Streptococcus (GAS). Using a combination RNAseq/ChIP‐seq approach, we found that CcpA affects transcript levels of 514 of 1667 GAS genes (31%) whereas direct DNA binding was identified for 105 GAS genes. Three of the directly regulated genes encode the key GAS virulence factors Streptolysin S, PrtS (IL‐8 degrading proteinase), and SpeB (cysteine protease). Mutating CcpA Val301 to Ala (strain 2221‐CcpA‐V301A) abolished interaction between CcpA and HPr and impacted the transcript levels of 205 genes (40%) in the total CcpA regulon. By ChIP‐seq analysis, CcpAV301A bound to DNA from 74% of genes bound by wild‐type CcpA, but generally with lower affinity. These data delineate the direct CcpA regulon and clarify the HPr‐dependent and independent activities of CcpA in a key pathogenic bacterium.Catabolite control protein A (CcpA) is a master regulator of carbon source utilization and contributes to the virulence of numerous medically important Gram‐positive bacteria. Most functional assessments of CcpA, including interaction with its key co‐factor HPr, have been performed in nonpathogenic bacteria. In this study we aimed to identify the in vivo DNA binding profile of CcpA and assess the extent to which HPr is required for CcpA‐mediated regulation and DNA binding in the major human pathogen group A  The carbon catabolite protein, CcpA, regulates carbohydrate utilization and virulence in concert with its phosphorylated co‐factor histine containing protein (HPr~P). In this study we engineered a CcpA isoform that does not interact with HPr~P. Using a combination of ChIPseq and RNAseq we report the in vivo binding sites of CcpA in group A Streptococcus. We also demonstrate that CcpA retains the ability to bind and regulate genes even when it cannot interact with HPr~P. To this end, we expressed and purified recombinant CcpA and CcpAV301A along with HPr. We used recombinant HPrK/P to phosphorylate HPr at Ser46 as previously described  compared to genes (153) whose transcript levels were lower in 2221∆ccpA , which is in accordance with previous observations  operon, the nga‐slo operon, and prtS . For the 12 virulence factor encoding genes/operons impacted by CcpA inactivation, only four were HPr~P‐dependent . Conversely, of the 17 CcpA‐regulated genes/operons encoding carbohydrate transport systems, 14 were HPr~P‐dependent , and S (function unknown) relative to HPr~P‐independent genes  of the 205 HPr~P‐dependent genes, which we subsequently considered as fully HPr~P‐dependent   were semi‐HPr~P‐dependent  comparison of the HPr~P‐dependent genes. If the CcpA‐HPr~P complex is essential for CcpA‐regulation of a particular gene, then, there should be no significant difference in transcript levels between strains 2221Δn) Table . For thes Figure . We desis Figure . All fount Table . Taken t2.3ccpA strain was used as a control to ensure the specificity of our findings.There are limited genome wide assessments of CcpA binding to DNA in pathogenic bacteria and none for a strain in which CcpA‐HPr~P interaction has been abrogated  in predicted promoter regions   of the 514 genes whose transcript levels were significantly impacted by CcpA inactivation in strain MGA2221were identified as being directly regulated by CcpA . By COG analysis, genes directly regulated by CcpA were significantly more likely to be in category C (energy production and conversion) and G (carbohydrate transport and metabolism)  Figure .pulA gene encoding a cell‐surface pullulanase important for both nutrient acquisition and adherence , and mannose, which stands in contrast to the 13 PTS gene/operons whose transcript levels were affected by CcpA inactivation. We also observed CcpA binding sites in genes/operons encoding three ABC carbohydrate transport systems for maltose, maltodextrin, and sialic acid. The central role of maltose/maltodextrin utilization in GAS pathophysiology is reflected by the five distinct CcpA binding sites for genes involved in the acquisition of these prevalent carbohydrates including that in the promoter region of the e Figure . In addiStreptococcus suis did not identify in vivo CcpA binding for arcA  which was previously reported to be directly regulated by CcpA  was CcpA‐bound. As previously noted, there was also CcpA enrichment in the intergenic region between malE and malR , which was identified to be directly regulated by CcpA in a previous study , were significantly affected by CcpA inactivation Table . Of thesR Figure , which e2.4prtS, sagA, and speB, also evidenced binding in strain 2221‐CcpA‐V301A  or the operon encoding the ATP synthase genes (Table p < .001 by Fisher's exact test). To validate the findings from our ChIP‐seq, six sites that were enriched in both the wild type MGAS2221 and the 2221‐CcpA‐V301A strain were confirmed by SYBR qRT PCR  were also enriched in strain 2221‐CcpA‐V301A Figure . All of A Tables . SimilarA Tables  bound bycA Table . In contR Figure .2.5pgi, which encodes a glucose utilization protein, plr, which encodes an aldehyde dehydrogenase, ldh, which encodes an enzyme that converts lactate to pyruvate, and the previously mentioned pulA. Of these 29 CcpA enriched sites that were not associated with transcript level variation after CcpA inactivation, 11 were also enriched in strain 2221‐CcpA‐V301 including three genes encoding carbohydrate utilization proteins  as well as one site in a CRISPR operon and the salY gene, which encodes an ABC transport protein involved in lantibiotic secretion , suggesting that the cre‐containing enriched DNA sites were more biologically impactful under the studied conditions. The cre sites that were associated with genes whose transcript levels were affected by CcpA inactivation, were significantly more likely to be located within 500 nt upstream and 50 nt downstream of the start codon compared to cre sites that were near genes not impacted by CcpA inactivation (p < .001 by Fisher's exact test)  Figure . Relative Figure ), the ma2 Figure . We compcre consensus derived from DNA loci in strain 2221‐CcpA‐V301A compared to that derived from MGAS2221. Of 57 cre sites in MGAS2221, 37 were bound in strain 2221‐CcpA‐V301, and we did not identify any enriched cre containing sites that were present in strain 2221‐CcpA‐V301 but not in MGAS2221. For cre sites that were bound only in strain MGAS2221 but not in the strain 2221‐CcpA‐V301A  was associated with a cre  strains were routinely grown in Todd‐Hewitt media supplemented with 0.2% yeast extract (THY) at 37°C with 5% CO2. A single amino acid change in ccpA was engineered into the wild type MGAS2221 strain by homologous recombination using the pBBL740 plasmid as described previously  analysis was performed at 25°C using BIAcore T200 instrument . Wild type CcpA and V301A mutant proteins were immobilized on sensor chip CM5 via amine coupling. PBS  was used as running buffer for immobilization and binding experiment. Sensor chip surface was activated by injecting a 1:1 mixture of 0.4 M of 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide hydrochloride and 0.1 M of N‐hydroxysuccinimide over the flow cell surface at 5 µl/min for 7 min. After the surface was activated, CcpA (10 µg/ml in 10 mM NaOAc pH 5.0) and CcpAV301A (20 µg/ml in 10 mM NaOAc pH 5.0) were injected over different flow cells. Sensor surface was deactivated with an injection of 1.0 M ethanolamine‐HCl pH 8.5 at 5 µl/min for 7 min. Approximately, 3,700 response units (RU) of CcpA and 4200 RU of CcpAV301A were immobilized. A reference flow cell was prepared with activation and deactivation steps but no protein was immobilized. Two‐fold dilutions of HPr or HPrSer46~P from 1.25 to 80 µM in PBS were flown over the immobilized proteins at 20 µl/min for 30 s. All SPR responses were baseline corrected by subtracting the response generated from the corresponding reference surface. Double‐referenced SPR response curves (with the buffer blank run further subtracted) were used for affinity determination. The equilibrium response of each injection was collected and plotted against the concentration of injected protein. A one‐site binding (hyperbola) model was fitted to the data (GraphPad Prism 4) to obtain the equilibrium dissociation constant 4.4Recombinant HPr protein was phosphorylated and purified as described previously ] and formaldehyde and harvested. Pellets were sonicated and CcpA‐containing complexes were immunoprecipitated using a polyclonal anti‐CcpA antibody, and then, analyzed for the presence of HPr by western blotting using a polyclonal anti‐HPr antibody and the Odyssey imaging system as described previously  were generated for individual comparisons of the wild‐type (MGAS2221) and the isoallelic strain 2221‐CcpA‐V301A to the ∆ccpA deletion strain. For each strain, TPM values from replicate samples for each gene within the reference MGAS5005 genome were averaged. Generated plots in combination with differential gene expression values produced by CLC Genomics Workbench (version 20) were used to identify targets with significantly altered transcript levels in individual comparisons. For Taqman real‐time qRT‐PCR, strains were grown in duplicate on two separate occasions to mid‐exponential phase in THY and processed as described previously  using the RNeasy kit (Qiagen) and processed as previously described  at MD Anderson Cancer Center. Briefly, Illumina compatible Indexed libraries were prepared from 12 ng of Diagenode Bioruptor Pico sheared ChIP DNA using the KAPA Hyper Library Preparation Kit (Roche). Libraries were enriched with 2 cycles of PCR then assessed for size distribution using the 4200 TapeStation High Sensitivity D1000 ScreenTape (Agilent Technologies) and quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher). Equimolar quantities of the indexed libraries were multiplexed, 9 libraries per pool. The pool was quantified by qPCR using the KAPA Library Quantification Kit (Roche) then sequenced on the Illumina NextSeq500 sequencer using the 75 nt high‐output flow cell. Raw FASTQ files from DNA sequencing were trimmed through Trimmomatic v. 0.36  using Bowtie2 v. 2.3.0  to remove sequencing noise background based on a coverage ≤2 of fold change. Finally, we applied MACS2 on the clean data using a cutoff q‐value of 0.05 with nonmodel option and MFOLD of “2 50.” The effective genome size was set to 1,838,562. The sequences of assembled peaks were retrieved and submitted to Multiple Em for Motif Elicitation v. 5.0.0 (MEME)  (Thorvaldsdottir et al., For ChIP analysis, MGAS2221, 2221∆To verify selected sites of enrichment observed by ChIP‐seq, ChIP DNA was analyzed by SYBR qRT PCR. Primers Table  were desThe authors have no conflict of interests.S.D., S.A.S., V.A., G.G., M.L., and M.H. designed the research, S.D., S.A., N.H., and S.A.S. did the experiments, S.D., X.L., S.A.S., A.R.F, V.A., G.G., V.M., M.L., and M.H. performed data analysis and S.D., X.L., M.L., M.H., and S.A.S. wrote the manuscript. All authors reviewed the manuscript.Fig S1‐S4Click here for additional data file.Table S1Click here for additional data file.Table S2Click here for additional data file.Table S3Click here for additional data file.


