. The reaction was set up by mixing2 μL of diluted cDNA, 2.5 μL of primer mix , 12.5 μL of QuantiFast SYBR green PCR master mix (Qiagen)and 8 μL of nuclease-free d.H2O. This was aliquotedin a MicroAmp fast optical 96-well plate . Triplicate reactions were prepared per sample alongside two negativecontrols, the absence of cDNA template and of reverse transcriptase(for genomic/plasmid contamination). The amplification process was95 °C for 5 min, followed by 94 °C for 15 s and 60 °Cfor 60 s over 40 cycles. Melting curve analysis was performed from60 to 95 °C. The cyclic threshold (Ct) was measured using the7500 fast real-time PCR system (Applied Biosystems). The mean Ct wascalculated per triplicate sample and normalized by comparison againsttwo internal reference gene controls (mmadhc and Fkbp1a).70 The primer amplification efficiencies for eGFP,mCherry, and tagBFP mRNA were calculated from standard curves . Efficiency was determined by usingthe equation E = 10 (−1/gradient) and wasbetween 93.1 and 97.9% .Transfected cells were harvested foreach biological replicate by centrifugation at 20071Transfected CHO cells expressingeGFP, mCherry, and tagBFP either as cotransfected SGVs or a MGEV foreach biological replicate were harvested after 24 h. The expressionwas determined by fluorescence using an Attune NxT flow cytometer(Thermo Fisher Scientific). A total of 10 000 viable cellswere analyzed per sample. The excitation lasers used to detect fluorescenceof tagBFP, eGFP, and mCherry were 405, 488, and 561 nm, respectively,and the emission filters were 440/50, 530/30, and 620/15, respectively.FlowJo software  was employed to apply fluorescentcompensation and analyze the data generated. The fluorescent expressionwas depicted as integrated median fluorescent intensity (iMFI) whichis calculated by multiplying the population frequency by the medianfluorescent intensity (MFI) for each fluorescent protein.


Chromatin consists of a complex of DNA and histone proteins as its core components and plays an important role in both packaging DNA and regulating DNA metabolic pathways such as DNA replication, transcription, recombination, and chromosome segregation. Proper functioning of chromatin further involves a network of interactions among molecular complexes that modify chromatin structure and organization to affect the accessibility of DNA to transcription factors leading to the activation or repression of the transcription of target DNA loci. Based on its structure and compaction state, chromatin is categorized into euchromatin, heterochromatin, and centromeric chromatin. In this review, we discuss distinct chromatin factors and molecular complexes that constitute euchromatin—open chromatin structure associated with active transcription; heterochromatin—less accessible chromatin associated with silencing; centromeric chromatin—the site of spindle binding in chromosome segregation. Chromatin is a complex of DNA and histone proteins with many functions, including packaging genetic material to fit inside the cell and regulating gene expression. Walther Flemming first described chromatin around 1880 to describe the stainable threads he observed in the nucleus during mitosis ,3,4. In Chromatin is divided into different categories based on the higher-order packaging of nucleosomes, histone post-translational modifications (PTMs) and histone variants ,11,12. HThe majority of chromatin is present as heterochromatin, a condensed chromatin structure that is transcriptionally less active and is mainly present at pericentric regions, telomeres, and other transcriptionally repressed regions. The second type of chromatin called euchromatin is more open, highly transcriptionally active and present in gene-rich genomic loci. The third specialized chromatin is present at centromeres where canonical histone H3 is replaced by its variant CENP-A. In addition to the core chromatin components, each type of chromatin is tightly associated and regulated by chromatin modifying proteins and protein complexes such as chromatin remodelers, histone-modifying enzymes, and architectural proteins that alter chromatin compaction ,20,21,22Euchromatin is characterized by active genes, wider spacing between nucleosomes, higher accessibility to transcription machinery, histone modifications and variants that facilitate active transcription ,34,35,36Euchromatin is mainly present on actively transcribing genes and regulatory elements such as promoters and enhancers, which regulate the access of transcription factors and chromatin remodelers to facilitate the opening of the chromatin and subsequent activation of transcription . Transcriptionally active genes constitute a minor fraction of the genome and are usually located in the interior of the interphase nucleus where euchromatin is enriched. The gene body is the transcriptional region, including introns and exons, from the start site to the end of the transcript and is marked by activating histone modifications to help keep gene bodies in a more open chromatin state . DNA metPromoters are regions where transcription is initiated and are found typically upstream of or at the 5’ end of a protein-coding gene or non-coding genes. Promoters of RNA polymerase II (RNAPII) contain a core promoter, the minimal sequence required to properly initiate transcription, such as the TATA box and additional motifs . The corEnhancers are DNA sequences that activate transcription independent of their distance or orientation with respect to the promoters of genes to be transcribed by binding to a variety of transcription factors. Enhancers regulate transcription in a tissue-specific manner, thereby playing an important role in development and differentiation ,53. EnhaEuchromatic histone PTMs include lysine acetylation and methylation. While lysine acetylation is almost always associated with chromatin accessibility and transcriptional activity, methylation can have different effects depending on which amino acid residue is modified. Histone modifications have been shown to play an important role in many biological processes including memory formation and storage, the aging of tissues, and differentiation ,62,63.The trimethylation of histone 3 at lysine 4 (H3K4me3) PTM is a characteristic feature of chromatin present at promotor regions near TSS . In mammESR1 (ERα) gene by interacting with its scaffold protein AFF4, suggesting that the TSS H3K27ac is involved in bridging the transition from transcriptional initiation to elongation [The acetylation of histone H3 at lysine 27 (H3K27ac) PTM is a well-recognized marker for active but not poised enhancers and is imparted by histone acetyltransferase (HAT) complexes . Howeverongation . Gene bodies of transcriptionally active genes are enriched mainly for activating methylation of histone H3 at lysine 36 (H3K36me3) PTM, which is imparted by SET2, the first H3K36 methyltransferase. SET2 is recruited for depositing H3K36me3 on gene bodies during transcription elongation by RNAPII phosphorylated at serine 2 . SWT2 maHistone variants associated with euchromatic regions are found at regulatory elements such as enhancers and promoters as well as gene bodies. Histone variants play roles in cell fate decisions and development and are found to be deregulated in cancers at the transcriptional level .H2A.Z is a replication-independent variant of canonical histone H2A. The C-terminal region of the yeast H2A.Z protein interacts with RNAPII, promoting its recruitment at promoters . H2A.Z iH3.3 is a conserved histone variant found at euchromatic actively transcribed regions and is incorporated into the nucleosome throughout the cell cycle, unlike canonical histones that are primarily deposited during replication ,81,82,83H2A.B status of both the father and mother, but not the zygote, affects embryonic viability and growth during gestation [The histone variant H2A.B (formerly named H2A.Bbd in humans) binds preferentially to H4 hyperacetylated regions and was named Barr body-deficient due to its exclusion from the inactive female X chromosome (Xi) ,90. Whenestation .Histone chaperones are a group of proteins that bind to soluble histones and regulate nucleosome assembly. Histone chaperones can be categorized based on their assembly mechanism, either as replication-dependent (replication coupled) or replication-independent . FollowiCoupling of DNA replication to nucleosome assembly was established early on ,97. SubsNucleosomes pose as barriers for transcriptional machinery and therefore they must be remodeled and reassembled following transcription through DNA replication-independent assembly . ReplicaAfter H3-H4 tetramer deposition, H2A-H2B dimers are incorporated to complete the nucleosome with the help of chaperone Nap1 . While HChromatin remodelers catalyze a variety of nucleosome restructuring reactions, including nucleosome sliding, conformation changes of nucleosomal DNA, and histone variant exchange, that play a role in chromatin accessibility and are necessary for all DNA-dependent biological processes . ChromatThe first ATP-dependent chromatin remodeler described was the yeast SWI/SNF complex. The complex