Signaling via B cell receptor (BCR) and Toll-like receptors (TLRs) results in activation of B cells with distinct physiological outcomes, but transcriptional regulatory mechanisms that drive activation and distinguish these pathways remain unknown.Two hours after ligand exposure RNA-seq, ChIP-seq and computational methods reveal that BCR- or TLR-mediated activation of primary resting B cells proceeds via a large set of shared and a smaller subset of distinct signal-selective transcriptional responses. BCR stimulation resulted in increased global recruitment of RNA Pol II to promoters that appear to transit slowly to downstream regions. Conversely, lipopolysaccharide (LPS) stimulation involved an enhanced RNA Pol II transition from initiating to elongating mode accompanied by greater H3K4me3 activation markings compared to BCR stimulation. These rapidly diverging transcriptomic landscapes also show distinct repressing (H3K27me3) histone signatures, mutually exclusive transcription factor binding in promoters, and unique miRNA profiles.Upon examination of genome-wide transcription and regulatory elements, we conclude that the B cell commitment to different activation states occurs much earlier than previously thought and involves a multi-faceted receptor-specific transcriptional landscape.The online version of this article (doi:10.1186/s13072-015-0012-x) contains supplementary material, which is available to authorized users. Deregulated activation can have disastrous effects resulting in immune disorders and several B cell malignancies, some of which resemble activated B cell phenotypes , 2. Matue output . Upon ene output –6 or vare output . Exposure output , 8. A pre output .0 state and subsequently in proliferation and effector function [Splenic B cell differentiation can begin as early as 4 h and fully develop by 48–72 h . While mfunction . Regulatfunction , 15. ButActivation of resting splenic B cells ex vivo provides a tractable model to explore this transition in a ligand-specific manner . B cellsCD69 and the PRGs c-fos, c-jun, and c-myc, were increased compared to the resting state by exposure to either anti-IgM/BCR or LPS/TLR4 as early as 30 min  or LPS (engages TLR4). Many expected genes, including the early activation marker cr4 Fig. . Smallercr4 Fig. . For exantiation , while LFigure We also analyzed 1315 genes whose transcription was not significantly altered by BCR or TLR4 stimulation. These genes, such as Polr2a, Max, ActB, and Dicer1, collectively annotated to biological processes, including maintenance of homeostasis, cell cycle, and apoptosis management  during BCR activation was also observed Fig. . These eChromatin modification is a well-defined transcriptional regulatory mechanism , 23. IncPolycomb repressor complexes (PRCs) catalyze repressive chromatin marks –28. In mIrf4 and Myc) as well as BCR-responsive genes (Il7r and Egr3) showed an increase in RNA Pol II and a decrease in H3K27me3  showed a preferential increase in RNA expression and an increase in RNA Pol II at downstream sites, while the H3K27me3 mark around the TSS remained largely unchanged. Lastly, although H3K4me3 was increased by both stimuli, it was clearly more pronounced at Il12b and Tnf after LPS stimulation. Hence, the global transcriptional patterns observed with each category of genes are also observed at individual target genes.Having observed ligand-selective regulatory signatures at shared and response selective genes, we examined two representative genes from each category  based on known functions in B cell activation and immunity and on the presence or absence of a Myc signature (for the shared and BCR-selective groups) or a NF-κB signature (for the TLR4/LPS-selective group). These genes collectively illustrated the characteristics observed at the global level. For example, the shared genes , we observed that many high-frequency motifs were common between Shared and BCR, while neither overlapped with the 30 lowest P value motifs found in LPS preferentially induced gene promoters  motifs within the promoter region (−1000 to +1000 of the TSS) were examined. TF motifs at induced gene promoters were clearly different between the two activation states. Most TF motifs were highly overrepresented in both the Shared- and BCR-induced gene sets, as shown by the large number of data points along the diagonal of the scatterplot Fig. . A much ers Fig. . Direct ers Fig. . The geners Fig. . The dirBecause miRNAs are known to play a pivotal role in various B cell processes such as development, inflammation, and tolerance , 38, we Deregulation of B cell activation can result in autoimmune disorders, chronic inflammation, and lymphoma. Although B cells express both BCR and TLR4, the functional consequences to these stimuli are distinct, at least under ex vivo conditions. While BCR engagement (triggered by anti-IgM stimulation) leads to proliferative expansion of activated B cells, engagement of TLR4 (triggered by LPS stimulation) leads to proliferation and eventual production of plasma cells. However, the transcriptional signatures and molecular mechanisms that distinguish these responses are relatively unknown. Here, we employed ex vivo activation of resting murine splenic B cells to examine these molecular signatures and define the transcriptional and regulatory landscape during early activation by high-resolution RNA- and ChIP-seq. We observe that at 2 h post stimulation, most genes (~90 %) induced by the two pathways are shared, despite being triggered through distinct receptors. This observation is consistent with previous studies , 45. HowWhile genes induced by both responses (shared) are greatly dominated by Myc targets and Myc-associated processes, groups of genes preferentially induced by LPS, as expected, show a strong correlation with NF-κB Fig. . SurprisGiven the distinct gene sets noted in each of the responses, we examined mechanisms that could drive these observed differences. While preexisting, paused, RNA Pol II is associated with a large number of genes in diverse cell types, new RNA Pol II recruitment indicates transcriptional activity . AlthougEpigenetic regulation of gene expression plays crucial roles in lymphoid differentiation and homeostasis , 52, 53.Myc and Il7r expression, both of which exhibit increased transcription in response to BCR in our experiment, also suggests Myc plays an important role during early phases of B cell activation [Given that helix-loop-helix (HLH) transcription factors play an important role in B cell development and differentiation , 60, it tivation . The factivation  further As deregulation of B cell activation is related to malignancies such as B cell lymphomas, our studies may also provide insights into lymphogenesis. Myc and NF-κB are well-established master regulators of initiation of transcriptional programs, but when deregulated, they function as oncogenic drivers in B cell lymphomas. Deregulated and increased Myc and Bcl proteins, such as Bcl2 and Bcl6, are associated with particularly aggressive lymphoma types , 65. HerTargeting of miRNAs is complex with an average miRNA having approximately 100 target sites in addition to non-canonical miRNA binding , 67. HerBCR responses are slower and presumably more precise , 45, 71,We conclude that the B cell commitment to different activation states is dependent upon rapid regulatory mechanisms and occurs much earlier than previously thought. Different RNA Pol II recruitment and transition from initiation to elongation, distinct activating (H3K4me3) and repressing (H3K27me3) histone signatures, mutually exclusive transcription factor binding in promoters and highly selective miRNA profiles distinguish these responses.