Chromatin accessibility of a promoter is fundamental in regulating transcriptional activity. The histone variant H2A.Z has been shown to contribute to this regulation, but its role has remained poorly understood. Here, we prepare high-depth maps of the position and accessibility of H2A.Z-containing nucleosomes for all human Pol II promoters in epithelial, mesenchymal and isogenic cancer cell lines. We find that, in contrast to the prevailing model, many different types of active and inactive promoter structures are observed that differ in their nucleosome organization and sensitivity to MNase digestion. Key aspects of an active chromatin structure include positioned H2A.Z MNase resistant nucleosomes upstream or downstream of the TSS, and a MNase sensitive nucleosome at the TSS. Furthermore, the loss of H2A.Z leads to a dramatic increase in the accessibility of transcription factor binding sites. Collectively, these results suggest that H2A.Z has multiple and distinct roles in regulating gene expression dependent upon its location in a promoter. Histone variant H2A.Z has been suggested to contribute to the regulation of promoter accessibility. Here, the authors present high-depth maps of the position and accessibility of H2A.Z-containing nucleosomes for human Pol II promoters and provide evidence that H2A.Z has multiple and distinct roles in regulating gene expression dependent upon its location in a promoter. The fundamental subunit of chromatin is the nucleosome core, ∼150 base pairs of DNA wrapped 1.65 times around a histone octamer5. The prevailing functional view of a nucleosome (the nucleosome core plus linker DNA) is that it acts as a barrier preventing DNA access8. Therefore, the current model holds that the specific positioning of nucleosomes within a promoter plays an important role in controlling the accessibility of key regulatory DNA elements and the transcription start site (TSS) to transcription factors and RNA Pol II, respectively10.The structure and dynamics of promoter chromatin is a principal driver of regulated gene expression20. Such promoter mapping studies have revealed an apparent common nucleosome arrangement with a nucleosome free or depleted region (NFR/NDR) at an active transcription start site (TSS) followed by a high occupancy and strongly positioned ‘+1’ nucleosome immediately downstream of the TSS. This strongly positioned +1 nucleosome then establishes a uniform or phased arrangement of nucleosomes downstream into the gene body. Similarly, in most cases, these nucleosome mapping studies revealed a strongly positioned ‘−1’ nucleosome located immediately upstream of an active TSS. A repressed gene, on the other hand, lacks this phasing of nucleosomes around the TSS and, as expected, the TSS is also covered by a nucleosome19. Therefore, transcriptional activation has been proposed to involve nucleosome eviction from the TSS followed by the phasing of downstream and upstream nucleosomes10.Genome-wide mapping of nucleosomes for several unicellular and multicellular organisms has provided an unprecedented amount of information concerning how nucleosomes are organised along the DNA of promoters, and how this organisation changes prior to and following transcriptional activation21. On the other hand, in humans and other complex multicellular organisms, promoters possess nucleosome attracting-regions21. This suggests that nucleosomes may be utilised in more complex ways to control the output of a promoter in complex organisms22. Indeed, new data are starting to emerge that challenge the current model; more recent information suggests that, rather than removing or repositioning a nucleosome to gain DNA access, the stability of a nucleosome may be modulated to enhance DNA accessibility23.It has been proposed that strong intrinsic nucleosome-repelling regions are encoded in the DNA sequence of gene promoters from simple unicellular organisms, such as budding yeast1. It is well established that the biochemical properties of nucleosomes vary significantly throughout the genome, which would affect their inherent stability and thus their sensitivity to MNase digestion24. In other words, a single level of MNase digestion may over-digest active unstable nucleosomes or under-digest stably repressed chromatin1. This is illustrated by the recent observation that the TSS may in fact not be nucleosome-free but contain a highly unstable nucleosome, which was not detected earlier because of its extreme sensitivity to MNase digestion and/or salt conditions employed26. However, this remains controversial27. By employing more than one level of MNase digestion, we and others previously showed that, despite nucleosomes displaying a similar level of occupancy in promoters, they exhibited differential sensitivity to MNase digestion30. Combined with a more recent study23, the emerging view is that high nucleosome occupancy does not necessarily exclude high accessibility.Current approaches for mapping nucleosome occupancy often employ micrococcal nuclease (MNase) to produce mononucleosome-size DNA fragments (∼150 bp) for subsequent sequence analysis. However, the majority of studies to date have only used a single digestion time point31. The existence of such sub-nucleosomal particles would also play an important role in regulating the accessibility of genomic DNA in cells32. However, again, most studies to date have only selected the canonical ∼150 bp nucleosomal-sized DNA fragment for high-throughput sequence analysis and not included smaller sized DNA fragments1. DNA fragments <150 bp in size derived from MNase digestion may arise from sub-nucleosomal particles or from the protection of DNA by non-histone DNA binding proteins such as transcription factors30. Therefore, sequencing sub-nucleosomal DNA fragments may be as important as sequencing canonical nucleosome DNA fragments for the understanding of promoter structure and function.Along these lines, it has become clear that the nucleosome is not a static structure but exists as a highly dynamic family of interconverting partially assembled or disassembled structural states33. However, despite considerable effort to understand the functional relationship between H2A.Z and transcription, its function remains enigmatic because the data supports both positive and negative roles in transcription33. To our knowledge, no high-resolution map of H2A.Z at mammalian promoters has been produced, but composite H2A.Z ChIP-seq studies in humans and mice have revealed the formation of two well-positioned H2A.Z nucleosomes flanking each side of the TSS (the −1 and +1 nucleosome)35. It also has been reported that an unstable H2A.Z-containing nucleosome occupies the TSS of an active promoter35. Also controversial is whether the +1 H2A.Z-containing nucleosome facilitates transcriptional elongation or creates a strong barrier contributing to the pausing of RNA Pol II in cells40. In vitro biochemical studies have shown that mammalian H2A.Z stabilises the nucleosome core and inhibits transcription43.A major player in shaping the chromatin organisation of a promoter is the evolutionarily conserved histone variant H2A.Z37. Moreover, we found that a +1 H2A.Z-containing nucleosome could repress the expression of some key epithelial genes while other H2A.Z promoter nucleosomes were correlated with gene activation. However, given the limitation of this study as well as other mammalian ChIP-Seq experiments as outlined above, an understanding of the role of H2A.Z in EMT and promoter function in general will require high-resolution H2A.Z-nucleosome position and MNase sensitivity maps. Additionally, analyses that includes mapping sub-nucleosomal particles will create a more complete picture of the role H2A.Z in regulating promoter architecture and transcription.Previously, we demonstrated that H2A.Z is a major regulator of the epithelial-mesenchymal transition (EMT), which is a critical de-differentiation process required for early mammalian development and malignancy13 to produce high-resolution H2A.Z-containing  nucleosome maps of a 2KB region surrounding the TSS of the  open reading frames of the human genome. To begin to understand how H2A.Z can facilitate or inhibit transcription, we have conducted our study in the context of gene expression changes associated with different epithelial cellular states, utilised a differential MNase digestion approach to obtain information on nucleosome stability, and analysed sub-nucleosomal-sized fragments to examine non-canonical nucleosomal and regulatory factor binding. These experiments have allowed us to redefine the nucleosomal architecture of active and inactive promoters, characterise nucleosome dynamics under different physiological contexts, and define the multiple roles of H2A.Z in these processes.Here, we have employed an MNase-Transcription Start Site Sequence Capture method (mTSS-seq) established previously13 to map both total MNase protected nucleosome footprints and specifically immunoprecipitated H2A.Z-containing nucleosomes as well as sub-nucleosomal particles. In addition, we correlated different promoter organisations with gene expression by RNA-Seq  data at the TSS with the coverage obtained from conventional, genome-wide H2A.Z ChIP-Seq (and mTSS-seq) using the same sequencing libraries (see Methods section). For this and subsequent experiments, we used MCF-10A cells , a human mammary epithelial cell line that are normal and near diploid.mTSS-seq combines in-solution targeted enrichment of 2 kb surrounding TSSs of 21,857 human protein-coding genes, as curated by NCBI RefSeqIn order to demonstrate the performance of the mTSS-seq capture approach, libraries were subsampled to 5 and 10 million reads  using the lentiviral vector shH2A.ZpLVTHM in MCF-10A cells46. To test this concept, first, we produced heat maps displaying total  and differing biological processes. For example, cluster 1 is linked with mitotic cell cycle process, cluster 3 is associated with nucleic acid metabolism and clusters 5 and 6 are associated with ubiquitin-dependent processes and cellular localisation, respectively. Clusters 4 and 7 are