is targeted to acetylated histones through the bromodomain subunit . The SWIThe ISWI family of remodelers contains an ATPase domain containing two RecA-like lobes separated by a small insertion sequence . They alThe CHD remodeler family is unique due to the presence of tandemly arranged chromodomains ,127. CHDThe INO80 subfamily of remodelers contains a large insertion between its RecA-like lobes. The N terminus of INO80 remodeler ATPases contains a HAS domain that nucleates actin and ARPs . The INOA significant fraction of nuclear RNA associates with chromatin and has been suggested to plan an architectural role in chromatin structure ,136,137.Drosophila is doubled to ensure equal levels of X-linked genes in males and females. This dosage compensation process is carried out by the dosage compensation complex . Upregulation of the X chromosome transcription is partly due to a key component of DCC, the MOF , which hyperacetylates histone H4 at lysine 16 [roX1 and roX2, that, while functionally redundant, loss of both dramatically affects male viability [roX genes. The sites serve as nucleation sites for spreading the DCC complex into the flanking chromatin [roX RNAs play an essential role in targeting the DCC to the X chromosome, and deletion of them both results in mislocalization of DCC and acetylation of histone H4 at Lys 16 by the MOF histone acetyltransferase [Transcription of the male X chromosome in ysine 16 . Two noniability . The DCChromatin . The roXnsferase ,147. Heterochromatin is transcriptionally less active and associated with gene silencing, less accessible than euchromatin. Emil Heitz first coined the term ‘heterochromatin’ when preparing cytological samples of chromosomes to distinguish regions that remained strongly stained throughout the cell cycle from those that became invisible during interphase . HeterocDrosophila, and Clr4 in yeast [Constitutive heterochromatin is a more static structure that ensures a condensed and transcriptionally less active chromatin conformation. Constitutive heterochromatin is characterized by the presence of histone marks such as Histone 3 Lysine 9 trimethylation (H3K9me3) and occurs at highly repetitive and gene-poor regions such as pericentric regions, sub-telomeric regions and transposable elements ,154. H3Kin yeast ,156. Clain yeast . For exain yeast . SatelliDrosophila, most rearrangements that have been shown to result in PEV map to pericentric regions and the genes involved in PEV are involved in constitutive heterochromatin formation.PEV is a phenomenon caused when a gene normally located in euchromatin undergoes abnormal juxtaposition to heterochromatic regions by genomic rearrangements is silencing in some cells . PEV silS. pombe, where the RNAi pathway plays the key role in the establishment of heterochromatin. S. pombe genome contains only one homolog each of the core RNAi machinery, Dicer (Dcr), Argonaute (Ago), and RNA-dependent RNA polymerase (RdRP) [S. pombe consist of inverted repeats called dg and dh repeats that transcribe into ncRNAs. Resulting ncRNAs are processed by the Dicer ribonuclease into small interfering RNAs (siRNAs), which then recruit the RNA-induced transcriptional silencing (RITS) complex in cis onto pericentric regions [S. pombe [The mechanism of constitutive heterochromatin formation is well characterized in the fission yeast e (RdRP) ,161. In e (RdRP) . Deletioe (RdRP) ,163. Per regions . The RITS. pombe . HP1 is S. pombe ,167. HP1S. pombe . PericenThe most striking feature of constitutive heterochromatin is highly compact packaging and a variety of functions impacted by heterochromatin, including heritable gene repression and maintenance of chromosome integrity, are due in part to compaction of the underlying chromatin. HP1 uses oligomerization to compact chromatin into phase-separated condensates ,170,171.While there are many histone variants associated with heterochromatic regions, a well-characterized one is MacroH2A, a variant generally considered transcriptionally repressive and associates with the inactive X-chromosome and inactive genes ,179. MacRecently it has been demonstrated that heterochromatin forms phase-separated liquid condensates ,171,181.Facultative heterochromatin is assembled on developmentally regulated loci, which can switch between transcriptionally active and inactive chromatin states during development and differentiation. As a result, not all cell types will have an identical pattern of facultative heterochromatin distribution in the genome. Facultative heterochromatin is regulated by polycomb group (PcG) proteins that impart histone 3 lysine 27 trimethylation (H3K27me3) marks. Facultative heterochromatin can be found on an entire chromosome, as seen in X-chromosome inactivation, or on discrete domains that are distributed genome-wide . trans-acting factors, including the lncRNA Xist (X-inactive specific transcript) that appears on the Xi. Xi territory occupies the perinucleolar location [Xist transcribes and spreads in cis on the entire Xi and recruits PcG complexes, such as Polycomb repressive complex 1 (PRC1), which catalyzes ubiquitylation of histone H2A at lysine 119 (H2AK119ub1) and PRC2 complex, which catalyzes trimethylation of H3K27me3 [Xist uses 3D proximal contacts to spread across the entire chromosome and recruits machinery for catalyzing repressive chromatin modifications [X chromosome inactivation (XCI), is a classic example of the developmentally controlled silent facultative heterochromatin formation, allowing only one X chromosome to be expressed in female mammals. . One ranlocation . In the H3K27me3 ,185,186.H3K27me3 . The forH3K27me3 ,189. Xisications ,191. Drosophila, establishment and maintenance of facultative heterochromatin over HOX gene clusters via PcG machinery involves ncRNA transcribed from polycomb response elements (PREs) [Xist and Tsix [cis or in trans with PcG proteins to provide context specificity to PcG protein recruitment and function in facultative heterochromatin formation [Facultative heterochromatin is also present at discrete genomic loci, including autosomal imprinted genomic loci and HOX gene clusters. Facultative heterochromatin of imprinted autosomal genes is characterized by histone hypoacetylation, H3K9me2, H3K27me, and DNA methylation with the inactive alleles . Notablys (PREs) . The PREs (PREs) . Dicer-2s (PREs) . PREs has (PREs) . A lncRNs (PREs) . The muland Tsix ,198,199.and Tsix ,201. Mulormation ,204,205.ormation . Once PRormation .Centromeres are the sites of spindle binding, where replicated chromatids or homologous chromosomes are pulled apart in chromosome segregation during meiosis and mitosis. While the overall chromosome segregation machinery is highly conserved across eukaryotes, the DNA and protein components of centromeric chromatin are evolving rapidly. The incompatibilities generated by evolving centromeric components may be responsible for the reproductive isolation of emerging species . CentromIn the absence of conserved centromeric DNA sequences, CENP-A chromatin has been considered as the epigenetic mark of centromeres ,213,214.Unlike histone H3, which is highly conserved across eukaryotes, CENP-A is strikingly divergent likely because H3 interacts with the entire genome while CENP-A interacts with centromeric DNA, which is rapidly evolving ,223,224.Drosophila and human cells and early S phase in S. pombe. Unlike canonical H3 nucleosomes deposited during the rS phase, CENP-A nucleosome assembly is replication-independent and, in humans, occurs in late mitosis and throughout G1 [Depletion of CENP-A results in mislocalization of kinetochore proteins, but depletion of kinetochore proteins does not affect CENP-A localization suggesting that CENP-A assembly acts upstream of the kinetochore formation ,232. Wheghout G1 ,237,238.ghout G1 ,239,240.Saccharomyces cerevisiae, which acts as a deposition factor for the CENP-A ortholog Cse4, and the only sequence similarity of Scm3 and HJURP is that binding domain [CENP-A is recruited to centromeres by a CENP-A-specific histone chaperone called Holliday Junction Recognition Protein (HJURP). HJURP stabilizes soluble CENP-A-H4 dimers before incorporation into centromeric nucleosomes ,242,243.g domain ,246,247.g domain . While Hg domain . HJURP eg domain .Several centromeric proteins control the timing and localization of HJURP recruitment. To deposit new CENP-A to centromeres, soluble HJURP-CENP-A complex associates with the Mis18 complex, which consists of centromeric proteins Mis18α, Mis18β, and M18BP1  213,251,259,260.Many approaches have been used to study centromeric chromatin, including biochemistry, biophysics, and genomics and a significant number of these studies have reported alternative CENP-A nucleosomal forms in centromeres, leading to different findings. Using elegant, functional centromeric plasmid-based genetic assays in the budding yeast, Furuyama and Henikoff demonstrated that centromeric DNA that assembles CENP-A nucleosomes undergoes positive supercoiling, which was proposed to arise due to the right-handed wrapping of centromeric DNA superhelix around CENP-A nucleosomes . The