+ by flow cytometry , and resuspended in cold media with either 10 ug/ml anti-mouse IgM goat IgG Fab fragments (Jackson Immunology) or 25 ug/ml Salmonella typhimurium typhus LPS (Sigma) were added. The cells were rested on ice for 30 min following a previously published method [2 for the experimental times. Animal care and use in this study are covered under the “Assurance of Compliance with PHS (USA) Policy on Humane Care and Use of Laboratory animals by Awardee Institutions” and approved by the Institutional Animal Care and Use Committee of Tufts University .Naïve resting B cells from splenocytes of 8-week-old male C57BL6 mice were isolated with anti-CD43 beads (Miltenyi), confirmed as 95 % CD19d method  and incuSample preparation was performed using common techniques. In general, single end, 100 bp , and 50 bp  reads were mapped against the mm9/ENSEMBL build 67 genome reference using Tophat v2.0.0  and for Differential expression (DE) was identified by a minimal twofold difference in log ratios of normalized reads generated with Cufflinks v1.3.1. Preferentially induced or reduced genes sets included genes that were either changed by either a single response or when affected by both responses changed only two- to fourfold by one response and were changed by the preferred response at a ratio of at least twofold more than the non-referred response. A spreadsheet of the differential expression list can be found in Additional file After Trizol isolation of RNA, TruSeq Small RNA Sample Preparation Kits were used to produce material for generating 50 bp single end reads which were then analyzed with miRDeep2  using thFor histograms of TSS coverage, custom R scripts were used to produce bedgraphs from mapped bam files, which were converted to BigWig files with bedgraphToBigwig for UCSC Genome Browser presentation , 81. ReaPredicted CpG island locations were from preloaded USCS Genome Browser tracks and produced by common methods . ProximiReal-time PCR was performed with specific primers .The sequences have been deposited to the GEO database (NCBI/NLM/NIH)—accession number (GSE61608) (


We investigated the role of the modified histone H3K4me3 (trimethylated lysine 4 of histone H3) in gene expression during the early stages of wood formation using ChIP-seq in Eucalyptus grandis, a woody biomass model.Histone modifications play an integral role in plant development, but have been poorly studied in woody plants. Investigating chromatin organization in wood-forming tissue and its role in regulating gene expression allows us to understand the mechanisms underlying cellular differentiation during xylogenesis (wood formation) and identify novel functional regions in plant genomes. However, woody tissue poses unique challenges for using high-throughput chromatin immunoprecipitation (ChIP) techniques for studying genome-wide histone modifications E. grandis trees. A “nano-ChIP-seq” procedure was employed for ChIP DNA amplification. Over 9 million H3K4me3 ChIP-seq and 18 million control paired-end reads were mapped to the E. grandis reference genome for peak-calling using Model-based Analysis of ChIP-Seq. The 12,177 significant H3K4me3 peaks identified covered ~1.5% of the genome and overlapped some 9,623 protein-coding genes and 38 noncoding RNAs. H3K4me3 library coverage, peaking ~600 - 700 bp downstream of the transcription start site, was highly correlated with gene expression levels measured with RNA-seq. Overall, H3K4me3-enriched genes tended to be less tissue-specific than unenriched genes and were overrepresented for general cellular metabolism and development gene ontology terms. Relative expression of H3K4me3-enriched genes in developing secondary xylem was higher than unenriched genes, however, and highly expressed secondary cell wall-related genes were enriched for H3K4me3 as validated using ChIP-qPCR.Plant chromatin fixation and isolation protocols were optimized for developing xylem tissue collected from field-grown E. grandis xylem, H3K4me3 enrichment is an indicator of active transcription, consistent with its known role in sustaining pre-initiation complex formation in yeast. The H3K4me3 ChIP-seq data from this study paves the way to understanding the chromatin landscape and epigenomic architecture of xylogenesis in plants, and complements RNA-seq evidence of gene expression for the future improvement of the E. grandis genome annotation.In this first genome-wide analysis of a modified histone in a woody tissue, we optimized a ChIP-seq procedure suitable for field-collected samples. In developing The online version of this article (doi:10.1186/s12870-015-0499-0) contains supplementary material, which is available to authorized users. A rich diversity of histone modifications affect chromatin structure and/or gene activation and repression in eukaryotes reviewed by ,2. ChromTrithorax protein complex COMPASS in yeast  . Crossing points (Cp) were calculated using the second derivative maximum method and quantities relative to the input sample calculated using the formula EΔCp(input) - Cp(sample), where E is the efficiency calculated from a standard curve of the relevant primer set.The V11 ChIP-seq samples (prior to library preparation) were used for ChIP-qPCR analysis. Primers targeting selected genomic regions are listed in Additional file http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE67873).The datasets supporting the results of this article are available in the NCBI’s Gene Expression Omnibus , Series


Osteogenesis is a highly regulated developmental process and continues during the turnover and repair of mature bone. Runx2, the master regulator of osteoblastogenesis, directs a transcriptional program essential for bone formation through genetic and epigenetic mechanisms. While individual Runx2 gene targets have been identified, further insights into the broad spectrum of Runx2 functions required for osteogenesis are needed.By performing genome-wide characterization of Runx2 binding at the three major stages of osteoblast differentiation - proliferation, matrix deposition and mineralization - we identify Runx2-dependent regulatory networks driving bone formation. Using chromatin immunoprecipitation followed by high-throughput sequencing over the course of these stages, we identify approximately 80,000 significantly enriched regions of Runx2 binding throughout the mouse genome. These binding events exhibit distinct patterns during osteogenesis, and are associated with proximal promoters and also non-promoter regions: upstream, introns, exons, transcription termination site regions, and intergenic regions. These peaks were partitioned into clusters that are associated with genes in complex biological processes that support bone formation. Using Affymetrix expression profiling of differentiating osteoblasts depleted of Runx2, we identify novel Runx2 targets including Ezh2, a critical epigenetic regulator; Crabp2, a retinoic acid signaling component; Adamts4 and Tnfrsf19, two remodelers of the extracellular matrix. We demonstrate by luciferase assays that these novel biological targets are regulated by Runx2 occupancy at non-promoter regions.Our data establish