linked with biological processes unrelated to epithelial function . Indeed, when we repeat the K-means clustering whereby two instead of seven clusters are produced, the classical view of a promoter with phased nucleosomes upstream and downstream of the nucleosome is recapitulated  . Moreover, promoter nucleosome MNase sensitivity but not nucleosome positioning changes following an alteration in cell fate. These results demonstrate that nucleosome positioning and MNase nucleosome accessibility are not coupled.Next, we investigated the relationship between total and H2A.Z nucleosome occupancy, and their respective MNase sensitivities, with gene expression by producing these four profiles for genes that are highly expressed , comparable results are obtained to that of TGF-β induction Fig. . In otheHaving defined a variety of different types of active promoter structures, we next investigated their histone modification status. To do this, we used publicly available ChIP-Seq data for MCF10A cells obtained from NCBI’s Sequence Read Archive  to the TSS for MCF-10A cells, MCF-10A cells induced with TGF-β, MCF-10CA1a cells, and shH2A.Z MCF-10A cells, respectively Fig. . SignifiNext, we examined whether these SFs originate from the same promoters in all the different cellular states, or are generated from different promoters in a cell type specific manner Fig. . DiffereIn each different experimental condition, cluster 1 contains promoters that have an increase of SFs at the TSS. In TGF-β induced cells, this cluster contains 22% of the top Signficantly differentially expressed genes as compared to MCF10A. In MCF-10CA1a cells, this cluster contains 16% of the top Signficantly differentially expressed genes as compared to MCF10A. Finally, in the H2A.Z knockdown cells, this cluster contains 21% of the top differentially expressed genes as compared to MCF10A. Cluster 6, which represents promoters that contain more SFs in the MCF10A cells, but less SFs than in each experimental condition, contains only 9–12% of the top significantly differentially expressed genes.To further explore the correlation between changes gene in expression and the increase in SFs following H2A.Z knockdown, we identified common promoters among: (1) the top quartile of promoters with the most MNase-sensitive nucleosome at the TSS (5454 genes); (2) the top quartile of promoters that yield the most SFs at the TSS following H2A.Z knockdown  and (3) the top 1% of genes whose expression increases following H2A.Z knockdown (1348 genes). This revealed that 110 promoters share all of these characteristics. A gene ontology analysis of these genes shows that they are indeed involved in epithelial related functions such as epidermal cell and keratinocyte differentiation, and regulation of morphogenesis of an epithelium Fig. .SFs derived from MNase digestion can arise from either the internal digestion of an unstable nucleosome core particle and/or by the protection of DNA by non-histone proteins such as transcription factors. Our final aims were to investigate: (1) whether SFs occupy other important promoter DNA elements i.e. transcription factor binding sites (TFBS) and (2) if the abundance of these SFs also increase when H2A.Z is depleted.49  and MCF-10CA1a (1002) increased compared to MCF-10A cells (597). However, the most dramatic increase is found in promoters in shH2A.Z MCF-10A cells . In other words, a substantial proportion of SFs arise from sites previously occupied by H2A.Z nucleosomes. More striking, 100% of these SF peaks overlap with transcription-factor binding sites which have the matched transcription factor expressed . The major H2A.Z isoform expressed in the different cell lines examined here is H2A.Z.1. Our data suggests that H2A.Z may have several roles in regulating the accessibility of promoters dependent on where it is located within a promoter e.g. upstream or downstream of the TSS, at the TSS or at transcription factor binding sites.Specifically, we show that: (1) there are many different types of H2A.Z-containing active and inactive promoters structures that differ in their nucleosome organisation and sensitivity to MNase digestion; (2) a surprising feature of different types of active promoters is the presence of MRNs nucleosomes that are positioned either upstream or downstream of the TSS including a H2A.Z MRN at the +1 position. Other types of active promoters have a H2A.Z MSN occupying the TSS while less active and repressed promoters contain a H2A.Z MRN at this position. All the different types of active promoter nucleosomal configurations display the expected active histone marks thus confirming our classification of the different types of active and inactive promoter nucleosome arrangements; (3) the loss of H2A.Z results in a dramatic increase in the formation of SFs that map to important functional DNA elements (TFBS and the TSS). This suggests that H2A.Z has an important role in preventing promiscuous DNA access and (4) while promoter nucleosome positioning does not change between different cellular states, nucleosome MNase accessibility does, perhaps suggesting the latter feature is more important in regulating promoter activity.Concerning the nucleosome arrangement of an active promoter, one important observation is that, rather than a single consensus active promoter organisation with a high occupancy −1 and + 1 nucleosome flanked by phased nucleosomes  nucleosome either at the −2, −1, +1 or +2 positions. Conversely, inactive promoters appear to be more disordered lacking positioned nucleosomes which are also more MNase sensitive.Our data suggests that changes in nucleosome accessibility rather than positioning play a more important role in regulating transcription when the phenotype of the cell changes. This indicates that nucleosome positioning and accessibility are not coupled, which confirms and extends previous studies50, a H2A.Z+1 nucleosome greatly lengthens RNA Pol II crossing time and in Drosophila S2 cells, this H2A.Z nucleosome is anticorrelated with nucleosome turnover39. Further, H2A.Z intrinsically forms a stable histone octamer51. Intriguingly, the presence of a less well positioned, and more MNase sensitive H2A.Z nucleosome centred around the +1 position is correlated with strong gene repression  would also produce sub-nucleosomal DNA fragments at promoters58.Finally, our data suggests that H2A.Z may have another important role at promoters by preventing promiscuous DNA access at the TSS and transcription factor binding sites because when H2A.Z is depleted in MCF-10A cells, there is a dramatic increase in subnucleosomal-sized particles at these important promoter DNA elements. The transition of nucleosomes into subnucleosomal particles, critical for the subsequent invasion by non-histone proteins, was first shown to occur during spermatogenesis involving the testis specific histone variant H2A.L.230. Another possibility, however, that cannot be excluded is that the loss of H2A.Z may allow other histone variants e.g. CENPA to be incorporated into promoters, which is known to wrap less DNA59. Future proteomic studies will elucidate the nature of these subnucleosomal fragments.The nature of the sub-nucleosomal sized particles observed here is unknown but could be nucleosomal hexamers or tetramers and/or reflect the binding of non-histone proteins. Significantly, sub-nucleosomal sized particles form at transcription factor binding sites only when their matched transcription factor is expressed suggesting that these small particles are transcription factors as indicated previouslyIn conclusion, the results of this investigation suggest that H2A.Z has multiple roles in regulating promoter chromatin architecture and transcription dependent upon where it is located, and whether it exists in a stable or unstable nucleosomal form Fig. . This mal-glutamine, 20 ng/mL human EGF, 500 ng/mL Hydrocortisone and 100 ng/mL Cholera Toxin. MCF 10A cells were obtained from the ATCC (CRL-10317) and MCF10Ca1a were obtained from the Barbara Ann Karmanos Cancer Institute . For EMT induction, MCF-10A cells were treated with 5 ng/ml TGF-β1 (Redsystems) for 8 days. MCF-10A cells were transduced with the lentiviral vector pLVTHM shH2A.Z37. GFP-positive cells were sorted 2 days post-transduction and further cultured for 8 days before being processed. RNA was purified using the RNeasy Mini Kit (Qiagen) and cDNA synthesised using the QuantiTect Reverse Transcription Kit (Qiagen). The knockdown of H2A.Z expression was analysed by qPCR on the 7900HT Fast Real-Time PCR system using SYBR Green I master mix (applied biosystems) and 0.15 mM primers37. Relative expression values were normalised to pooled housekeeping genes Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Beta-actin (B- actin), Ribosomal Protein S23 (RPS23) and Splicing Factor 3a Subunit 1 (SF3A1)60. Primer sequences are shown in Supplementary Table MCF-10A and MCF-10Ca1a cell lines were grown in DMEM/Nutrient F12 (DMEM/F12) media supplemented with 5% Horse Serum, 14 mM NaHCO3, 10 µg/mL insulin, 2 mM 37. The UCSC hg19 “knownGene” set of transcripts were used as reference transcriptome against which transcript abundance estimation was performed, followed by collapsing of transcript abundances on gene level. Detailed analysis scripts implemented in “snakemake” and R scripts can be found at: https://github.com/JCSMR-Tremethick-Lab/MCF10APromoterAnalysis66.All RNA-Seq experiments were performed in triplicate. Total RNA was isolated using the Qiagen RNeasy kit following manufacturer’s instructions. Stranded mRNAseq libraries were constructed according to Illumina TruSeq stranded mRNA protocol with poly-A enrichment (Illumina). Libraries were sequenced with 2 × 75 bp paired-end on Illumina NextSeq500 instrument in high-output mode at Biomolecular Research Facility of the Australian Cancer Research Foundation, Canberra, Australia. Base-calling was performed using the “bcl2fastq2” script (V2.16) of the CASAVA pipeline. Technical aspects of the gene-level differential expression analysis were performed2, 0.3 M sucrose, 1 mM CaCl2 and 1% Nonidet P-40. The nuclei were then pelleted