posDrosophilia CID arrays were found to contain all four histones in a heterotypic tetramer or “hemisome” configuration [Drosophila CID particles that were octamers [The widely assumed octameric nature of CENP-A nucleosomes where two copies of each of the four core histones form the nucleosome octamer was revisited when native guration . Immunopguration . The lacguration . Hemisomguration . Subsequoctamers . Howeveroctamers ,272. Moroctamers . Although AFM is well suited to visualize the DNA and protein components of nucleosomes to probe the structures of molecular complexes at the single-molecule level both label-free and with sub-nanometer resolution, the measurements of structural parameters by AFM suffer from (1) the convolution of the molecular and AFM tip geometry, (2) the typically small sample sizes, and (3) inconsistencies associated with manual data analysis ,278,279.Given that CENP-A chromatin is considered to contain an epigenetic component, analysis of in vivo chromatin organization is crucial to understand the native structure of centromeric chromatin, especially in the light of conflicting evidence regarding the histone composition and stoichiometry of CENP-A nucleosomes. However, native centromeric regions have been difficult to study due to the presence of long arrays of highly homogeneous satellite repeat sequences and remain one of the last frontiers of the human genome. Although centromeric maps have been recently generated for human centromeres, utilization of these maps for functional studies remains to be attempted ,281,282.The regulation of chromatin is essential for controlled gene expression, development, and cell identity. Abnormal chromatin regulation can lead to pathological conditions, including cancer . ChromatEpigenetic dysregulation and aberrant transcription of centromeric and heterochromatic satellite DNAs have been shown to contribute to chromosome instability, aging, and cancers ,300,301.Chromatin is indispensable for both packaging DNA and regulating gene expression. The core chromatin components remain mostly the same throughout the genome, but the higher-order structure and chromatin-associated regulatory proteins and protein complexes differ strikingly on different chromatin types designated to perform distinct functions. The primary outcome of these differences is a change in the accessibility of DNA to the transcription machinery leading to the activation or repression of target DNA loci. Although euchromatin forms only a small fraction of the total chromatin, it is associated with a variety of chromatin remodelers, histone modifications, histone variants and chaperones possibly to regulate the active transcription of different genes differentially and tightly. Additionally, long-range interactions between enhancers and promoters have appeared to play an important role by bringing together the core transcription machinery with chromatin-associated factors that enhance the transcription and provide tissue-specificity to the gene expression. Heterochromatin forms the major fraction of the total chromatin and is associated with two sets of heterochromatic proteins depending upon its constitutive and facultative nature. Due to its ability to undergo strong compaction and silence the transcription, the constitutive chromatin packages a large amount of our genome and preserves genome integrity. Therefore, constitutive heterochromatin-associated factors such as H3K9me3 modifying enzymes and HP1 homologs and ncRNAs are mainly involved in establishing the permanent compact state of their target substrate chromatin ,308,309.Finally, centromeric chromatin is the most unique among all types of chromatin as it is dedicated to performing a single yet essential function of mediating chromosome segregation and is characterized by the presence of histone variant CENP-A, one of the least conserved histone variants. CENP-A chromatin marks the underlying genomic loci for centromere function and provides the structural platform for kinetochore formation . Unlike Due to their fundamental role in gene expression, mechanistic understanding of chromatin complexes is crucial for both their biological function and biomedical relevance. Chromatin factors including histone modifications, histone variants, and chromatin remodelers are important in several biological processes including differentiation, memory formation, and cell fate, and altered chromatin regulation has been implicated in many human diseases including PD, ICF, and many cancers ,304,305.In summary, the chromatin field has made significant progress and facilitated the understanding of both fundamental mechanisms underlying the regulation of gene expression and genome packaging and involvement of chromatin components in several diseases.


Culture expansion of primary cells evokes highly reproducible DNA methylation (DNAm) changes. We have identified CG dinucleotides (CpGs) that become continuously hyper- or hypomethylated during long-term culture of mesenchymal stem cells (MSCs) and other cell types. Bisulfite barcoded amplicon sequencing (BBA-seq) demonstrated that DNAm patterns of neighboring CpGs become more complex without evidence of continuous pattern development and without association to oligoclonal subpopulations. Circularized chromatin conformation capture (4C) revealed reproducible changes in nuclear organization between early and late passages, while there was no enriched interaction with other genomic regions that also harbor culture-associated DNAm changes. Chromatin immunoprecipitation of CTCF did not show significant differences during long-term culture of MSCs, however culture-associated hypermethylation was enriched at CTCF binding sites and hypomethylated CpGs were devoid of CTCF. Taken together, our results support the notion that DNAm changes during culture-expansion are not directly regulated by a targeted mechanism but rather resemble epigenetic drift. Julia Franzen et al. investigate if changes in DNA methylation at specific genetic loci during cell culture expansion are due to a specific mechanism or gradual deregulation of an epigenetic state. Their results suggest that changes in CpG methylation are due to indirect epigenetic drift, rather than a consequence of targeting by DNA methyltransferases. This is of particular relevance for the more than 1000 registered clinical trials with mesenchymal stem/stromal cells , which usually use high cell doses (often about 106 cells/kg body weight) and therefore necessitate excessive culture expansion4. Furthermore, long-term culture of cells is often required for basic research and this may hamper the reproducibility of results. Tracking the impact of long-term culture is therefore an important aspect for quality control of cell preparations.Cell preparations are often expanded in vitro for many passages to achieve enough material for basic research or cellular therapy. However, culture expansion has a severe and continuous impact on the growth, morphology, gene expression, metabolomics, and function of primary cells, until they ultimately enter a state of replicative senescence6. We have previously demonstrated that DNAm levels at only six cytosine/guanine dinucleotides (CpG sites) can be used to estimate passage numbers and cumulative population doublings (cPD)8. It therefore provides a reliable biomarker to estimate the state of culture-associated modifications. In our previous studies, we referred to these epigenetic modifications as senescence-associated DNAm changes, albeit it is unclear if the culture-associated DNAm changes are linked to the state of cellular senescence—we therefore changed the terminology into culture-associated DNAm changes. Furthermore, it is so far unclear how DNAm patterns evolve during culture expansion and why they occur at specific genomic regions.Long-term culture is reflected by highly reproducible DNA methylation (DNAm) changes at specific sites in the genome9. In fact, there is some overlap with epigenetic clocks for aging, but the two processes can be clearly discerned and it is yet unclear how the DNAm changes are governed. It is generally anticipated that DNAm changes during development are regulated by epigenetic writers—such as the de novo methyltransferases DNMT3A and DNMT3B, or TET methylcytosine dioxygenases10. If an epigenetic writer is targeted to a specific site in the genome the neighboring CpGs will most likely also be modified. Alternatively, the DNAm changes that accumulate during culture expansion might not be directly regulated but rather reflect dysregulation, as suggested for epigenetic drift during aging of the organism13. A better understanding of how DNAm changes evolve in culture expansion might shed light into the underlying process.At first sight, the culture-associated DNAm changes seem to be related to the DNAm changes that are acquired during the aging of the organism14. Using hairpin-bisulfite PCR15 several studies have demonstrated that, despite the general preference for concordant DNAm on both strands, certain sites are specifically methylated on only one strand18. Such hemimethylation can be stably