that Runx2 interactions with chromatin across the genome reveal novel genes, pathways and transcriptional mechanisms that contribute to the regulation of osteoblastogenesis. Osx (osterix), Ocn , and Bsp , and represses the expression of non-osteoblast genes such as PPAR-γ (peroxisome proliferator-activated receptor gamma) and MyoD (myogenic differentiation), which are required for adipogenic and myogenic commitment, respectively [The development of bone tissue, new bone formation in the adult, mineral homeostasis and maintenance of bone mass are mediated by cells of the osteoblast lineage. These bone forming cells progress through a highly regulated differentiation program, with each subpopulation of cells acquiring stage-specific phenotypes that are characterized by distinct profiles of expressed genes . Osteoblectively -9. Studiectively ,3,10. Inectively . A signiectively , histoneectively , and SWIectively .cis-regulatory DNA elements, the Runx motif (TGTGGT) located in or near gene promoter regions [cis-regulatory networks that are critical for cell lineage control in adipocytes, myocytes and T cells, respectively [cis-regulatory network through which Runx2 regulates the transcription program for bone formation.Runx2 has been shown to activate or repress gene expression through binding to  regions . Genome-ectively -19. Thesin vitro model for osteoblastogenesis [Hence, we have characterized the genome-wide occupancy of Runx2 by ChIP-Seq in MC3T3-E1 preosteoblasts, a well-studied ogenesis . Runx2 oin vitro differentiation of MC3T3-E1 preosteoblasts  500 ChIP-Seq peaks and was then scanned using FIMO [We next isolated and sequenced DNA from chromatin bound by Runx2. Sequence reads from Runx2 ChIPs and input controls were mapped to the mouse genome. Statistically significant enrichments of Runx2 were identified by MACS   Additio. RefSeq ing FIMO   to the JASPAR MA0002.2 RUNX motif [The NX motif   | > 1.5, and a false discovery rate (FDR) <0.05, where I is the measured normalized probe-set intensity. These genes included 115 up- and 44 down-regulated genes  short hairpin RNA (shRNA) or a Runx2-specific shRNA (shRunx2) that knocks down Runx2 expression. Runx2 protein levels decreased by 80% in cells treated with shRunx2   was usedFinally, downregulated and upregulated genes were compared on the basis of their evolutionary conservation Figure F. FunctiTaken together, the complementary methods described above suggest that Runx2 employs different mechanisms to regulate gene expression: 1) shRunx2 downregulated genes show increased Runx2 binding at promoter and far upstream regions; and 2) based on EMBER, intronic binding does not imply Runx2-mediated gene regulation to the same degree as Runx2 binding at promoter, exon and upstream regions. Thus, during the normal course of osteoblast differentiation, Runx2-activated genes (shRunx2 downregulated) are regulated through both promoter and non-promoter regions; and regulation of Runx2 repressed genes (shRunx2 upregulated) also occurs in promoter and other genomic regions, but with less Runx2 binding.Tnfrsf19 , which is involved in bone formation as a Wnt-responsive regulator of mesenchymal stem cell commitment to osteoblastic lineage [Tnfrsf19 exhibited enrichment of Runx2 binding to the promoter as well as intronic regions  that have established roles in bone formation, and more importantly, have extended the understanding of Runx2-mediated gene regulation to a broader range of cellular functions during osteoblast differentiation.Through comprehensive genomic analysis of Runx2 by ChIP-Seq, we describe widespread Runx2 binding throughout the genome of differentiating osteoblasts. In addition to Runx2 interaction at promoters, we find Runx2 binding in non-promoter regions regulating novel targets that are silenced or expressed at different stages of osteoblast differentiation. Runx2 peaks cluster into temporal and functional categories associated with genes in a broad range of cellular programs, including bone development, negative regulation of proliferation, active matrix formation and mechanisms for mineral deposition that reflect the progression of osteogenesis. Our data have identified new Runx2-regulated genes  and more dynamic binding groups . Steady-state binding of Runx2 persists from proliferative pre-osteoblasts to differentiating osteoblasts with binding signals plateauing during matrix deposition and mineralization stages. Peaks in this category represent genes related to housekeeping processes such as protein folding, negative regulation of mitotic cell cycle, and mRNA catabolism and processes not previously related to Runx2  was obtained from ATCC  and maintained in ascorbic acid-free alpha-MEM  supplemented with 10% fetal bovine serum , 2 mM L-glutamine, 100 U/ml of penicillin and 100 μg/ml streptomycin . To induce osteogenic differentiation, complete alpha-MEM was supplemented with 280 μM ascorbic acid and 10 mM beta-glycerophosphate . Cells were maintained at 37°C in a humidified 5% COHprt1 using 2C-ΔΔT method. Experiments were performed in triplicate and results are presented as mean values ± SEM. Primers for qPCR reactions were designed by FoxPrimer [Total RNA was isolated using Trizol reagent (Invitrogen) according to the manufacturers’ specifications. Total cellular RNA treated by DNaseI  was primed with random hexamers and reverse transcribed into cDNA using Superscript First-strand cDNA Synthesis Kit (Invitrogen) according to the manufacturer’s instructions. Gene expression was determined by quantitative real time PCR (qPCR) using iQ™ SYBR Green PCR Master Mix  in an ABI Prism 7300 thermocycler . For each gene, the expression level was normalized to that of oxPrimer ,62, and Lentiviruses carrying Runx2-shRNA and previously described control Scramble-shRNA  were uset-test. The Benjamini-Hochberg FDR [Microarray samples were handled following the manufacturers’ recommended protocols . Briefly, RNA isolated from MC3T3-E1 cells  were reversely transcribed into cDNA using WT Expression Kit , labeled and fragmented with GeneChip WT Terminal Labeling and Controls Kit. Labeled cDNAs were then hybridized to GeneChip Mouse Gene 1.0 ST Array rev.4 using a GeneChip Hybridization, Wash, and Stain Kit. Hybridization signals were obtained by GeneChip Scanner (Affymetrix). Microarray data were analyzed using Bioconductor (version 2.11) packages affy and limma in R (version 2.15.1) -67. Brieberg FDR  was applberg FDR . These eet al. [Nuclei extracts were prepared from MC3T3-E1 cells using a protocol modified from Dignam et al. . Primary8 MC3T3-E1 cells were washed with PBS  and then fixed on a plate with 1% formaldehyde for 8 minutes to crosslink DNA-protein complexes. The fixed cells were washed with ice-cold PBS, harvested, and pelleted. Nuclei extraction was performed using a protocol modified from Dignam et al. [At days 0, 9, and 28 of differentiation, approximately 1 × 10m et al. . Isolatem et al.  or immunP < 10-10 level were retained for subsequent analysis. For each of the three time points in our data, read counts were normalized to 10 million reads. The UCSC genome browser [Single-end 36 base sequences from Runx2 ChIP-Seq and input libraries were mapped to the mouse genome (assembly mm9) using Bowtie (version 0.12.8) . Runx2 p browser  was usedRunx2 binding regions were classified on the basis of genomic location categories and annotated to known RefSeq genes . Runx2 