by centrifugation at 1000 × g for 5 min at 4 °C.In all, 2.5 × 107 cells were harvested at, cross-linked in 1% formaldehyde in PBS, and incubated for 10 min at room temperature. After the 10 min incubation, the cross-linking reaction was stopped by addition of 125 mM glycine. Next, the nuclei were isolated in nucleus isolation buffer containing: 10 mM HEPES at pH 7.8, 2 mM MgOAc2, 5 mM KCl, 50 mM Tris-Cl (pH 7.4), 1 mM CaCl2, 12.5% glycerol). The MNase digestion reactions were stopped with 50 mM EDTA. Next, the protein-DNA crosslinks were reversed by treating the MNase-digested nuclei with 0.2 mg/mL proteinase K and 1% sodium dodecyl sulfate, and incubating overnight at 60 °C. The samples were then run and the nucleosomal ladder was separated on a 2% agarose gel. Following the separation of the DNA fragments, mononucleosome-sized and subnucleosomal-sized fragments (<200 bp) were isolated from the agarose gel. Next, the mononucleosomal- and subnucleosomal-sized fragments for all MNase conditions were combined for each respective sample. Following the combination of all fragments per sample, DNA was extracted with phenol-chloroform and precipitated with alcohol for 10 min at −20 °C. The DNA was then pelleted by centrifugation at 3000 × g for 10 min at 4 °C, and dissolved in TE .All experiments were performed as biological replicates. Nuclei were digested under either Light MNase conditions (40 gel units for 15 min at 37 °C) or Heavy MNase conditions  (NEB M0247S) in MNase cleavage buffer . An uncropped western blot is shown in Source Data File 1.H2A.Z ChIP assays employing either low or high MNase-digested chromatin and the preparation of ChIP-Seq libraries for conventional genome-wide ChIP-Seq were carried out using our in house H2A.Z antibody, as extensively used previouslyUsing the NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (NEB #E7370S/L), DNA sequencing libraries were prepared for the mononucleosomal-sized and subnucleosomal-sized fragments for each sample. DNA was end-prepped using NEB Prep enzyme mix, end-repair reaction buffer (10X), and 30 ng of DNA for each sample, then held at 30 °C for 30 min and then at 65 °C for 30 min. Adaptors were ligated onto the end-repaired samples by adding NEB Blunt/TA Ligase Master Mix, NEBNext Adaptor for Illumina, and Ligation Enhancer and incubating at 20 °C for 15 min. The adaptor-ligated DNA was cleaned up using AMPure XP beads to remove any unwanted ligated products. The universal and indexed sequences were added by PCR using 23 μl of adaptor-ligated DNA fragments, NEBNext High Fidelity 2X PCR Master Mix, index primers provided in NEBNext Multiplex  Oligos for Illumina, and Universal PCR Primers provided in NEBNext Multiplex  Oligos for Illumina. Then PCR was done for 8 cycles . The adaptor-ligated DNA was cleaned up using AMPure XP beads to remove any unwanted products. The libraries were quality-checked using Agilent 2100 Bioanalyzer High-Sensitivity. Across the libraries, the samples ranged between 200 and 400 bp and there were no adaptor or primer dimers.13, as curated by NCBI RefSeq44. We termed this approach mTSS-seq. Size selected fragments (∼50–200 bp) were used to prepare Illumina sequencing libraries and subjected to targeted enrichment utilising the custom-designed Roche Nimblegen SeqCap EZ Library. DNA fragments were captured according to the Roche Nimblegen protocol. Paired-end reads (see below) were aligned to the hg19 genome assembly (IHGSC 2001) and read densities were visually inspected with the UCSC genome browser. Globally, each data set contained an average of 85.7% of TSS-seq reads uniquely aligned to the genome (∼10.4 million of uniquely mappable reads per experiment). Whole genome mapping data sets contain an average of 77.4% of TSS-seq reads in the absence of sequence capture. Of the reads that uniquely aligned to the genome, >98% overlapped with the targeted TSS regions compared to 2% in the absence of sequence capture.Previously, we combined MNase-seq with in-solution targeted enrichment of 2 kb surrounding TSSs of 21,857 human genesWe compared the TSS region coverage of the TSS-Seq libraries with the same libraries sequenced without TSS capture for three conditions: subsampling 5 M and 10 M reads, and including the fully sequenced libraries . See Supplementary Table Using a PE-50 single lane on an Illumina HiSeq 2500, HiSeq Flow Cell v3, the samples were loaded at 12 pM. The libraries were sequenced using standard Illumina sequencing protocols. Two kits were used: the TruSeq SBS Kit v3 and the TruSeq PE Cluster Kit v3 -cBot – HS. The reads were demultiplexed using the Casava Software (v1.8.4), and the library adaptors were removed using the cutadapt software. The sequenced fragments were aligned to the HG19 assembly of the human genome using bowtie2 2.1.0. Using samtools, non-unique and non-paired fragments were removed from the sequenced fragments. Nucleosome occupancy profiles were obtained by calculating the fragments per million that mapped at each base-pair in the SeqCap regions (bedtoolsCoverage). Midpoints ±30 bp (i.e. 60 bp centred on the nucleosome dyad) were used to plot nucleosome distributions and were determined through the calculation of centre fragments in 60 bp windows at a 10 bp step in the 2 kb surrounding each TSS. Further analysis of the nucleosome distributions was done in the R environment, R 2.15.1, using our lab-developed software, genmat . matHeatmap was used to plot normalised rpm mTSS-seq data for all RefSeq genes ±2 kb surrounding the annotated TSSs. Average plots represent the average value of all promoters in a respective cluster in 10 bp windows across the 2 kb SeqCap region. K-means clustering of nucleosomal data was performed using the matHeatmap function, using the Hartigan and Wong algorithm. Dinucleotide periodicity was calculated from total 147–150 bp nucleosomal fragments using the bedWords function. Gene ontology analyses based on heatmap gene classifications were performed using two unranked sets of genes, the target set of genes and the total mTSS-seq gene list as background65. Alluvial diagrams were created using the R package “alluvial” (Bojanowski M and Edwards R (2016). alluvial: R Package for Creating Alluvial Diagrams. R package version: 0.1–2, https://github.com/mbojan/alluvial), based on the results of the k-means clustering analyses.Analysis of nucleosome distributions was done in the R environment using our custom tools  of each peak, and a cut-off of IDR 0.1 was chosen to select the most reproducible set of peaks for further analysis. The genome size parameter was adjusted to 40,999,507, as explained above.In order to determine if specific DNA motifs, which could represent transcription factor binding sites (TFBS), are associated with the observed subnucleosomal fragments, and if furthermore the subnucleosomal fragments originate from known H2A.Z peak sites, we performed our analysis in following steps: First, we used the aligned subnucleosomal fragments as input for peak calling using MACS2 v.2.1.2-0. MACS2 was run without control/input libraries, as these fragments are the result of an MNase digest of DNA without immuno-precipitation. Instead the local background distribution (“lambda”) was imputed from the treatment/MNase libraries. The genome size parameter was adjusted to 40,999,507 which represents the total number of nucleotides covered under the promoter capture array, and thus represents the maximum mappable genome. For H2A.Z containing nucleosomes, MACS2 peak calling was performed with independently for each biological replicate H2A.Z ChIP library. MNase-digested DNA libraries were pooled and used as the control. The https://github.com/JCSMR-TremethickLab/MCF10APromoterAnalysis/blob/master/snakemake/rules/shortFragments_macs2_peak_calling.py66.A “snakemake” implementation of the peak calling pipeline can be found at q-value < 0.1 and a fold-enrichment of >4 as the central point of subnucleosomal peaks. For each summit passing the filter threshold, we exported 500 bp of DNA sequence surrounding the summit site (single nucleotide) as FASTA files using the BSgenome package .The lists of subnucleosomal fragment peaks and summits were loaded into R/Bioconductor using functions from the “data.table” package. We chose the summits of peaks with a The FASTA files were used as input for “meme” and motif discovery was performed using the objective functions “centrally enriched” as well as “centrally distributed”. Minimum word size was set to 8, maximum to 15. The background hidden markov model was estimated from the actual promoter sequences, as captured by our promoter capture array using the “fasta-get-markov” utility supplied as part of the MEME suite (v5.0.5). E-value threshold for motif discovery was set to 0.05 and mode was set to “zoops”.https://github.com/JCSMR-Tremethick-Lab/MCF10APromoterAnalysis/blob/master/snakemake/rules/shortFragments_meme_processing.py66.The results from the “meme” search were used as input for the “tomtom” search in the “HOCOMOCO v11 full HUMAN” database of human transcription factor binding sites. The “snakemake” script driving this abridged MEME analysis pipeline can be found at https://github.com/JCSMR-Tremethick-Lab/MCF10APromoterAnalysis/tree/master/R/TFBSAnalysis66.Both “meme” and “tomtom” search result files were loaded into R using the fread function from the “data.table” package and further analysis was performed using the information from peak calling and differential gene expression analysis. All scripts in the integrative analysis can be found at 67. Publicly available ChIP-Seq data for MCF10A cells were obtained from NCBI’s Sequence Read Archive using “sra-tools”  downloaded to our local HPC system using “prefetch” and exported in FASTQ format using “fastq-dump”. Raw FASTQ sequencing data for each library were processed with fastp version 0.19.568 in order to remove potential adaptor sequences, trim low-quality bases and to perform quality control. The trimmed and quality-checked reads were then aligned to the unmasked human reference genome build GRCh37/hg19 (UCSC annotation) using bowtie2 2.3.569 with minor