inherited over several passages and has been associated with CCCTC-binding factor (CTCF)/cohesin binding sites19. CTCF has been linked with changes in chromatin conformation and cellular senescence21. We have demonstrated that senescence entry upon extensive culture expansion is associated with a reorganization of CTCF into large senescence-induced CTCF clusters (SICCs)20. However, it remains unclear if and how the binding of CTCF changes during long-term culture.Furthermore, it is generally thought that DNAm patterns are identical on the forward and the reverse DNA strand (Watson and Crick strands)In this study, we further investigated if culture-associated DNAm changes are caused by a targeted regulatory mechanism or rather by epigenetic drift.7. In continuation of this work, we utilized the meanwhile available 450k Illumina BeadChip datasets, which interrogate ~16 times more CpGs than the 27k version of the chip, and we also considered datasets of additional types of primary cells. We compiled 63 DNAm profiles of human primary MSCs (n = 45), fibroblasts (n = 5), and human umbilical vein endothelial cells (n = 13) with precise information on passage numbers  to select 15 hypermethylated and 15 hypomethylated sites  based on 27k Illumina BeadChip datasets, and thereby established a 6 CpG predictor to estimate passage numbers specifically for MSCsArachidonate 12-Lipoxygenase , Docking Protein 6 , Leukotriene C4 Synthase , and TNNI3 Interacting Kinase . Samples of the training set were all cultured until growth arrest and senescence was checked by staining with senescence-associated β-galactosidase  of 3.9 passages. Subsequently, our epigenetic predictor for long-term culture was validated on an additional independent set of samples  levels, which cannot be distinguished from 5-methylcytosine (5mC) by bisulfite treatment. To address this question, we used the TrueMethyl Array kit on three MSC donors in early (passage 4) and late (passage 10) passages to specifically look at the percentage of 5hmC at our 646 hyper- and 2442 hypomethylated culture-associated CpGs. Overall, we did not detect high levels of hydroxymethylation at early or late passages (mean estimated 5hmC levels about 0.3%) and neither hyper- nor hypomethylated culture-associated CpGs showed higher mean levels of 5hmC than other CpGs .Importantly, culture-associated DNAm changes are distinct from age-associated DNAm changes, which correlate with chronological age rather than with passage numbersics Fig.  and the 23  there is an inverse switch in epigenetic patterns of pluripotency and culture expansion around day 7. While the long-term culture-associated DNAm changes are then continuously acquired upon differentiation of iPSCs, this is not observed for age-associated signatures23 Fig. . Convers 23 Fig. . Taken t30, which might contribute to culture-associated DNAm changes. Therefore, we aimed for a better understanding how DNAm patterns at neighboring CpGs evolve during culture expansion and how this is affected by the clonal composition of MSCs. We anticipated that tracking of DNAm patterns over several passages would provide insights into the changing composition of subclones within MSC preparations. To address this question, we used samples from a previously published study:30 Umbilical cord-derived MSCs from two donors were transduced with lentiviral vectors containing random barcodes and three different fluorescent proteins. Flow cytometry and deep sequencing demonstrated that the diversity of cellular subsets declines and that senescent passages became oligoclonal 7. The combined BBA-seq measurements were then used to predict passage numbers. Taking all passages into account, the epigenetic estimations correlated well with the number of passages —the other amplicons hardly comprised neighboring CpGs and could therefore not be included into this analysis. DNAm patterns overall remained stable or became even more diverse during culture expansion, which is also reflected by a moderate increase of the Shannon index in most amplicons  in the loop region to adjust for potential PCR bias . As a control, we considered an additional genomic region that was generally methylated (associated with the genes C12orf12). The accuracy of epigenetic predictions of passage numbers was similar when reads with the same UMI were only considered once  using circular chromatin conformation capture (4C). Two independent MSC preparations at early (P2 and P3) and late (P7 and P9) passages revealed overall reproducible interaction profiles : A similar number of interactions revealed such significant gains and losses during culture expansion  was previously shown to have an exceptionally large differentially methylated region during culture expansion, which may explain this enrichment in the differentially methylated nearbait region22. However, this finding might also be coincidental since the significant gains of interaction at KRTAP13.3 included only 6 CpGs of the Illumina Bead Chip. Overall, our results did not indicate that the four culture-associated genomic regions revealed clear enrichment of interaction with other culture-associated CpGs at nearbait or cis-contacts.Next, we investigated if chromatin interactions preferentially occurred at other genomic regions that become methylated or demethylated during culture expansion. As mentioned above, we used the 63 DNAm datasets of cells at different passages . In contrast, such overlap with LADs was rather depleted for hypermethylated regions  we used a publicly available dataset of fibroblasts20. When we analyzed the distribution of CTCF in MSCs by fluorescence microscopy we also observed increased co-localization of CTCF in the larger nuclei of senescent MSCs . Overall, the ChIP seq peaks were in line with previous data of embryonic stem cell-derived MSCs35  in HUVEC and IMR90 cellsSCs Fig. . We thenALOX12 and LTC4S) exhibited significant enrichment of binding motifs for CTCF and CCCTC-binding factor like  for all three donors  Resetting of culture-associated DNAm during reprogramming into iPSCs seems to occur synchronously with the epigenetic changes in pluripotency genes, which is directly linked to the epigenetic transition itself. Furthermore, it occurs simultaneously with resetting of age-associated DNAm, which has also been attributed to epigenetic drift13. (2) The culture-associated DNAm patterns do not reflect MSC clonality and become even more diverse in oligoclonal cell preparations at late passages. If these epigenetic modifications were evoked by targeted regulation in individual subclones specific patterns should become dominant, too. (3) BBA-seq analysis demonstrated that culture-associated DNAm patterns do not develop in an additive manner at neighboring CpGs and this was further supported by hairpin sequencing of the complementary DNA strands. If an epigenetic writer is targeted to a specific site in the genome the neighboring CpGs will most likely also be modified, as observed for CRISPR-guided approaches of epigenetic writers that coherently modify neighboring CpGs37. (4) There was no evidence that culture-associated DNAm is coherently modified at interacting chromatin domains. It is known that DNMTs accumulate in replication foci or punctate heterochromatic foci39 and hence, it might be speculated that a targeted mechanism of an epigenetic writer would also involve the interaction of culture-associated chromatin domains. If culture-associated DNAm changes are not governed by targeting of epigenetic writers, there needs to be another—yet unknown— mechanism that disposes specific genomic regions to epigenetic drift.The continuous and highly reproducible nature of culture-associated DNAm changes may suggest that this process is tightly controlled41. Nuclear depletion of HMGB2 and its induction of CTCF clustering are early events on the path to replicative senescence, which disturb the chromosomal 3D organization20. Our 4C analysis supports the notion that the 3D chromatin structure undergoes highly reproducible changes during culture expansion, while these changes do not seem to be associated with changes in DNAm. It has been demonstrated that CTCF occupancy, which is to some extent cell-type specific, is also linked to differential DNAm42. Furthermore, some CTCF sites may function as a bifurcation point defining the differential methylation landscape43. In this regard, it was unexpected that we did not observe clear differences in the CTCF ChIP-seq data of early versus late passages. On the other hand, hypermethylation seems to occur preferentially at CTCF binding sites, whereas hypomethylation occurs apart from CTCF binding sites and preferentially at LADs. This might partly be attributed to the fact that the hypermethylated CpGs have rather low DNAm levels at early passage, which may support binding of CTCF to the corresponding motive at early passage43.Cell culture is evidently associated with major changes in chromatin structure. The nucleus becomes much larger at later passages, while chromatin volume decreases due to extensive reorganization of hetero- and euchromatin conformation44. Thus, the binding