pRunx2 shRNA. Runx2 binding and Runx2-mediated transcription control were also evaluated by EMBER [PeaksToGenes ,62 Addi was usedby EMBER . Analogoby EMBER , EMBER oFunctional genomic elements can be characterized by evolutionary conservation and have been shown to distinguish functionally verified from unverified transcription factor binding sites on human promoters . For eacThe raw sequences and peak-related files in BED and WIG formats representing processed data have been deposited in the GEO database under accession number GSE54013.Selected Runx2 peak regions were cloned with MluI combined with BglII or XhoI into a pGL2-SV40-Luc reporter . Primers used in cloning are listed in Additional file bp: base pair; ChIP: chromatin immunoprecipitation; FDR: false discovery rate; GEO: Gene Expresion Omnibus; GO: Gene Ontology; qPCR: quantitative real time polymerase chain reaction; shRNA: short hairpin RNA; TSS: transcription start site; TTS: transcription termination site; UTR: untranslated region, SEM, standard error of mean.The authors declare that they have no competing interests.HW and JARG conceived and designed the experiments. HW performed the experiments. TWW, JRD, and HW performed bioinformatics data analysis. HW, JARG, TWW, and JRD interpreted the data with insights from PWLT, JBL, JLS, AVW, and GSS. HW, TWW, JARG, JRD, PWLT, JBL, and JLS wrote the paper with help from AVW and GSS. All authors read and approved the final manuscript.Summary of MC3T3 Runx2 ChIP-Seq. This table lists the read numbers and genome coverage of Runx2 ChIP-Seq libraries.Click here for fileDistribution patterns of Runx2 peaks across genomic locations. This table is related to Figure Click here for fileDetailed description of ChIP-PCR, ChIP-Seq with bioinformatics analysis, and supplemental figure legends.Click here for fileGREAT Gene Ontology terms. This table contains the top GO terms assigned by GREAT to clusters 1 to 7 defined in Figure Click here for fileGO term analysis from GREAT for clusters 1 to 3 and 5 to 7 in Figure Click here for fileValidation of Runx2 peaks by ChIP-PCR. This figure is related to Figures Click here for fileBsp gene during differentiation. This figure is related to Figure Runx2 peaks associated with Click here for fileDetailed annotation of Runx2 peaks. This table is a meta-spreadsheet of Runx2 peaks annotated to RefSeq genes.Click here for fileValidation of Runx2 knockdown in MC3T3 cells. This figure is related to Figure Click here for fileAdditional characteristics of Runx2 binding in shRunx2 responsive genes. This figure is related to Figure Click here for fileRunx2 shRNA-responsive genes. This figure is related to Figure PeaksToGenes analysis of Runx2 occupancy in Click here for fileEMBER analyses of Runx2 binding in the genes differentially regulated by Runx2 knockdown. This figure is related to Figure Click here for fileTnfrsf19. This figure is related to Figure Validation of novel Runx2 target Click here for fileRunx2 shRNA. This table lists the genes that are up- or down- regulated by shRunx2 treatment in MC3T3 cells differentiated for 9 days.Genes responsive to Click here for fileqPCR and ChIP-PCR primers.Click here for fileCloning primers. This table contains the primers used for plasmid construction.Click here for file


Neurospora circadian oscillator, the White Collar Complex (WCC) formed from White Collar-1 (WC-1) and White Collar-2 (WC-2) drives transcription of the circadian pacemaker gene frequency (frq). Although FRQ-dependent repression of WCC has been extensively studied, the mechanism by which the WCC initiates a circadian cycle remains elusive. Structure/function analysis of WC-1 eliminated domains previously thought to transactivate frq expression but instead identified amino acids 100–200 as essential for frq circadian expression. A proteomics-based search for coactivators with WCC uncovered the SWI/SNF (SWItch/Sucrose NonFermentable) complex: SWI/SNF interacts with WCC in vivo and in vitro, binds to the Clock box in the frq promoter, and is required both for circadian remodeling of nucleosomes at frq and for rhythmic frq expression; interestingly, SWI/SNF is not required for light-induced frq expression. These data suggest a model in which WC-1 recruits SWI/SNF to remodel and loop chromatin at frq, thereby activating frq expression to initiate the circadian cycle.In the negative feedback loop comprising the  Neurospora, a fungal model for these clocks, the proteins that activate expression of the clock gene “frequency” bind to DNA far away from where the coding part of the gene begins, and a mystery has been how this action-at-a-distance works. The answer revealed here is that the activating proteins recruit other proteins to unwrap the DNA and bring the distal site close to the place where the coding part of the gene begins.Circadian clocks govern behavior in a wide variety of organisms. These clocks are assembled in such a way that proteins encoded by a few dedicated “clock genes” form a complex that acts to reduce their own expression. That is, the genes and proteins participate in a negative feedback loop, and so long as the feedback has delays built in, this system will oscillate. The feedback loops that underlie circadian rhythms in fungi and animals are quite similar in many ways, and while much is known about the proteins themselves, both those that activate the dedicated clock genes and the clock proteins that repress their own expression, relatively little is known about how the initial expression of the clock genes is activated. In  Neurospora has been for several decades an excellent model for studies of the eukaryotic circadian clock characteristic of fungi and animals. In this organism, the White Collar Complex (WCC), a heterodimer comprised of WC-1 and WC-2, serves as the transcriptional activator for the pacemaker gene frequency (frq) by binding to one of two DNA elements, the Clock box (C box) Proximal Light-Response Element (PLRE) in the light frq expression in a circadian cycle is still poorly understood. WC-1 has two predicted transactivation domains located close to the N- and C- termini respectively in vivo functions.Circadian clocks are key cellular mechanisms regulating a wide variety of physiological and molecular activities. in vivo, the histone acetyltransferases and the ATP-dependent remodeling enzymes Saccharomyces cerevisiae as a 2 MDa complex that alters chromatin structures for essential functions such as transcriptional activation, DNA repair, recombination, and chromosome segregation swi2 knockouts are defective in sporulation and display slow growth on nonfermentable carbon sources swi1 null strains, defective in mating-type switching, display sporulation defects and slow growth snf5 null mutants show reduced growth on glucose and sucrose per se without their disruption, by sliding histone octamers to other sites on the same DNA molecule, or transferring histone octamers to other DNA molecules Nucleosome and chromatin structure play critical roles in transcriptional regulation. To overcome nucleosomal barriers in transcription, different transcriptional complexes coordinate to make genomic DNA accessible to RNA polymerase II  Neurospora, histone acetylation of the frq promoter oscillates over a circadian cycle and chromatin structure at frq is rhythmically remodeled in a circadian fashion, accompanied by rhythmic binding of WC-2 C box peaks when frq transcription is