modifications to the default parameters (‘’—no-mixed –no-discordant”), and allowing for a maximum insert size of 500 bp. The resulting SAM output was further processed using samtools version 1.970 and picardTools version 2.20.1 . In a first step, alignments with a quality score <10 were removed, followed by marking and removal of duplicate reads using picardTools ‘MarkDuplicates’. The sorted, quality filtered and de-duplicated BAM files were than indexed using samtools ‘index’ and insert size distributions estimated with picardTools ‘CollectInsertSizeMetrics’. Aligned ChIP-Seq and input data were further inspected using a set of quality control tools provided as part of the ‘deepTools’71 Pearson correlation of read coverages were calculated and used to estimate the technical variability between replicates. DeepTools was further used to create bigwig files (using bamCoverage), and these bigwig files were used for calculating enrichment scores over input coverage (bigwigCompare) which were then visualised for selected genomic regions using computeMatrix and plotHeatmap/plotProfile. We used the set of genes defined by our k-means clustering for plotting coverage for the individual histone marks in the promoter regions as defined by our TSS arrays. A snakemake workflow implementing this pipeline can be accessed at https://github.com/skurscheid/mcf10_promoter_profiling72.All software packages were installed using the pythonic package manager “conda” through the “bioconda” channelFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Addition Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Reporting Summary


NLRP6 is a member of the NLR (nucleotide-oligomerization domain-like receptor) family of proteins that recognize pathogen-derived factors and damage-associated molecular patterns in the cytosol. The function of NLRP6 has been attributed to the maintenance of epithelial integrity and host defense against microbial infections. Under some physiological conditions, NLRP6 forms a complex with ASC and caspase-1 or caspase-11 to form an inflammasome complex cleaving pro-interleukin-1β(IL-1β) and IL-18 into their biologically active forms. Here, we summarize recent advances in the understanding of the mechanisms of activation of the NLRP6 inflammasome and discuss its relevance to human disease. Inflammation is a host response against microbial infections and tissue damage to limit harm to the body. Inflammation is initiated following the sensing of microbial components and signs of acute damage or disturbances of the steady state . SeveralNLRs are a group of cytosolic sensors of both PAMPs and DAMPs that are activated by both endogenous and exogenous triggers . They shNLRP6 mRNA expression was also shown to be induced by the encephalomyocarditis virus (EMCV), polyinosinic:polycytidylic acid (poly(I:C)), and interferon-α (IFN-α) in mouse fibroblasts -induced colitis compared with that of wild-type (WT) controls , NLRP6 containing only the PYD (NLRP6PYD), and NLRP6 containing both the PYD and NBD (NLRP6PYD+NBD) and tested their ability to induce ASCPYD polymerization. All NLRP6 constructs were able to promote ASCPYD polymerization, but to a different extent. NLRP6PYD+NBD was the strongest nucleator of ASCPYD polymerization (Hill coefficient of 0.33), followed by NLRP6PYD (Hill coefficient of 0.71). NLRP6FL was found to be the weakest nucleator among all, with the highest dependence on concentration. It also showed the highest Hill coefficient of 4.2 in promoting ASC polymerization. The low Hill coefficients for NLRP6PYD and NLRP6PYD+NBD and their high ability to polymerize ASC compared with NLRP6FL might be related to the presumed autoinhibited conformation of the FL protein. The NLRP6PYD filaments possess a hollow cylindrical architecture that assembles through a right-handed helix, forming multiple layers. The NLRP6-PYD makes a filamentous structure that provides the stage for recruitment of the ASC-PYD and oligomerization through NLRP6-PYD:ASC-PYD interaction. The polymerized ASC nucleates caspase-1 filaments via a CARD-CARD interaction, leading to caspase-1 activation. Activated casase-1, in turn, facilitates the maturation of pro-IL-1β and pro-IL-18.The cryo-EM and crystal structure of NLRP6 has recently been solved . The autPYD is surrounded by the NBD and LRR domain  and mitogen-activated protein kinase (MAPK) activity. Interestingly, there was no differences in the level of IL-1β or caspase-1 activation in Nlrp6−/− mice, suggesting an inflammasome-independent mechanism. Similarly, NLRP6 acted as a negative regulator of pulmonary host defense during Gram-positive bacteria (S. aureus) infection of the lungs (C.-rodentium-induced colitis, NLRP6 deficiency resulted in impaired host defense. Intestines from Nlrp6−/− mice were extensively colonized with C. rodentium and displayed extensive mucosal ulceration, edema, and hyperplasia compared with WT mice (Listeria. LTA from Listeria binds to NLRP6 and activates the NLRP6 inflammasome via ASC to regulate host defense. Interestingly, NLRP6 activated both caspase-11 and caspase-1 upon binding of LTA or Listeria for processing of IL-1β and IL-18. Upon infection with Listeria, Nlrp6−/− mice showed reduced bacterial burdens compared with WT mice. This protection was abolished when these mice received recombinant IL-18, but not IL-1β, suggesting that the NLRP6 inflammasome exacerbates Listeria infection via IL-18 (Inflammasomes play a critical role in the innate immune response against microbial infections . Anand ehe lungs . By cont WT mice . Consistia IL-18 .Nlrp6−/− mice exhibited no difference in survival when infected with EMCV. However, Nlrp6−/− mice had higher viral loads in the intestine, suggesting that NLRP6 may play an important role in viral clearance from the intestine. Interestingly, Nlrp6−/− mice displayed increased susceptibility to EMCV when administered orally; similar results were obtained for oral infection with murine norovirus (In addition to the role of NLRP6 in bacterial infections, it also plays a crucial role in viral infections, as shown by orovirus . Mechaniorovirus .Thus, NLRP6 plays a protective role in the host against bacterial and viral infections in the intestine, where it is highly expressed. However, in systemic and pulmonary infections, NLRP6 expression appears to have negative effects . It is pNlrp6 aggravates DSS-induced colitis or colitis-associated tumor growth due to deregulated regeneration and proliferation of intestinal epithelial cells (Nlrp6−/− mice compared with WT mice. Interestingly, co-housing of Nlrp6−/− mice transferred microbiota to WT mice, resulting in enhanced susceptibility of WT mice to colitis (Nlrp6−/− mice. Porphyromonadaceae and Bacteroidaceae, but not Prevotellaceae, were differentially represented in these mice. These differences were due to mother and cage covariates rather than Nlrp6 deficiency. In support of this finding, Nlrp6−/− and Nlrp6+/+ mice, suggesting that Nlrp6 does not regulate microbiota composition. On the contrary, Nlrp6−/−/IL-10−/− and Nlrp6+/+/IL-10−/− littermate control mice, supporting the notion that Nlrp6 influences the composition of gut microbiota. One possible explanation for these discrepancies are non-genetic factors such as familial transmission and stochastic events. In support of this possibility, NLRP6 is predominantly expressed in the small and large intestine, especially in enterocytes, colonic goblet cells, and myofibroblasts , suggestal cells . Since a colitis . HoweverNlrp6−/− mice exhibited reduced autophagy and hyperplasia of goblet cells and a failure to exocytose mucin granules. This resulted in a thin mucus layer over the epithelium, leading to increased susceptibility to enteric infections (Cyld−/− mice (NLRP6 is also linked to epithelial integrity through the regulation of goblet cell function and secretion of antimicrobial peptides . It was fections . Our grofections . IL-18 hfections . Also, gfections . In linefections . Howeverfections . This su−/− mice . These d−/− mice . FurtherNLRP6 alterations in mRNA expression in IBD patients (CYLD, which deubiquitinates NLRP6, is downregulated in UC patients (CYLD expression are negatively correlated with IL-18 expression in the colonic mucosa of UC patients (Consistent with the mouse model data, the transcriptomic analysis showed abundant expression of NLRP6 in the human intestine, suggesting that NLRP6 has an important role in maintaining gut homeostasis in humans . Howeverpatients . We and patients . Furtherpatients . This suNLRP6 (Colonic inflammation increases the risk of developing colon cancer among IBD patients . AlthougNLRP6 . It is pNLRP6 , the invNLRP6 . In anotNLRP6 . SimilarNLRP6 . An antiNLRP6 . IntriguNLRP6 . Thus, aNLRP6 exhibits diverse functions in the regulation of responses against pathogenic infections and gut homeostasis. Conflicting observations in different studies suggest that NLRP6 harnesses context-reliant inflammasome-dependent and -independent functions. Similarly, NLRP6 seems to have both protective and detrimental effects against microbial pathogens in the intestine and other mucosal surfaces. Studies involving deletion of NLRP6 in specific cell compartments, such as myeloid cells, epithelial cells, or lymphocytes, could provide more conclusive findings.Since NLRP6 recruits both caspase-1 and caspase-11 to form an inflammasome, future biophysical and biochemical studies are essential to understand how these caspases are recruited during NLRP6 inflammasome assembly. Similarly, how NLRP6 function and stability are regulated remain to be investigated. It is likely that post-translational modifications such as phosphorylation, ubiquitination, and sumoylation could modulate its function. Also, complexity might exist in the upstream regulators of NLRP6. Furthermore, the discrepancies regarding the role of NLRP6 in the regulation of gut microbiota need careful evaluation. Finally, the majority of the functions of NLRP6 are currently studied in mouse models, and exploring the full spectrum of cellular functions of NLRP6 in humans could lead to novel therapeutic strategies for human diseases.