of these transcription factors might be affected by DNAm or vice versa44. It is also conceivable that DNAm changes during long-term culture are indirectly mediated by the histone code, insulators, chromatin loops, and the overarching nuclear structure. Furthermore, the culture-associated differentially methylated regions might be related to nucleosome size. However, for such analysis it needs to be considered that the Illumina Bead Chip data has the limitation that it does not address all CpGs of the genome. Furthermore, we have only exemplarily analyzed few amplicons with BBA-seq, hemimethylation analysis, and 4C. It is therefore unclear if the findings of our study are really representative of the entire genome. Importantly, the culture-associated DNAm changes might also be influenced by specific culture conditions or cell types and this deserves further analysis in the future.Taken together, the results of this study support the notion that the given chromatin conformation favors site-specific epigenetic drift over subsequent passages—but they also provide room for additional hypotheses, which have not been addressed with our current study. We have previously demonstrated that some transcription factor binding motifs  were enriched in senescence-associated differentially methylated regions and in the promoters of differentially expressed genes8. Thus, a direct link to a specific gene promotor is often not possible. It has been shown that culture-associated DNAm is only partly associated with gene expression changes of the corresponding genes46. Yet, it would be an oversimplification to only consider transcriptional regulation as being functionally relevant. Chromatin conformation, loop structures, histone modifications, and LADs may favor modulation of DNAm at specific sites in the genome—and on the other hand, culture-associated DNAm may stabilize such chromatin features. In future research, different levels of chromatin organization should be considered to fully understand the underlying mechanism that drives epigenetic drift during long-term culture.Last but not least, the functional relevance of DNAm changes is still unclear. Overall, culture-associated CpGs are rather depleted from CpG islands and their shore/self-regions, while they are enriched in open sea, 3′UTR and intergenic regions47, respectively. An outlier test using the R package car was performed resulting in the exclusion of one sample (GSM1004625). Initially, we selected culture-associated CpGs based on Pearson correlation of DNAm levels  and passage numbers at a threshold of R > 0.7 or R < −0.7 22 as well as DNAm profiles during re-differentiation of iPSCs to iMSCs (GSE54767)23. We focused on three different sets of culture-associated CpGs: (1) 30 CpGs ; (2) 3088 CpGs ; and (3) 9 CpGs of our epigenetic signatures 7. Furthermore, we focused on 1432 pluripotency-associated CpGs27 and 99 age-associated CpGs25 of the Illumina 450k BeadChip. Heatmaps were produced with R package gplots, Pearson correlation, and corresponding p-values were calculated with R package stats.The kinetics of culture-associated DNAm changes during reprogramming of fibroblasts into iPSCs were investigated using the dataset of Ohnuki et al. n = 11; BM-MSC RWTH Aachen)48, from bone marrow aspirates of allogeneic hematopoietic stem cell donors 49, from subcutaneous adipose tissue lipoaspirates 50 and from umbilical cord pieces 30. Fibroblasts were isolated from dermis 7. Human umbilical vein endothelial cells were isolated from umbilical cords of healthy donors after cesarean sections 51 or obtained from Lonza 8. All samples were taken after informed and written consent and the study was specifically approved by the ethics committees of the corresponding Universities. All cell preparations were thoroughly characterized  and culture conditions were used as described in detail in our previous work50. In addition, HUVECS were cultured on 0.1% gelatin in M199 medium  supplemented with 20% fetal calf serum (FCS) (Gibco Thermo Fisher), 1% penicillin/streptomycin (PAA), 0.1% heparin  and 50 µg/ml endothelial cell growth supplement (ECGS) . For long-term culture all cells were passaged at ~90% confluency and reseeded at 10,000 cells/cm2. Beta-galactosidase  staining was performed using the Senescence Detection Kit ab65351 . cPD were calculated as described before50.All human cell samples were taken after informed and written consent was obtained from donors and the study was specifically approved by the ethics committees of RWTH Aachen University Medical School , University of Heidelberg, and Hannover Medical School. MSCs were isolated from the bone marrow of donors undergoing orthopedic surgery  according to the manufacturer’s instructions. In short, three MSC donors of early passage (passage 4) and late passage (passage 10) were each divided into two samples: one sample was oxidized and bisulfite converted (OxBS), the other sample was just bisulfite converted (BS). Oxidation prior to bisulfite treatment leads to the conversion of 5hmC to 5-formylcytosine (5fC), which is decarbonylated and deaminated to uracil upon bisulfite treatment, similar to non-methylated cytosines. Both OxBS and BS samples were finally investigated by Illumina 450k BeadChip microarrays . Beta values of the microarrays were provided by Illumina’s GenomeStudio software and hydroxymethylation was calculated as the difference of beta values of BS samples subtracted by the beta values of OxBS samples.β-values) at the four CpGs in α = ALOX12 (cg03762994), β = DOK6 (cg25968937), γ = LTC4S (cg26683398) and δ = TNNI3K (cg05264232) was as follows:52 to perform 10-fold cross-validation on the training dataset.Genomic DNA was isolated with the NucleoSpin Tissue kit  and bisulfite converted using the EZ DNA Methylation kit . Pyrosequencing was performed on a PyroMark ID System . Primers for pyrosequencing were designed with the PSQ assay design software  and by deep sequencing of PCR amplified barcodes using ion torrent sequencing. Area plots were produced with the R package ggplot2. The Shannon-Index was calculated with the following formula:Primary MSCs in umbilical cord tissue were transduced with three different lentiviral vectors containing the fluorescent proteins mCherry (red), Venus (green) or Cerulean (blue) and a barcode of 16 random and 15 vector-backbone-specific nucleotides, as previously published8. Amplicons were pooled and sequenced on an Illumina MiSeq lane with the v2 nano reagents (Illumina) in 250 PE mode. Bisulfite converted sequencing data were analyzed using TrimGalore, Bismark53 and bowtie254. Mean sequencing coverage of amplicons was ~3900 reads per amplicon  based on their binary sequel of methylated and unmethylated CpG sites. Probabilities of passage numbers were based on linear regression models at each CpG site, retrieved from the training dataset. Finally, we calculated the mean passage number for each sample based on all sequencing reads. Further details on the rational and derivation of the mathematical model are provided in our previous work31.Bisulfite-converted DNA was used for a nested PCR using the PyroMark PCR kit  was digested with restriction enzymes that cut close to our CpG of interest: AccI , DdeI  and CviQI . Hairpin linkers  and cis interaction analysis and k = 20 for trans chromosomal interactions. We focused particularly on the high-interacting reads that were called in both replicates. Differential interactions were called with 4Cker, which uses DESeq2 and a FDR corrected p-value < 0.5 to call significant differential interactions. Circos-plots were generated with the R package RCircos58. For comparison we used DNAm profiles of MSCs at early (n = 5) and late passages 22. CTCF and CTCFL enrichment in interacting regions was tested with the RGT motif enrichment tool . Motif enrichment was tested within high-interacting regions called by 4Cker ranging approximately from 1.1 to 336 kilobases and a mean region length of 23 kilobases within nearbait and from 1.3 to 950 kilobases with a mean region length of 86 kilobases within cis regions. Enrichment of interactions with LADs of human fibroblasts32 were analyzed in comparison to randomly chosen regions of similar sizes by Fisher’s exact test using R stats.Ten million cells from two MSC preparations at early (P2 and 3) and late (P7 and 9) passages were cross-linked with 4% paraformaldehyde  for 10 min., harvested into ice cold PBS with 0.125 M glycine, and frozen under a protease inhibitor cocktail . 4C-seq was performed as described before20. For image acquisition, a widefield LeicaDMI 6000B with a HCX PL APO 63x/1.40 (Oil) objective was used.Staining of CTCF  and counterstaining with DAPI was performed as described in detail beforen = 3) and late passage  were cross-linked with 1.2% formaldehyde  for 15 min at room temperature. After cell lysis, chromatin fragmentation was performed for 20 min  with a Biorupter® Pico Sonicator Device (Diagenode). 