repressed and decreases just before frq transcription starts C box to terminate frq transcription. Another chromatin remodeler, CHD-1, plays a role in methylation and rhythmic expression of frqfrq transcription and participate in chromatin remodeling at frq, how the WCC interacts with remodeling factors to relieve chromatin-mediated repression of the C box is still elusive. Consistent with this, there is a several hour lag between the turnover of FRQ and the rapid increase in frq mRNA frq expression than simply relief of repression.Rhythmic histone modifications and chromatin remodeling over a circadian cycle have been reported for a variety of genes in different circadian systems. For example, the promoter regions of frq transcription; notably, elimination of the N- and C- terminal polyQ stretches does not influence frq circadian transcription and such strains showed a WT circadian phenotype. The SWI1 subunit in the SWI/SNF complex is essential for initiation of frq transcription in a circadian context although interestingly not in response to light, and in a Δswi1 strain chromatin structure adjacent to the C box is less remodeled and the oscillation of the nucleosomal density is abolished, as is the circadian clock.In this study, we identify a previously undescribed N-terminal domain of WC-1, close to but not including the prominent polyglutamine stretch, that acts as the transactivation domain for recruiting SWI/SNF and driving Neurospora, WC-1 has two polyQ domains located at N- and C- termini respectively that were hypothesized to be transactivation domains (AD) frq expression, we eliminated both of them (aa 16-57 and aa 1097-1128)   and surp97-1128) . We furtfrq transcription, and circadian phenotypes of these mutants were monitored by race tube assays  and only a weak FRQ band or no FRQ at all appeared at DD28   ; levels  than WT . Thus, ir period  and redur period  retainedectively , indicatet al., in preparation); these preliminary mass spectrometry data showed that SWI/SNF subunits copurified with the Neurospora WCC. To validate this interaction in vivo, SWI1 was C-terminally tagged with V5. Immunoprecipitation using WC-2 antibody revealed the anticipated strong interaction with WC-1 as well as an interaction with SWI1 that was dependent on the presence of WC-1 . The acidic nature of aa 100-300 is consistent with reported acidic activation domains of the VP16 protein, yeast Gcn4, and yeast Hap4, which are able to recruit the SWI/SNF complex to release chromatin-mediated repression of transcription To confirm that the association between this region of WC-1 and SWI/SNF is still robust in vitro, GST alone, GST-WC-1 aa 1-300-6xHis and GST-aa 100-300-6xHis  were cloned, expressed and purified from peptides . To pullpeptides . NegativNeurospora SWI/SNF, V5-tagged SWI1 was purified using a single V5 antibody step and the result showed that several proteins were specifically co-purified with SWI1-V5 in a stoichiometric manner  according to their homology with yeast SWI/SNF subunits . FRQ protein expression was examined by Western blot , the Δswp82 strain had a normal FRQ level and circadian expression whereas Δswi1 did not  of frqswi1, Δsnf5, and Δswi59 backgrounds respectively, such that the activity of the complex of WC-1 and WC-2 on the frq promoter could be analyzed in vivo. Consistent with frq promoter:luc transcription was dramatically impaired in Δswi1 while Δswi59 showed a WT phenotype  expression needed for the overt rhythm. Of particular note is that Δswi1 completely lost rhythmic transcription of frq and overall expression was about 1% of WT. Taken together, these data indicate that SNF5 contributes significantly to frq transcription whereas SWI1 is essential for the transcriptional oscillation of frq over a circadian cycle.To check the circadian significance of rhythmic , grew moeast see , but alshenotype . frq tras strain  suggestsfrq, it does not itself bind to DNA, but instead relies on dedicated transcription factors. Two reported models for SWI/SNF recruitment by transcription factors have been advanced: (1) Transcription factors that have a strong affinity for a promoter associate first with an Upstream Activation Sequence (UAS) and then recruit SWI/SNF Neurospora, WCC binds to the PLRE adjacent to the transcription start site (TSS) of the frq promoter under light conditions while it associates with the C box of the same promoter located ∼1.2 kilobases 5′ of the TSS (transcription start site) in the dark. To test whether SWI/SNF is recruited by WC-1 to the C box, in addition to determining which model fits best for WC-1, chromatin immunoprecipitation was performed at DD16, a time when frq expression should be near maximal, using the WT and WC-1 Δ1-300 strains  steadily increases over this period and this is accompanied by a slightly delayed and steeper increase in SWI/SNF association (using SWI1 as a proxy). Loss of nucleosome components from the region of the C box was tracked by loss of histone H3 which shows a lag of 3–4 hours followed by a rapid disappearance from the C-box region. A working model for initial events in activation of frq transcription consistent with these data posits WC-1 and WC-2 forming an active transcriptional complex on the C box DNA  digested, and mononucleosomal DNA was gel-purified and quantified for real-time PCR (C box is closed and frq transcription repressed), DD12 , DD24 , and DD32 ; these times were chosen based on the peaks and troughs of nucleosome occupancy reported by Belden et al. C box and the region of NucB on the mononucleosomal gel-purified DNA. As previously reported swi1 and NucB density was always higher than WT. To better gauge whether NucB was being moved aside or displaced to truly open up the chromatin at the C box we applied a nuclease sensitivity assay previously used to probe chromatin structure at this locus swi1 strains at the same 4 subjective times (corresponding to two successive peaks and troughs in frq expression in WT and the corresponding times in Δswi 1) and subjected to limited digestion with micrococcal nuclease (MNase), an enzyme that will cut open DNA but not DNA bound within nucleosomes (C box (NucB) appears to be rhythmically present in WT but continually present in the Δswi1 background, consistent with the PCR analysis of frq transcription in a circadian cycle and that SWI1 plays an essential role in this process. As controls for specificity, the nucleosome near NucB and bracketing the C box  partially occludes the time PCR . Four daleosomes . The nucs tested .frq promoter to aid in remodeling the nucleosome environment of the C box and thereby to initiate a circadian cycle of frq transcription. To identify potential transactivation domains on WC-1 required for frq transcription, a series of WC-1 deletions were generated and studied. Among these, all deletions covering amino acids 101-200 had normal WC-1 levels but displayed severely impaired frq mRNA and FRQ expression in the dark and arrhythmic circadian phenotypes. These data suggest aa 101-200 of WC-1 is a transactivation domain that is essential for circadian expression of frq. A search for coactivators recruited by WCC identified SWI/SNF, components of which interact with WC-1 in vivo and in vitro. In addition, the portion of WC-1 containing the transactivation domain is required for recruiting SWI/SNF to the C box.In this study, we showed that WC-1 in the WCC recruits SWI/SNF to the