Long non-coding RNAs were once considered as “junk” RNA produced by aberrant DNA transcription. They are now understood to play central roles in diverse cellular processes from proliferation and migration to differentiation, senescence and DNA damage control. LncRNAs are classed as transcripts longer than 200 nucleotides that do not encode a peptide. They are relevant to many physiological and pathophysiological processes through their control of fundamental molecular functions. This review summarises the recent progress in lncRNA research and highlights the far-reaching physiological relevance of lncRNAs. The main areas of lncRNA research encompassing their characterisation, classification and mechanisms of action will be discussed. In particular, the regulation of gene expression and chromatin landscape through lncRNA control of proteins, DNA and other RNAs will be introduced. This will be exemplified with a selected number of lncRNAs that have been described in numerous physiological contexts and that should be largely representative of the tens-of-thousands of mammalian lncRNAs. To some extent, these lncRNAs have inspired the current thinking on the central dogmas of epigenetics, RNA and DNA mechanisms. The advancement of next generation sequencing and bioinformatic techniques in the last 20 years has led to the detection of genome-wide transcriptional events within non-coding regions and the subsequent discovery of thousands of long non-coding RNAs (lncRNAs). Countless studies have already revealed that lncRNAs are relevant for many physiological and pathophysiological processes Fig.  and thatOnly a small fraction of the human transcriptome is translated into proteins since the majority of RNA transcripts are non-coding. These non-coding RNAs can be further divided into small non-coding RNAs, such as miRNAs, tRNAs, snoRNAs and snRNAs, and long non-coding RNAs (lncRNAs) which are longer than 200 nucleotides Fig.  61]. Cu. Cu61]. cis- or trans-acting lncRNAs, Fig. An official and appropriate classification that encompasses all lncRNAs does not yet exist . One proLncRNAs usually enact their functions by interacting with proteins, metabolites, DNA or even other RNAs. Additionally, important regulatory elements may be embedded within the gene body of lncRNA genes where transcriptional activity can influence genome structure, chromatin accessibility and neighbouring gene activity . lncRNAshttp://r2.amc.nl)), provide an abundance of lncRNA data which can be exploited by researchers working in many different areas of physiology.Despite the fact that most lncRNAs are so far uncharacterised, there already exists an exhaustive list of lncRNAs that have been investigated across almost all physiological systems. Bioinformatic databases, as reviewed in , or RNA Here, we will present examples of highly physiologically relevant lncRNAs and describe their mechanisms of action Fig. . It shouFendrr , Peril, and Mdgt mutant strains and growth defects were reported for linc–Brn1b and linc–Pint. Fendrr−/− neonates displayed defects in multiple organs. Linc–Brn1b−/− mutants showed distinct abnormalities in the generation of upper layer II–IV neurons in the neocortex [Lincpint was observed in parallel with a reduction in body weight, probably due to reduced total body fat and lower femur bone mineral density, and the development of lordokyphosis. Knockout of Fendrr led to an abnormal lung morphology, Hotair knockout mice displayed a homeotic transformation in the 4th caudal vertebra and Hottip knockout showed hindlimb malformations [As early as 2013, a study with 18 lncRNA knockout mouse strains revealed that lncRNAs are important for viability and are involved in the development of lungs and the cerebral cortex. Peri- and postnatal lethal phenotypes were observed in lncRNA eocortex . Lai et eocortex . An incrrmations . In a larmations .X-inactive specific transcript (XIST) was one of the earliest described lncRNAs and, as such, studies on this lncRNA have provided an invaluable framework for research in the lncRNA field. Xist is responsible for X-chromosome inactivation; a process which achieves dosage compensation of the sex chromosomal genes between females and males. Both X chromosomes, the Xa (active) and Xi (inactive) chromosomes, contain the XIST gene, with the Xi gene initiating X-chromosomal inactivation during early development. Xist is transcribed and spreads in cis across the X-chromosome to coat Xi, but not Xa. The lncRNA triggers gene silencing by recruiting chromatin modifying factors including the polycomb-repressive complex 2 (PRC2), which results in a huge structural reorganization of the X-chromosome [romosome , 44.Tug1 lncRNA is important for male fertility. Tug1-knockout mice were sterile with underlying defects in spermatogenesis as indicated by a low sperm count and abnormal sperm morphology. Molecular characterisation revealed that Tug1, however, functions beyond its role as a lncRNA: the locus acts as a cis-DNA repressor regulating neighbouring gene expression, whereas the lncRNA itself has a trans-regulatory function. Furthermore, the overexpression of an evolutionarily conserved open reading frame of Tug1 encoded a protein identified to be important for mitochondrial membrane potential [lncRNA loci can be functionally highly complex as demonstrated in a study by Lewandowski et al. The authors identified that the widely expressed and highly conserved otential .Peg13, which is part of a complex of imprinted genes on chromosome 15 in mice, showed distinct behavioural differences: they prefer to associate with their own sex after losing interest in the opposite sex. They also develop a higher level of anxiety, lowered activity and curiosity, and a deficiency in pup retrieval behaviour. The authors analysed whole-brain RNA of 16-week-old Peg13-deficient mice and revealed that expression of genes involved in the serotonergic system, formation of glutamatergic synapses, olfactory processing, and estrogen signalling and several others of the imprinted genes on chromosome 15 were changed. It was concluded that Peg13 is part of a regulatory network that governs the female–male differentiation of the brain, as well as the neurobiology of social interactions [Mice lacking the 3′ half of paternally expressed gene, ractions .Fendrr which regulates the development of the heart and body wall of developing mouse embryos [Fendrr binds chromatin-associated protein complexes, specifically PRC2, and recruits it to genes where PRC2 deposits Histone3 Lysine27 trimethylation (H3K27me3) marks, leading to transcriptional repression. Concomitantly, Fendrr represses the Trithorax group/mixed lineage leukaemia complex (TrxG/MLL) at the same gene targets to prevent H3K4me3 deposition, normally associated with gene activation. This work highlights the cell-type specificity yet crucial roles of lncRNAs, where their perturbation can have drastic consequences on fundamental physiological processes such as organ development.LncRNAs are also known to be central in developmental processes where their altered regulation often promotes disease. Grote et al. identified the lateral mesoderm-specific lncRNA  embryos . In a siMAENLI (master activator of engrailed-1 in the limb) is a lncRNA whose transcriptional activity is important for the deposition of active histone marks  on human chromosome 2 in patients with a severe, recessively inherited congenital limb malformation featuring mesomelic shortening, syndactyly and ventral nails . These deletions led to the loss of En1 expression in the limbs of mice and a similar phenotypic outcome. Interestingly, they identified an unknown limb-expressed lncRNA within the deleted region, which they termed Maenli and which is part of an En1 topologically associated domain, the central locus control element during embryonic limb development. The Maenli locus itself is essential to drive limb-specific En1 activation in cis simply through its transcriptional activity. Mechanistically, Maenli transcription led to the deposition of H3K4me3 epigenetic marks on the En1 and Maenli loci and the surrounding regulatory landscape. A similar effect on limb malformation was also seen after depletion of lncRNA Hottip in mice [HOXA cluster was controlled by Hottip RNA [rks Fig. . Its del in mice  and chic in mice , where Wttip RNA .Figure 4FIRRE is a trans-acting lncRNA that regulates lymphopoiesis. Mice with Firre mutations have been shown to exhibit cell-specific lymphocyte phenotypes displayed by a reduction in the abundance of CD4 and CD8 T cells. Moreover, upon exposure to lipopolysaccharide, mice overexpressing Firre exhibited increased levels of the pro-inflammatory cytokines TNFα, IL12-p40, and MIP-2 and impaired survival [LncRNA survival .Morrbid is a cis-acting lncRNA tightly controlling the lifespan of neutrophils, eosinophils and classical monocytes in response to pro-survival cytokines in mice. Mechanistically, the lncRNA promotes the enrichment of PRC2 and H3K27me3 at the promoter of its neighbouring gene, the pro-apoptotic gene Bcl2l11 (BIM), which leads to repression of transcription. The lncRNA was found to be upregulated in individuals with hypereosinophilic syndrome, which is characterised by a persistently elevated eosinophil count [il count .Sarrah (SCOT1-antisense RNA regulated during aging in the heart), also known as OXCT1-AS1, as downregulated in aged mice and infarcted hearts [Sarrah was responsible for cardiomyocyte survival as its silencing led to apoptosis while its overexpression in mice improved their recovery from acute myocardial infarction. Mechanistically, the authors suggested that SARRAH forms RNA-DNA triplexes at gene promoters, which were downregulated after SARRAH silencing. An induction of NRF2 and the binding of CRIP2 and p300 facilitated transcriptional activation of SARRAH target genes.Aging is one of the main risk factors for numerous diseases. Trembinski et al. identified the conserved lncRNA d hearts . Loss ofNorad-deficient cells reflected by faster aging in animals. Such a relationship is putatively seen since PUMILIO levels increase with age while those of NORAD decrease [Norad by Kopp et al. revealed that Norad depletion led to a degenerative phenotype characterised by increased alopecia, gray fur, kyphosis and aging-associated pathologies within the central nervous system, which is