5–33 µg of sonicated chromatin was incubated with 4 µg ChIP-validated CTCF antibody (Active Motif) overnight on an end-to-end rotator at 4 °C. For immunoprecipitation, samples were incubated with protein G agarose beads (Active Motife) at 4 °C for 3 h. Finally, the cross-link was reversed, the DNA was proteinase K digested and purified.Chromatin Immunoprecipitation of CTCF was performed using the ChIP-IT PBMC kit with the fixation protocol of the ChIP-IT High Sensitivity kit . In brief, MSCs of early passage  before reads were aligned to the hg19 genome assembly using bowtie254. Reads approximately exhibited a 97% concordant alignment rate in all samples and sequencing depth ranged from 38 to 170 million reads. We used MACS259 for peak calling on each sample with the default parameters against the input control. All the peaks were filtered by signal >50 and selected with the overlapping of potential CTCF binding motif, which was obtained by RGT motif analysis . Enrichment of interactions between 4C high-interacting regions and CTCF ChIP-seq peaks of early and late passages were analyzed in comparison to randomly chosen regions of similar sizes by Fisher’s exact test using R stats. The normalized coverage of each sample was calculated by Deeptools60 bamCoverage with normalization of Reads Per Kilobase per Million mapped reads (RPKM), these normalized coverages were used for down-stream analyses. The correlation heatmap across the samples was generated by Deeptools multiBigwigSummary. All the potential CTCF binding sites were evaluated by the normalized coverage profiles and shown in MA plot by comparing early passages with late passages. The lineplots were calculated by a sliding window (size = 200 bp with step = 100 bp) on the normalized read counts on the extended sites (length = 2000 bp for each direction) of either CTCF motifs or various sets of CpGs. Statistical analysis was performed against these random CpG sites using the Kruskal–Wallis test with post hoc Dunn’s test and Benjamini & Hochberg adjustment.Sequencing and library preparation were conducted by the IZKF Sequencing Core Facility of RWTH Aachen University Medical school. The samples were sequenced in paired-end mode on the Illumina NextSeq 500. The quality of the resulting reads was checked by FastQC (Available online at: Statistical tests were performed as described in the corresponding methods sections. All experiments were performed for at least two independent cell donors and at least four culture-associated CpGs.Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Reporting Summary


Shigella dysenteriae type 1 and Stx-producing Escherichia coli (STEC). Infection with these bacteria causes bloody diarrhea and other pathological manifestations that can lead to HUS (hemolytic and uremic syndrome). At the cellular level, Stxs bind to the cellular receptor Gb3 and inhibit protein synthesis by removing an adenine from the 28S rRNA. This triggers multiple cellular signaling pathways, including the ribotoxic stress response (RSR), unfolded protein response (UPR), autophagy and apoptosis. Stxs cause several pathologies of major public health concern, but their specific targeting of host cells and efficient delivery to the cytosol could potentially be exploited for biomedical purposes. Moreover, high levels of expression have been reported for the Stxs receptor, Gb3/CD77, in Burkitt’s lymphoma (BL) cells and on various types of solid tumors. These properties have led to many attempts to develop Stxs as tools for biomedical applications, such as cancer treatment or imaging, and several engineered Stxs are currently being tested. We provide here an overview of these studies.Shiga toxins (Stxs), also known as Shiga-like toxins (SLT) or verotoxins (VT), constitute a family of structurally and functionally related cytotoxic proteins produced by the enteric pathogens  Shigella dysenteriae, in 1897  fluoropyridine-based maleimide coupled to StxB and injected into the retro-orbital sinus of the control mice, was delivered to normal tissues in the urinary tract, kidney, spleen, lungs and liver, but that the abdominal region was not labeled. By contrast, in adenocarcinoma-bearing mice, [18F]-StxB was able to reach tumor sites in the digestive tract [The specific targeting capacity of StxB was initially evaluated in digestive tumors. Confocal laser endoscopy showed that orally administered FITC-coupled StxB reached intestinal tumors in mice. PET imaging showed that the contrast reagent, [ve tract . Viel etve tract .STxB has also been used to functionalize microbubbles for ultrasonography. Microbubbles are used as contrast agents but, because of their size, their biodistribution is limited to the blood after injection. Functionalized microbubbles can be targeted to blood vessels and to vascular endothelial cells if the target is present on the accessible surface. Gb3 is overexpressed in the neovasculature of tumors, and STxB-functionalized microbubbles are therefore able to bind specifically to cells expressing Gb3 in vitro and in vivo, suggesting that they could be used for the ultrasound imaging of angiogenesis .These experiments, although promising, showed that StxB binding is not limited to tumor area, but also found in normal tissues, raising questions about how to distinguish between background and specific signals, particularly for small tumors. They also highlight the importance of studying the possible adverse effects on normal tissues of the use of StxB to deliver drugs.8 StxB binding sites, whereas antibodies typically have, at most, 106 binding sites per target cell. StxB is, therefore, likely to be more efficient than antibodies as a vector for the tumor-specific targeting of cytotoxic drugs and could be a carrier of choice for targeting Gb3-positive cancers [50 values in the nanomolar range [Cancer cells may carry up to 10 cancers . Moreove cancers . StxB haar range . The cytar range ,103. Stxar range . Moreovear range . HoweverPseudomonas aerugina exotoxin A fused to monobodies targeting the Lck tyrosine kinase has been shown to be delivered to cells and to inhibit the kinase signaling pathway [Interest in the StxB fusion protein has recently increased. Notably, Stx2B has been used to overcome the inability of small engineered proteins called monobodies to cross cellular membranes. Monobodies are small proteins engineered with a fibronectin type III domain (FN3) as a molecular scaffold, which specifically bind and inhibit various cytosolic oncoproteins. A chimeric toxin composed of Stx2B and the translocation domain of  pathway . Mohseni pathway .However, most of these studies on StxB-drug conjugates were performed in vitro, with the notable exception of studies on the N8A (MDM2 inhibitor)-StxB fusion protein, which was shown to suppress tumor growth in hepG2-xenografted mice following intraperitoneal injection every two days . BatisseStxB has also been used in antigen presentation studies, to induce protective cell-mediated immunity to improve the clearance of certain tumors. StxB can deliver antigen directly to dendritic cells, which then express it on their surface, inducing a specific T-cell response. Furthermore, the vaccination of mice with StxB coupled to E7, a protein derived from HPV16, has been shown to inhibit the growth of E7-expressing tumors . StxB isNatural selection has made Stxs one of the most efficient carriers for the delivery of toxic proteins to cells through binding to a specific receptor on the cell surface. Stxs can be used as anticancer agents in their native form or as engineered variants. However, the attachment of a molecule to StxB or StxA can alter the chemical and physical characteristics of the original Stx, introducing variability in terms of stability, immunogenicity and biodistribution, with potential impacts on the off-target toxicity and efficacy of the complex. In addition, StxB is rather small, and its coupling to large molecules may impair its binding to Gb3. Stability and pharmacokinetics are therefore of crucial importance in the development of engineered Stxs.E. coli, but the overexpressed or recombinant proteins accumulate within inclusion bodies in this system, potentially limiting the amount of material obtained and necessitating further processing, leading to additional losses. Furthermore, production systems of this type require the removal of any other endotoxins produced by the bacterium before use in humans. As an illustration of this, the clinical use of Ontak has been discontinued due to issues relating to production in a bacterial expression system in which purification is difficult.One important issue concerning the use of Stxs, with or without modification, is the economic and environmental feasibility of their production. For example, the use of Stxs as anticancer tools will require large-scale (g or kg amounts) production of the Stx-derived protein. In most studies, Stx, StxA or StxB are produced in ETBs appear to be the most promising applications based on Stxs to date. Recent advances in genetic engineering have paved the way for the development of immunotoxins capable of killing target cells in a highly specific manner with only minor side effects and low levels of immunogenicity. However, preclinical and clinical studies are required to evaluate their efficacy and safety.