Neurospora SWI/SNF complex identified the expected subunits based on yeast homology predictions  and CHD-1 as chromatin modifiers required for the rhythmic opening and closure of the C box in the frq promoter that leads to rhythmic expression of FRQ frq (qrf) promoter and may play a more general role in maintaining chromatin structure at frq as without it expression levels never reach either peak or trough levels seen in WT C box preferentially during the time when frq is becoming active frq is always inactive and without CSW or CHD-1 frq is always moderately active. In the core negative feedback loop, FRQ helps to inactivate WC-1 and prevents WCC from binding to DNA. However, the fact that frq remains moderately active without CSW or CHD-1 indicates that activation/repression of FRQ is more than only binding/inactivation of WCC but also requires the active participation of other factors, perhaps to eject the WCC/SWI/SNF complex from the C box and un-loop the DNA. Although both CSW and CHD-1 actively remodel DNA at frq their role in the clock cannot be succinctly stated as activating or repressing; the timing of CSW binding, for instance, coincides with frq activation yet frq is still expressed without it. When present and active, WCC might dominate the competition between activation and repression, recruiting SWI/SNF to activate frq despite the presence of other factors. Not explicitly accounted for yet in this model is the involvement of the frq antisense qrf, whose promoter is also remodeled by CHD-1. A recent publication revealed a novel factor, CATP (Clock ATPase), involved in remodeling chromatin at the C boxfrq in Δcatp strains appears to be about a log order greater than in strains lacking SWI/SNF, consistent with CATP acting as an accessory factor to help to open the C box.Through the lens provided by this study we can now begin to understand how multiple proteins and complexes with opposing functions coordinate their activities to open/activate and close/repress the frq transcription starts, NucB mostly blocks the C box due to the action of chromatin remodelers that may include CHD-1; when with WC-2, WC-1 binds to the C box in its active form, it recruits SWI/SNF which aids in removing NucB from the C box, stabilizing the active state essential for frq transcription; CSW binds most strongly at this time. Based on the action of SWI/SNF in other systems we anticipate that its action involves DNA looping to bring the TSS into proximity with the C box and WCC. This configuration remains active, with active WC-1 being rapidly turned over, until FRQ depresses activity of WC-1 and reduces its affinity for DNA thereby also stabilizing it. Without WCC bound, the SWI/SNF-mediated looping is reversed by the action of other chromatin remodelers, and NucB returns to cover the C box.A working model based on these data is summarized in frq may represent a distinct function. In the light frq mRNA levels are much higher than that in the dark, FRQ does not inhibit this expression, swi1, Δsnf5 and Δswp59 that severely reduce circadian frq expression. This suggests that the WCC recruited to the PLRE in the light recruits functional coactivators other than SWI/SNF to modify the PLRE in frq transcription. Additionally and consistent with this, the PLRE is located adjacent to the TSS site and WCC acts on these regions neither by looping of the DNA to bring activators to the TSS, nor by wholesale remodeling of chromatin, although epigenetic modifications have been noted C box versus light activators at the PLRE. A variety of data suggest that light activation of bd Aras-1) and 74A (WT Aras-1) were each used as a clock-WT strain in this study. Race tube analyses were carried out as previously described bd; Δfrq::hph+; Δmus-52::hph+ aras-1). Neurospora transformation was done as previously described wc-1 {knock-in} (KIwc-1) targeting cassette pWB-1-6 was introduced into 21-9 and replacement mutants were backcrossed to 328-4 to obtain homokaryotic strains for race tube analyses. WC-1 and SWI/SNF deletion strains generated by the Neurospora genome project were obtained from Fungal Genetics Stock Center (FGSC) 328-4  and rotated for at least 2 hours. The supernatant was removed and the beads were washed three times with the pull-down buffer.WC-1 aa1-300 and 100-300 were each cloned into pGEX4T1 in-frame fused with an N-terminal GST tag, and a hexahistidine tag was added by PCR to the C-termini. The plasmids were expressed in bacteria grown in LB medium with Ampicillin at a concentration of 10 ug/mL for 3 hours and induced with 1 mM IPTG for 1 hour. For WC-1 aa1-300 and 100-300, a cobalt purification step (Pierce) followed by a subsequent glutathione (Thermo Pierce) step was carried out to obtain full length polypeptides. GST was purified with glutathione resin (Thermo Pierce) alone. Neurospora nuclei were isolated from tissues cultured for indicated dark time. For each sample, 80 µgs of nuclei were digested with micrococcal nuclease (Takara) at the final concentration of 0.1 unit/ml for 1.5 min at 37 degrees. The digestion reaction was stopped by adding a buffer containing 0.2 mg/ml protease K and incubated at 37 degrees overnight. Chromatin DNA was extracted using Gentra Puregene Cell Kit (Qiagen) and cut with NcoI. Southern blot was carried out with a digoxigenin-labeled probe as described in The micrococcal nuclease assay was performed as described in Chromatin immunoprecipitation experiments were done as previously described Figure S1wc-1 mutants between aa 100-200 and FRQ protein levels in Δ163-200 (A) Amino acid sequence alignment of four fungal WC-1s. When WC-1 sequences from four closely related fungi  were aligned, it is clear that the polyQ domains are not conserved. Amino acid sequences were downloaded from the NCBI website and the alignments were performed using the EMBI-EBI on-line tool ClustalW2  (B) Race tube analyses of wc-1 mutants in the region of aa 100-200. 328-4 (bdras-1) was WT for this assay. Period lengths were as shown +/- SEM. (C) Western blot analysis of FRQ and WC-1 in WT and WC-1 Δ163-200 in constant light, DD16, and DD28. (D) FRQ is normally induced in response to light in Δ163-200. After a light exposure of 15 minutes, WC-1 underwent hyperphosphorylation and light-induced FRQ was seen after 1 hour light pulse in WT and Δ163-200.Race tube phenotypes of (TIF)Click here for additional data file.Figure S2Protein levels of WC-1, WC-2, SWI1, and SWI2 in constant dark over one circadian cycle. WC-1, WC-2, SWI1, and SWI2 protein levels were examined by Western blot across 28 hours in the dark. WC-1, WC-2, SWI1, and SWI2 showed relatively even protein levels. 15 µgs of total protein lysate were loaded into each lane.(TIF)Click here for additional data file.Figure S3swi1, Δsnf5, and Δswp59 (A) FRQ and WC-1 levels in SWI/SNF deletion strains analyzed by Western blot. Samples were grown in 2% LCM medium, synchronized in the light, transferred to the dark, and harvested at indicated time points. Non-specific bands were shown to demonstrate equal loading. (B) Δswi1, Δsnf5, and Δswp59 strains with and without the bdras-1 mutation grown on standard race tube medium. Black lines marked daily growth fronts of the strains in race tubes. Δswi1 and Δsnf5 in the bdras-1 background displayed conidiation and growth defects.FRQ expression in SWI/SNF single subunit knockouts and race tube phenotypes of Δ(TIF)Click here for additional data file.