the consequence of genomic instability and mitochondrial dysfunction, explained by PUMILIO2 overexpression [Another lncRNA involved in aging is NORAD, which protects the genome by reducing the activity of PUMILIO proteins. NORAD depletion leads to overactivation of PUMILIO proteins with augmented repression of a program of target mRNAs that includes key regulators of mitosis, DNA repair, and DNA replication. Dysregulation of these genes could result in genomic instability in decrease . The invpression .Cardiovascular disease encompasses a diverse range of pathologies that make it the number one cause of death worldwide . It is tMALAT1 is one of the few very highly expressed lncRNAs. Initially identified as a cancer biomarker [MALAT1 has diverse roles in multiple different cancer types [Malat1 had pro-atherosclerotic effects, which resulted from an increased accumulation of haematopoietic cells at the murine carotid artery vessel wall [H19 was reduced and an H19 vector–based, cardiomyocyte-directed gene therapy was able to attenuate heart failure [The lncRNA iomarker , MALAT1 er types . In the er types . Reducedsel wall . In fail failure .ANRIL (antisense non-coding RNA in the INK4 locus). ANRIL is an example of a lncRNA locus transcribing multiple isoforms, two of which are highly physiologically relevant: a linear lncRNA and a circular RNA with opposing functions , a member of the PeBoW  complex. Inhibition of PeBoW leads to defects in rRNA maturation, increased nucleolar stress and activation of p53, which inhibits cell proliferation and increases apoptosis, whereas linear ANRIL confers overproliferation [Multiple studies have been performed on the Chr9p21 locus, where numerous CAD risk SNPs accumulated within the gene encoding the lncRNA ons Fig. . Linear feration .MANTIS is an example of a nuclear scaffolding lncRNA having a critical function in endothelial cells. Its depletion leads to a loss of the ability of endothelial cells to align in the direction of flow. Moreover, MANTIS is required to promote angiogenesis, limit inflammatory gene expression and maintain angiogenic capacity [MANTIS increased the adhesion of monocytes to endothelial cells in an ICAM-1-dependent manner. Statin therapy induced MANTIS in vitro and in patients with carotid artery disease. MANTIS also maintained the pleiotropic responses of endothelial cells to atorvastatin. Central to these effects is the interaction of MANTIS with the SWI/SNF chromatin remodelling complex member, BRG1. MANTIS guides BRG1 to its different effector genes, resulting in activation of pro-angiogenic gene expression and inhibition of inflammatory gene expression in endothelial cells [capacity , 37. ConMhrt is also connected to the chromatin remodeller BRG1. The lncRNA functions to limit cardiac hypertrophy by binding to and inhibiting the helicase domain of BRG1 to create a transcriptional feedback loop [LncRNA ack loop . The inhCaren (Cardiomyocyte-enriched transcript) is a prime example of a cytoplasm-enriched lncRNA with a pivotal role in the development of a highly complex disease. It protects against the development of heart failure in pressure-overloaded hearts by inhibiting Hint1. Hint1 is a tumour suppressor that activates the ATM kinase and the DNA damage response (DDR), a process that is prominent in cardiomyocytes of patients with heart failure. Caren’s protective effects are related to the inhibition of Hint1-mediated mitochondrial dysfunction and DDR signalling through ATM. At least in mice, the overexpression of Caren alone is sufficient to prevent heart failure [The lncRNA  failure .lncRNA studies in the central nervous system have revealed a striking specificity of lncRNA expression in nervous tissue. Neurogenesis, the differentiation of neural stem cells (NSC) to neurons is a highly complex but well-orchestrated process that involves multiple physiological, molecular and genetic pathways. It is becoming evident that lncRNAs provide an additional layer of molecular control and that this is particularly true in neurogenesis and the complex differentiation of the brain.Pnky  is an evolutionarily conserved and neural-specific lncRNA involved in embryonic and postnatal neurogenesis. Pnky interacts with the RNA splicing regulator PTBP1 to mediate the expression and alternative splicing of transcripts important for limiting neural differentiation and neurogenesis [Pnky. Other prominent lncRNAs such as RMST (rhabdomyosarcoma 2–associated transcript) [TUNA (Tcl1 Upstream Neuron-Associated lincRNA) [Dali (DNMT1-Associated Long Intergenic) [BDNF-AS [Malat1, mentioned above for its function in tumours and the cardiovascular system, also controls the expression of genes involved in synaptogenesis and synapse function [MALAT1 is ubiquitously expressed and has been shown to perform fundamental tasks in many cell types.ogenesis . PTBP1 hogenesis , exemplinscript) , TUNA (TlincRNA)  and Daliergenic)  also fun[BDNF-AS . Malat1,function . Many ofBACE1-AS , β-secretase (BACE1), produces the β amyloid plaques that are a hallmark of AD pathophysiology . Interes-AS Fig. . Diseaseack loop . This opresearch .As mentioned above already, lncRNAs are required for tissue and organ development, and this is also true for lung tissue. The lncRNA NANCI (Nkx2.1-associated non-coding intergenic RNA) associates with and promotes the expression of the Nkx2.1 transcription factor, which is crucial for pulmonary development and homeostasis. Loss of NANCI itself is not sufficient to limit development, since it is normally inhibited by Nkx2.1 in a negative feedback loop; however, mutations which impact both NANCI and Nkx2.1 lead to severe lung degeneration .The lncRNA TYKRIL (tyrosine kinase receptor-inducing lncRNA) was found to be strongly upregulated in the pulmonary arterial pericytes and smooth muscle cells (SMC) of patients with idiopathic pulmonary arterial hypertension (IPAH) . TYKRIL One study demonstrated that the lung tissue from smokers with chronic obstructive pulmonary disorder (COPD) expressed 120 upregulated and 43 downregulated lncRNAs compared to smokers without COPD [SPRY4-IT1 disrupted barrier integrity by reducing the stability of mRNAs encoding tight junction proteins claudin-1, claudin-3, occludin and JAM-1 [H19, heavily studied in the cardiovascular and cancer fields, is also involved in barrier integrity. H19 acts as a precursor for microRNA 675 (miR-675) which represses the expression and reduces the stability of tight junction proteins ZO-1 and E-cadherin through its interaction with the RNA-binding protein HuR [The intestinal barrier includes the chemical mucosal and physical epithelial layers that allow for selective absorption of nutrients, ions and water into the bloodstream. Barrier integrity is upheld by the tight junction proteins which are tightly regulated through multiple signalling pathways; dysregulation of which predisposes to inflammatory bowel diseases and metabolic disorders. lncRNAs also play a role in the context of intestinal barrier integrity. For example, depletion of the lncRNA nd JAM-1 . Similartein HuR .4- and bile duct ligation-induced liver fibrosis in mice. The authors revealed that Lnc-LFAR1 has multiple functions organising Smad2/3 binding, phosphorylation and induction to promote liver fibrosis leading to the activation of the TGFβ and Notch pathways [Zhang et al. identified the multi-functional lnc-LFAR1 (liver fibrosis-associated lncRNA1) that promotes liver fibrosis, a disease characterised by extracellular matrix component accumulation in the liver leading to hepatic dysfunction. Silencing of lnc-LFAR1 impaired hepatic stellate cells activation, reduced TGFβ-induced hepatocytes apoptosis and attenuated the CClpathways .Tug1 mentioned above, a peptide coded by the Myoregulin (MLN) lncRNA was shown to be important for skeletal muscle performance. MLN KO mice had improved skeletal muscle exercise performance and muscle Ca2+ handling. The conserved micropeptide of the lncRNA, which encodes a transmembrane alpha helix with strong structural resemblance to phospholamban and sarcolipin—direct interactors with SERCA in the sarcoplasmatic reticulum membrane—co-localises with SERCA and regulates Ca2+ handling by inhibiting SERCA pump activity [By definition, lncRNAs should lack protein-coding potential. However, as in the case for the lncRNA activity .ANRIL, physiologically important SNPs are not restricted to protein-coding genes. Yang et al. identified a novel mutation on chromosome 2 (rs3819316 C >T) in the lncRNA Reg1cp that is associated with elevated bone mass. Mutant Reg1cp increased the formation of the CD31hiEmcnhi endothelium in the bone marrow to stimulate angiogenesis during osteogenesis. They identified Krüppel-like factor 3 (KLF3) as a protein interaction partner of mutant, but not wild-type Reg1cp in human microvascular ECs, and showed that binding of KLF3 to its downstream target JUNB is reduced by mutant Reg1cp. Endothelial-specific Klf3 knockout mice also had increased CD31hiEmcnhi endothelium and bone formation [As already mentioned for ormation .Given the expression of lncRNAs across all tissues and their involvement in fundamental cellular processes, it is unsurprising that lncRNAs have been linked to cancer development and progression. Cancer is a highly complex disease whose hallmarks include, among others, immune evasion, genome instability, angiogenesis and sustained proliferation , 24. DatPANDA, linc-p21 and LINC-PINT are lncRNAs that function to alter transcription factor activity and binding [PANDA (P21-associated ncRNA DNA damage activated) is upregulated by p53. Fibroblasts with PANDA knockdown displayed increased DNA damage, and a higher sensitivity to DNA damage–induced apoptosis. Interestingly, PANDA interacted with the p53-downstream transcription factor nuclear transcription factor Y subunit alpha (NF-YA), which bound more to its target genes after PANDA knockdown. These data suggested that PANDA, in response to DNA damage, represses NF-YA target gene binding and thereby blocks apoptosis to promote cell survival. Huarte et al. identified a p53-repressor and pro-apoptotic lncRNA termed lincRNA-p21 [lincRNA-p21 functions as a mediator of other p53 gene targets. Independent knockdowns of p53 or lincRNA-p21 revealed an overlap of 930 differentially regulated genes, 80% of which were returned to normal expression levels