Sox proteins are known as crucial transcription factors for many developmental processes and for a wide range of common diseases. They were believed to specifically bind and bend DNA with other transcription factors and elicit transcriptional activation or repression activities in the early stage of transcription. However, their functions are not limited to transcription initiation. It has been showed that Sox proteins are involved in the regulation of alternative splicing regulatory networks and translational control. In this review, we discuss the current knowledge on how Sox transcription factors such as Sox2, Sry, Sox6, and Sox9 allow the coordination of co-transcriptional splicing and also the mechanism of SOX4-mediated translational control in the context of RNA polymerase III. Gene expression is a process that transforms genomic information into biological functions. Gene expression in eukaryotes can be regulated at multiple levels. Though each individual level can be studied in isolation in vitro, it becomes clear that they are intimately linked within cells. Transcription factors (TFs) are a large class of DNA-binding proteins that play a key role in gene transcription. Nevertheless, increasing evidence shows that they can function as cross-talkers in several levels of gene expression. 1T2T3T4G5T6C7-like motif. All bases of the core of the DNA motif are directly bound by amino acids in base-specific interactions, and amino acids engaged in base-specific DNA interactions are constant amongst all Sox TFs. The HMG box interacts with the minor groove of the DNA duplex and causes an overall bend of 60–70° towards the major grove, which may in turn regulate the functions of Sox proteins [Sox TFs exist in all metazoans and regulate many developmental processes and homeostasis in adult tissues. The 20 Sox proteins are characterized by a highly conserved Sry-related high-mobility-group (HMG) box. Depending on the sequence similarities of the HMG box, Sox TFs are classified into 9 groups  [Outside the HMG box, strong homology of amino acid sequences and overall organization of protein domains are found only within a group, although this does not mean that synonymous function only exists within each group. Sox2 (a member of the SoxB1 group) and Sox9 (a member of the SoxE group) are proposed to engage at regions exposed on nucleosomes and are defined as pioneer factors ,6,7. RecEukaryotes possess three distinguishing nuclear RNA polymerases that target different classes of genes. RNA polymerase I (Pol I) specifically aims to transcribe large ribosomal RNA (rRNA) genes, including 28S, 5.8S, and 18S cytoplasmic rRNA. RNA polymerase III (Pol III) is devoted to the transcription of abundant small RNAs containing 5S rRNA, all transfer RNAs (tRNAs), and some small RNAs involved in pre-mRNA splicing and protein transport that are not transcribed by Pol II . Pol II The splicing of pre-mRNAs in eukaryotes is tightly coordinated with transcription. Nascent RNAs are mostly spliced as soon as Pol II is downstream of introns during transcription elongation ,36. ThisSox2 is a determinant in the maintenance of embryonic and neural stem cells. It presents diverse regulatory roles, especially in chromatin organization and transcriptional regulation. In addition to the general function as a TF, there is strong evidence for its role as a pioneer factor that unwraps nucleosomal DNA to enable gene expression from areas of highly packed chromatin. Cryo-electron microscopy (cryo-EM) structure and SeEN-seq data show that the HMG domain of Sox2 binds to nucleosomal entry-exit sites ,40. TogeNg et al. found that long noncoding RNA ES1 (lncRNA_ES1) and long noncoding RNA ES2 (lncRNA_ES2) physically interact with Sox2 in hESCs and were suggested to maintain pluripotency in a Sox2-dependent mode . In addiLately, Sox2 was identified as an RNA-binding protein that contains no classic RNA-binding motifs. Sox2 is able to directly interact with RNA. It recognizes G/C-rich RNA via a 60-amino-acid RNA binding motif (RBM) C-terminally juxtaposed to the HMG box (amino acids 120–180) . The HMGSequence-specific DNA-binding TFs can also alter mRNA isoform production by regulating transcription elongation rates . Sox2 waSox9, Tcf21, and Ntf3 in testicular development [SAGA-associated factor 29 (SGF29) in hepatocarcinogenesis [Cbln4, rat renin (Ren), angiotensinogen (Agt), angiotensin-converting enzyme (Ace) and Ace2 in the nervous system and neuroendocrine regulation [Testis-determining factor SRY/Sry (sex-determining region Y), the gene of which is located on the Y chromosome, contains a conserved HMG box between species, but variant in other regions . It appeelopment ,63,64; Sogenesis ; and Cblgulation ,67. It igulation ,69. The HMG domain of Sry can also bind to the Holliday-like four-way junction DNA and RNA substrates . During M. m. domesticus subspecies  and the C-terminal transactivation domain , which abrogate the transcription ability, do not significantly affect the Sox9-associated splicing . Moreoveachinery ,83. By uachinery . Knockouachinery . Sox9 isachinery . Xenopus oocytes [Sox9 connects with RNP transcripts of lampbrush chromosomes (LBCs), in which the active transcription occurs, in  oocytes . However oocytes . It bind oocytes ,86,87. TSox4 is another key TF required for vertebrate tissue development and differentiation. It is overexpressed in many types of cancer cells. Sox4 interacts with p53, a pivotal tumor suppressor, through its HMG domain and stabilizes it by blocking Mdm2-mediated ubiquitination and degradation ,89. Sox4Met (located in Chromosome (Chr) 1, Chr 6, and Chr 17) and 6 genomic copies of tRNAPhe  [Met is one of the most significant targets of Sox4. All 8 genomic copies of its gene are regulated by Sox4 [Met inhibits glioblastoma cell proliferation and the replenishing of ectopic tRNAiMet partly rescues the Sox4-mediated proliferation inhibition [Pol III specifically aims to transcribe short, stable nonprotein-coding RNA transcripts, which play important roles in the modulation of transcription , RNA processing , and translation  ,89,90. P Chr 19) . On the  Chr 19) . It was  by Sox4 . CRISPR/hibition . p53, a partner of Sox4, is also believed to act as a general repressor of Pol III-mediated transcription. Endogenous cellular p53 interacts with TFIIIB and thereby severely impedes the function of Pol III. Since the function of p53 is retarded in the majority of cancers, e.g., HCC, it is probable that Pol III will be released from repression and induce the loss of growth control during the tumor’s development ,93,94. TGene expression is primarily regulated at the level of transcription, largely as a consequence of the binding of TFs to specific sites on DNA. The function of TFs is to turn genes ‘on’ and ‘off’ in order to ensure that they are expressed to adapt to environmental changes throughout the life of the cells. Emerging studies have shown that TFs, such as Sox TFs, can control gene expression both at the step of transcription initiation and also during co-transcriptional pre-mRNA splicing by altering transcription elongation rates and/or influencing the interaction between splicing factors and pre-mRNA, in concert with the fact that more than one-third of the alternative splicing regulators are TFs . More re


This family of proteins can be metaphorically compared to the Golden Snitch from the Harry Potter series, buzzing on and off several regions of the chromatin, in combat with competing transcription factors and chromatin remodellers, thereby critical to the epigenetic endgame on short and long temporal scales in the life of the nucleus. Here, we summarize recent efforts spanning structural, computational, genomic and genetic experiments which examine the linker histone as an unseen architect of chromatin fibre in normal and diseased cells and explore unanswered fundamental questions in the field.Histone H1s or the linker histones are a family of dynamic chromatin compacting proteins that are essential for higher-order chromatin organization. These highly positively charged proteins were previously thought to function solely as repressors of transcription. However, over the last decade, there is a growing interest in understanding this multi-protein family, finding that not all variants act as repressors. Indeed, the H1 family members appear to have distinct affinities for chromatin and may potentially affect distinct functions. This would suggest a more nuanced contribution of H1 to chromatin organization. The advent of new technologies to probe H1 dynamics  The wordiameter . With thdiameter   [Most of the studies above focused on the globular domains of H1 which does not take into consideration the primary determinant of H1 binding, the H1 CTD. The disordered domains are very challenging to model, but new efforts into defining the CTD for some H1 variants have been fruitful. Recent studies have showed that H1.0, H1.4 and H1.10 globular domains bind nucleosomes ‘on-dyad’ using specific residues of the NTD, the α3 helix, and L-1 loop of the globular domain. Here, the H1 CTDs play a predominant role in determining the linker DNA closeness in a nucleosome. H1.0 and H1.4 were found to simultaneously interact with both linker DNAs and bringing them closer to each other ,60. In afigure 1c) .. 