Diseased myocardium from humans and experimental animal models shows heightened expression and activity of a specific subtype of phospholipase C (PLC), the splice variant PLCβ1b. Previous studies from our group showed that increasing PLCβ1b expression in adult mouse hearts by viral transduction was sufficient to cause sustained contractile dysfunction of rapid onset, which was maintained indefinitely in the absence of other pathological changes in the myocardium. We hypothesized that impaired contractility alone would be sufficient to induce a compensatory transcriptional response. Unbiased, comprehensive mRNA-sequencing was performed on 6 biological replicates of rAAV6-treated blank, PLCβ1b and PLCβ1a (closely related but inactive splice variant) hearts 8 weeks after injection, when reduced contractility was manifest in PLCβ1b hearts without evidence of induced hypertrophy. Expression of PLCβ1b resulted in expression changes in only 9 genes at FDR<0.1 when compared with control and these genes appeared unrelated to contractility. Importantly, PLCβ1a caused similar mild expression changes to PLCβ1b, despite a complete lack of effect of this isoform on cardiac contractility. We conclude that contractile depression caused by PLCβ1b activation is largely independent of changes in the transcriptome, and thus that lowered contractility is not sufficient in itself to provoke measurable transcriptomic alterations. In addition, our data stress the importance of a stringent control group to filter out transcriptional changes unrelated to cardiac function. Atp2a2 .In the current study, contractile function was assessed as ejection fraction determined by echocardiography. Anesthesia was maintained with 1.7% isoflurane. Echocardiography was performed using a Philips iE33 ultrasound machine and a 15 MHz linear-transducer. After a short-axis 2-D image of the LV at the level of the papillary muscles was obtained, 2-D guided M-mode images were acquired digitally at a sweep speed of 133 mm/s. Ejection fraction [EF = (LVIDd7 paired reads per heart (mean ± SEM). Reads were aligned to the mouse transcriptome represented in the Illumina iGenomes mm10 UCSC release using Tophat [7 paired reads per heart (mean ± SEM); this represents a mean alignment to the transcriptome of 61%. All mRNA-sequencing reads and transcriptome-aligned read counts have been deposited in the NCBI GEO with accession number GSE73909.Total RNA from mouse heart left ventricle was prepared by Trizol extraction from flash-frozen tissue. Six (6) biological replicates were used for each of rAAV6-blank, rAAV6-PLCβ1a and rAAV6-PLCβ1b conditions. Ribosomal RNA depletion along with RNA fragmentation and conversion to paired-end, strand-specific Illumina sequencing libraries were performed with Illumina TruSeq preparation kits. Sequencing reads of 76 nt in length were obtained from multiplexed libraries on an Illumina NextSeq instrument at the Ramaciotti Centre for Genomics, Sydney, Australia with (3.02 ± 0.11) * 10g Tophat , achieviFor ejection fraction measurements in Myh6-driven expression of Gαq [vs blank virus, and 1.2-fold (20%) for Gαq vs wt or PKCα vs tet-off. In order to compare expression changes for all 781 mRNAs across all three experiments, the fold-change of each mRNA between ‘treatment’ and appropriate ‘control’ groups was computed, the mean mRNA abundance value of control hearts was adjusted to 1 , and the resulting variation across individual hearts from each group was displayed in a heatmap .Data sets for rAAV6-mediated adult cardiac expression of PLCβ1a and PLCβ1b were compared with existing RNA-Seq datasets of αMHC/n of Gαq –26 or ofn of Gαq  commencin of Gαq ), or betin vivo using rAAV6-mediated transduction  began immediately after birth (see Cardiac overexpression of either Gαq (upstream regulator of PLCβ1b ) or of Pirth see  Fig 4A)Myh6 promirth see .Our previous studies showed that heightened expression of PLCβ1b is sufficient to cause chronic contractile dysfunction, whereas expressing the closely related splice variant PLCβ1a had no discernible effect . Furtherper se does not lead to widespread compensatory transcriptional changes. Our previous report noted changes in canonical transcriptional markers of cardiac hypertrophy, once hypertrophy finally ensued in PLCβ1b hearts, presumably as a result to prolonged contractile depression. After only 8 weeks of chronically lowered contractility, we found no changes in the expressions of ANP and α-skeletal actin, collagen-1A1, -3A1, α-or β-myosin heavy chain or phospholamban as measured by RT-qPCR [Despite chronically lowered contractility, we detected only minor changes in gene transcription associated with PLCβ1b expression and furthermore, similar changes followed expression of PLCβ1a, even though PLCβ1a did not alter contractile function. Thus, depressed contractility  RT-qPCR , nor did RT-qPCR .2+ from the sarcoplasmic reticulum [The finding that PLCβ1b-induced contractile dysfunction does not involve substantial transcriptional changes is compatible with our previous data, which showed that the contractile deficit could be fully reversed by treatment with a PKCα inhibitor for only 5 days , 33. Thieticulum . Neithereticulum . HoweverWhilst we conclude that chronically depressed contractility does not meaningfully alter transcription, it must be considered that the PLCβ1b-expressing mice were unchallenged, caged and fed and so had limited requirement for flight or fight responses. It remains possible that the combination of chronically low contractility induced by PLCβ1b and a stressor requiring increased cardiac performance might result in an altered transcription response compared with stressed but otherwise untreated mice. As a result, stressing PLCβ1b-overexpressing mice might result in a more rapid development of compensatory hypertrophy, together with its associated transcriptional changes.In cardiac signal transduction, PLCβ1b is immediately downstream of Gαq  and upstper se does not initiate major transcriptional responses, our study also stresses the importance of a stringent control group in studies of this type. Although transcriptional changes were detected in response to PLCβ1b expression, these do not appear intuitively connected to pathways known to cause, or to be affected as a consequence, of blunted contractility. Because these alterations were similar to those in PLCβ1a-expressing hearts, we were able to define these as events not specific to PLCβ1b overexpression, but caused by the overexpression of the common molecular domains of PLCβ1a and PLCβ1b, which are 97% homologous  declines in accordance with increasing read depth-normalized number of counts per mRNA.(TIF)Click here for additional data file.S1 TableAssignment to Gene Ontology ‘biological process’ categories was performed using the Cytoscape plugin BiNGO . Chd6 (c(XLS)Click here for additional data file.