after a double knockdown of p53 and lincRNA-p21. Mechanistically, lincRNA-p21 recruited and enabled the localisation of hnRNP-K as a p53-dependent repressor that drives the apoptotic response to DNA damage. Cancer cells often develop the ability to depart the primary tumour and invade other tissues and organs to form metastases. The p53-regulated lncRNA LINC-PINT was shown to act as a tumour suppressor, owing to its ability to reduce the invasive and migratory potential of cancer cells [The lncRNAs  binding , 29, 47.cRNA-p21 . lincRNAtrans as indicated by its ability to regulate telomeres on other chromosomes in addition to that from which it was transcribed. TERRA is recruited to telomeres and stabilises the interaction of proteins with telomeres. As well as regulating telomerase directly, TERRA can also form RNA-DNA hybrid structures known as R-loops at telomeres to induce homologous recombination and delay senescence. This has been proposed as the mechanism by which telomeres are preserved in the 10% of cancers that do not have elevated telomerase levels [LncRNA TERRA is recruited to telomeres where it stabilises protein-telomere interactions . Telomere levels .PCAT19 (prostate cancer associated transcript 19) is another example of a lncRNA whose SNPs have been linked to pathophysiological effects  that differentially regulates the expression of a short and long isoform of PCAT19 [PCAT19-short promoter that switches to an enhancer and upregulates the PCAT19-long isoform. This long isoform of PCAT19 binds hnRNPAB to activate the transcription of genes involved in cell cycle and the growth and metastasis of prostate cancer tumours. Sustained proliferation is one of the central characteristics of cancer cells.cts Fig. . The genf PCAT19 . The preThe remarkable progress in the field of lncRNA research has provided evidence that lncRNAs are highly relevant for physiology, development and behaviour. As such, lncRNAs are implicated in a number of diseases, often by fine-tuning gene regulation. Due to their extensive number of features, functions and expression patterns, there is no doubt that lncRNAs offer a great potential and perspective for future interventions such as tissue regeneration and personalised medicine. LncRNA research could lead to innovations that engineer synthetically active lncRNAs for RNA-based therapeutics.As is the case for protein-coding genes or other ncRNAs, lncRNAs are more than just biomarkers; they are potentially highly specific therapeutic targets . AntisenOnly a relatively small fraction of lncRNAs has been studied so far with the physiological roles of the majority of lncRNAs remaining unknown. Current research is just beginning to examine whether lncRNA genomic loci are functionally independent of the RNA transcript and whether small peptide-coding RNAs add an additional layer of regulation to the system. This raises important questions surrounding the multiple functions of lncRNAs, e.g. on genomic locus-, transcript- and peptide-levels. Many lncRNAs are species- and tissue-specific, indicating that the role of transcriptional activities around the lncRNA loci, the chromatin architecture and regulatory sequence elements within or closely attached to lncRNA gene bodies could be important. Additionally, the structural features of lncRNAs need to be considered. Future studies will also reveal the physiological importance of lncRNA-containing phase-separated condensates, RNA modifications and certain lncRNA isoforms at the single-cell level. The classification of this large group of ncRNAs will also be simplified once a more complete picture of lncRNA biology exists. This could be based on their functionality, processing, structural features and interaction with other RNAs, DNA or proteins. Here, lncRNA databases should take centre stage.There is no doubt that the progress of lncRNA research will lead to a better understanding of physiological and pathophysiological processes, their fine-tuning and epigenetic control, which can then be used interdisciplinarily to advance therapies and to improve disease outcome.Alu elements   Short stretches of repetitive DNA that act as transposable elements (mobile genetic elements that can move around the genome). Alu elements are located across the genome and are believed to be functionally relevant, particularly in the generation of new genes in the process of evolution.cis-acting lncRNAs   lncRNAs that regulate gene activity at the same locus from which the lncRNA is transcribed.Functional elements   Regions of DNA that have a known regulatory function such as promoter regions where transcription factors bind to initiate gene transcription; and enhancers where molecules bind to potentiate the activation of an associated gene.Histone modifications   Covalent post-translational modifications of the histone proteins that comprise the nucleosome . Histone modifications include acetylation, methylation, phosphorylation, sumoylation and ubiquitylation to name a few and can regulate the transcriptional state of a gene.m7G 5′ cap   A methylated guanine nucleotide with a triphosphate linkage on the 5’ end of primary RNA transcripts. This 7-methylguanylate (m7G) functions in nuclear export, intron excision, inhibition of degradation and translation in the case of mRNAs.Oligonucleotide   Short stretches of DNA or RNA that have a wide range of applications in molecular biology research.Poly-A tail   Stretch of RNA consisting exclusively of adenine bases. Important for RNA stability, termination of transcription, nuclear export and translation.R-loop   Three-stranded DNA:RNA structure consisting of two antiparallel DNA strands and an RNA strand. R-loops are involved in gene regulation and genome stability.SNP   SNPs (single-nucleotide polymorphisms) are substitutions of a single nucleotide in the genome. The majority of SNPs have no functional consequence but some can influence gene regulation and ultimately disease development, depending on their precise location.Splicing   The process whereby introns are removed from a primary RNA transcript and the exons joined to form the mature RNA transcript.trans-acting lncRNA   lncRNAs that leave their site of transcription to function elsewhere in the cell.Triplex   Three-stranded oligonucleotide structures of DNA:DNA:RNA in which the RNA occupies the major groove of the DNA and binds through Hoogsteen pairing with the purines of the Watson-Crick basepairs.


Western blot analyses confirmed a strong reduction in the expression of the clusterin isoforms after corneal damage and suggest that repression of CLU gene expression might be a prerequisite to hTEC wound closure. Transfection with segments from the human CLU gene promoter revealed the presence of three regulatory regions: a basal promoter and two more distal negative regulatory regions. The basal promoter bears DNA binding sites for very potent transcription factors (TFs): Activator Protein-1 (AP-1) and Specificity protein-1 and 3 (Sp1/Sp3). By exploiting electrophoretic mobility shift assays (EMSA), we demonstrated that AP-1 and Sp1/Sp3 have their DNA binding site overlapping with one another in the basal promoter of the CLU gene in hCECs. Interestingly, expression of both these TFs is reduced (at the protein level) during hTEC wound healing, thereby contributing to the extinction of CLU gene expression during that process. The results of this study contribute to a better understanding of the molecular mechanisms accounting for the repression of CLU gene expression during corneal wound healing.In order to reduce the need for donor corneas, understanding of corneal wound healing and development of an entirely tissue-engineered human cornea (hTECs) is of prime importance. In this study, we exploited the hTEC to determine how deep wound healing affects the transcriptional pattern of corneal epithelial cells through microarray analyses. We demonstrated that the gene encoding  Visual acuity is directly correlated with the integrity of the anatomical structures of the eyes. One of these key components is the cornea, consisting of the outer layer of the eye. The cornea is a transparent structure, crucial for light refraction to the retina that accounts for three-quarters of the total eye refractive power. Hence, maintaining corneal transparency is of basic necessity; otherwise, visual acuity will be inevitably affected . MoreoveCorneal repair and regeneration mainly depend on the limbal stem cells located at the junction between the corneal and conjunctival epithelium. Indeed, a compromised limbal niche is a key factor of the injury’s severity, the prognostic of healing being poor when more than 50% of the limbal area is damaged . FurtherCLU gene promoter, including activator Protein 1 (AP-1), Specificity Protein 1 (Sp1), Nuclear Factor 1 (NFI), Signal Transducers and Activators of Transcription (STAT), MYCN Proto-oncogene and Heat Shock Factor (HSF), to name a few . DNA–protein complexes were next separated by gel electrophoresis through 6% non-denaturing, native polyacrylamide gels run against Tris-glycine buffer  at 120 V or 110 V  for 6.5 h, 4 °C. The position of the DNA–protein complexes was then revealed upon autoradiography of the dried gels at −80 °C. When specified in the legend of each figure, synthetic double-stranded oligonucleotides bearing the recognition sequence for known transcription factors (AP-1 or Sp1/Sp3), or that of either the wild type or mutated CLU-203/-153 50 bp DNA fragment  at passages 3 to 4 were seeded in the presence of iHFL at 5 × 10escribed ,99. Briefragment , were adt-test was performed for comparison of the groups for quantification of the western blot signals shown in p < 0.05. After a first t-test analysis, and when this one is significant, a comparison of means was also performed by an ANOVA test (Tukey) using GraphPad PRISM software  for the analysis of the transfection data presented in Student’s In this study, we demonstrated that the expression of clusterin is dramatically repressed (at both the gene and protein levels) during closure of hTEC wounds. This reduced expression of CLU gene transcription was found to be mediated, at least in part, by alteration in both the expression and DNA binding of the transcription factors AP-1 and Sp1/Sp3, which we demonstrated to bind overlapping target sites in the basal promoter of the human CLU gene.