1.1.3in vivo. However, powerful H1 binding to nucleosomal arrays have also been studied. For example, cryo-EM of chromatin arrays bound to H1 shows that under low salt conditions, H1 can drive the formation of a highly compact, twisted 30 nm fibre from a 12-nucleosome array. H1 shows a 1 : 1 stoichiometric association with the nucleosome cores and directly binds the nucleosome dyad and interacts with the entering and exiting linker DNA. They observe asymmetric or ‘off-dyad’ binding in tetranucleosomal units and mononucleosomes and posit that this domain localization of H1 plays a crucial role in the formation of a twist between tetranucleosomal units and thereby the 30 nm fibre [A cautionary point to consider is that the studies above were conducted using mononucleosomes, many with artificial DNA sequences. These provide powerful insights into binding probabilities, but likely do not accurately articulate the true state of chromatin fibre nm fibre ,51. A lanm fibre . It is nnm fibre .in vivo, the fundamental biophysical and biochemical properties driving H1 interaction with chromatin may be applicable. These principles governing H1 binding in vitro and in silico can help predict functional outcomes with regard to chromatin dynamics in diseases where H1 is altered.Although these conditions are not direct evidence of the chromatin state . 1.2. 1.2.1A fundamental concept that has gained traction in cancer research is that chromatin state, controlled by genetic and epigenetically alterations of histone proteins, histone-modifying enzymes and chromatin remodellers, modulates the initiation and progression of cancer. Here, we will focus on the implications of altered histone H1 which has emerged as a key player in regulating chromatin states by controlling the formation of higher-order chromatin fibres and may thereby facilitate cellular transformation.HIST1H1B-E have been reported in follicular lymphoma (FL) [Recent evidence of mutations in H1 variants leading to differences in H1 function has been found to have serious functional implications. For example, eight somatically acquired and seven germline mutations in oma (FL) . The higoma (FL) ,65. Thesoma (FL) .HIST1H1B-E which result in a disruption of H1 function [a). This, in turn, may give rise to less compact ‘6mer’ like zig-zag ladder-like conformation or the highly compact ‘12mer’ like conformation [Another set of mutations have been reported in the function . This drfunction . Recurrefunction –71. But,ormation ,56. Therormation ,57 that b. These data generated from the cBio portal can be used as a tool to visualize how subtle alterations to the structure of H1 may affect its interactions with chromatin. These data raise tantalizing possibilities that are ripe for experimental exploration—for instance, would these mutations prevent the H1 from binding at all? Will the altered H1 change the conformation of the linker DNA? Much like a kink in a garden hose at one end affects the outcome at the other end, do local conformational changes translate to genomic-scale alterations?The available 3D structures of the globular domains of H1 variants and known mutations for all of these, in addition to side chains affected, are presented in figure 2. 1.2.2H1 stoichiometry in mammalian chromatin is found to be in a variable ratio of nearly 0.4–1 H1 per nucleosome. Changes in this ratio were found to affect the NRL where a loss of H1 increased the NRL and an excess caused a drop ,73. AlthExpression analyses obtained from the Cancer Genome Atlas (TCGA) reveals that H1 variants are misexpressed in a variety of cancers . AdditioH1 genes are under tight transcriptional control with some being regulated at the post-transcriptional and post-translational levels. Therefore, mRNA expression may not always correlate with H1 protein levels. That being said, elucidating the content of H1 variant in a cancer is key to predicting possible functional outcomes. There is now an abundance of evidence that demonstrates distinct functions of H1 variants and variable binding affinities –90.c,d).The loss of protein or even the production of a mutant protein can trigger aberrant chromatin states. A recent study showed that the local density of H1 controls the balance of active or repressed domains by inducing genomic compaction and maintaining genome organizational states. A reduction in this stoichiometry can lead to a more diffuse interactive chromatin state leading to defects in cell survival pathways . Anotherfigure 3. 1.2.3H1 variants are made up of basic amino acids with an abundance of lysine which is responsible for its highly positive electrostatic charge . The lysPhosphorylation of H1 is the most commonly found PTM and has been studied in a fair amount of depth. This PTM is particularly interesting because it appears to have a modulatory role across the cell cycle where the phosphorylation of certain H1 variants like pT146H1.4 is critical for the condensation of mitotic chromosomes and thereby appropriate cell cycle progression. Other variants, like H1X, are phosphorylated to be precluded from the mitotic chromosome.During interphase, however, phosphorylation is associated with decondensed chromatin . Some stH1 PTMs associated with cancer are the subject of extensive study in order to identify potential biomarkers for cancer progression. pT145H1.2 and pT145H1.4 have been identified as cancer biomarkers for high-grade invasive bladder cancer and metastatic progression in hepatocellular carcinoma ,89. PhosIt is noteworthy that many PTMs occur in combination, primarily in the disordered regions of the H1 and some on the globular domain, which are responsible for appropriate H1 placement in a nucleosomal complex. This suggests that these PTMs can modify the manner of H1 binding and effectively modulate the chromatin state. This possibility presents an attractive challenge to structural biologists to investigate.. 1.3Interestingly, in addition to cancer-associated defects discussed above, there is a population of H1 predominantly found in the cytoplasm primarily associated with neurodegenerative diseases . The imp1–42 and α-synuclein. These amyloid plaques are a common feature of Alzheimer's and Parkinson's diseases [This non-nuclear fraction of H1 is usually phosphorylated, most likely phospho-H1.2, and preferentially binds to the common amyloid-like structure which is made of proteins like Aβdiseases . This stdiseases . Anotherdiseases . While s. 2a). Since tails are primarily the target of PTMs, these could contribute to different functional chromatin states. For example, in the pro-transcription environment, modified H1 might stay associated with the nucleosome to adjust the conformation such that it enables or guides transcription machinery towards it (b). Approaches that use genome-wide mapping such as CUT&RUN, or single-molecule tracking in living cells, will prove powerful allies in the quest to uncover how H1s are localized, what (if any) chromatin and DNA motif features they prefer, and the functional outcomes of their structural changes upon the nucleosome and fibre.Over the last few decades, the field of chromatin biology has made remarkable advances from first describing the ‘beads on a string’ structure to cutting edge computational modelling predicting the dynamics of chromatin in the nucleus. However, in this race to decode the details of chromatin organization, the linker histone family is now finally beginning to catch up, inevitably leading to many questions. We discuss here the various modes of H1 binding to chromatin in both mononucleosome form and arrays. But, since most of the predictions are based on the properties of globular domains, the effect of different H1 tail domains remains to be determined. How much of an effect would these different tails make? One possibility is that the addition of the H1 tail helps them bind to nucleosome conformations that are thought to preclude H1 binding due to the linker DNA proximity a. Since wards it b. ApproaInitially thought to be a merely a global repressor, it is now apparent that the H1 family of proteins have the potential to affect the genome architecture through not only their intrinsic properties but also by the addition of structural conformations. It is tempting to visualize H1 dynamics in the form of a high-energy Quidditch match in a dense nuclear forest, with H1 taking the form of the ever-buzzing golden Snitch zipping in and out and around Bludger-like chromatin remodellers and Quaffle-like transcription factors, to determine the final outcome of the epigenetic game of accessibility in the eukaryotic nucleus.