Recent work observed that while all var genes produce transcripts, only a few var genes exhibit high expression levels. However, the global regulation of var expression and the relationship between epigenetic and genetic control remains to be established.Understanding the regulation mechanism of var gene always maintain high chromatin accessibility, with high potential to bind transcription factors (TFs). The levels of transcripts for different var gene family members are associated with this chromatin accessibility. Any given var gene thus shows punctuated chromatin states throughout the asexual life cycle. This is demonstrated by three independent transcript datasets. Chromatin accessibility in the var intron and 2 kb upstream regions are also positively correlated with their GC content, suggesting the level of var genes silencing might be encoded in their intron sequences. Interestingly, both var intron and 2 kb upstream regions exhibit higher chromatin accessibility when the genes have relatively lower transcription levels, suggesting a punctuated repressive function for these regulatory elements.We have systematically reanalyzed the existing genomic data including chromatin configurations and gene expressions; and for the first time used robust statistical methods to show that the intron and 2 kb upstream regions of each endogenous var control. We found dynamic modulations of specific epigenetic marks around the var intron and distal upstream regions are involved in the general var gene expression patterns in malarial antigenic variation.By integrating and analyzing epigenomic, genomic and transcriptomic data, our work reveals a novel distal element in The online version of this article (doi:10.1186/s12864-016-3005-7) contains supplementary material, which is available to authorized users. Plasmodium falciparum is responsible for 85 % of malaria cases and kills over one million people each year  centered at different 5′ upstream sites TSS, 500 bp, 1 kb, 1.5 kb, 2 kb and 2.5 kb were extracted for chromatin accessibility investigation.In this work, two complementary high-throughput sequencing datasets, one created via formaldehyde-assisted isolation of regulatory elements sequencing (FAIRE-Seq)  and the eq) data  were usesing BWA . Using Msing BWA  and apprsing BWA , all thesing BWA . Three ising BWA , 27, 28 sing BWA  was usedvar intron and 2 kb upstream regions.For sequence motif discovery, the MEME algorithm  was usedThe reads per kilo-base per million reads (RPKM) is calculated for comparing the sequencing signal between different genome elements.R is the sequencing reads number in a specific genome segment, L represents the length of the segment and Rtotal is the total mapped reads in the experiment.Where NC) is calculated for plotting the sequencing signal around the var intron and 5′ upstream regions. It is expressed as the fraction of the highest coverage value among all bins within the plot window.The normalized coverage  is the average number of reads for a specific gene type t in bin i. t = 1,2maxi = 1 : n(Et(Ri)) is the maximum average reads number of all gene type t = 1,2 on all bins i = 1:n.P. falciparum genome  is divided into equal length bins of 1 kb. Each 1 kb bin is represented as Bi, where i = 1 to 23,292. Each Bi is divided again into smaller bins bi with equal length 100 bp.For the correlation between GC content and chromatin accessibility on the whole genome scale, the gc) and FAIRE-Seq normalized reads number (NRN) is calculated for each small bin bi. The GC content and FAIRE-Seq signal for each Bi can be represented as two vectors.The GC content (Bi can be calculated between Then the Pearson correlation value for each bin var genes and controls with replacement. Then the Wilcoxon signed-rank test is used to compare the median value for the two samples.To compare the correlation value with control genes, the bootstrap method  was implCi, i = 1…14 with length Li. We assume each nucleotide nij, j = 1… Li, independently follows the same distribution. This distribution comes from the percentage of each nucleotide type in P. falciparum.A Shuffled genome was generated for each chromosome


GAL1, GAL2, GAL7 and GAL10 in Saccharomyces cerevisiae cells deficient for topoisomerases I and II. We find that topoisomerases are required for transcriptional activation of the GAL genes, but are dispensable for ongoing transcription, eliminating a role of the enzymes in transcriptional elongation. Furthermore, we demonstrate that promoter chromatin remodeling of the GAL genes is unaffected in the topoisomerase deficient strain. However, the cells fail to successfully recruit RNA polymerase II due to an inability of the TATA-binding protein (TBP) to bind to the TATA box in these promoters. We therefore argue that topoisomerases are required for accurate assembly of the preinitiation complex at the promoters of the GAL genes.To investigate the importance of topoisomerases for transcription of the galactose induced genes, we have studied the expression of  Several studies have demonstrated that transcription and DNA supercoiling are tightly coupled , 2. The Saccharomyces cerevisiae DNA superhelicity is primarily influenced by topoisomerase I (Top1) and topoisomerase II (Top2), encoded by the TOP1 and TOP2 gene, respectively [DNA topoisomerases solve topological problems arising during DNA metabolism. In ectively . Top1 reectively . Thus, bectively .Chromatin structure adds another layer of complexity to DNA supercoiling. Approximately 80% of the genome is covered by nucleosomes in yeast , and nucIt has recently been suggested that chromatin is able to adapt to changes in DNA superhelicity by a slight conformational change, which is reverted upon relaxation by either Top1 or Top2 . This imSeveral studies have established a role of topoisomerases in transcription and transcriptional regulation. Accordingly, a genome-wide study in yeast showed a preferential localization of the enzymes to intergenic regions, i.e. promoter regions, of highly transcribed genes , 14, andPHO5 gene we demonstrated that topoisomerases were required during transcriptional activation, but not for reinitiation and transcription elongation. In the absence of topoisomerase activity, the Pho4 transcription factor failed to bind to the promoter, thus inhibiting eviction of nucleosomes from the promoter region.A recent study from our laboratory combined microarray gene expression analyses and single gene studies to investigate the role of topoisomerases for global gene expression . TopoisoGAL1, GAL2, GAL7, and GAL10 to investigate if topoisomerases have a similar effect on the transcriptional activation of these genes. As for the PHO5 gene, we find that topoisomerases are essential for activation of the GAL genes but not for ongoing transcription. However, we discover that nucleosome removal from the promoters is unperturbed during transcriptional activation of the GAL genes in a strain lacking functional topoisomerases, but the strain displays faulty RNA polymerase II recruitment to the GAL gene promoters. In correlation with this, we find that the TATA-binding protein (TBP) fails to bind to the TATA box in these promoters, suggesting an involvement of topoisomerases in an early step of preinitiation complex assembly. Thus, although the overall effect of topoisomerase deficiency is the same for different inducible genes, the specific step, where the enzymes exert their effect, may vary from gene to gene.In the present work we have studied transcription of the galactose inducible genes, S. cerevisiae strains used are derivatives of W303a and are listed in 7 cells/ml at 25°C in YP with 2% raffinose and arrested in G1 with α-factor . Top2 was subsequently inhibited for 15 min at 37°C before galactose was added to a final concentration of 2% for induction of the galactose responsive genes. A sample was taken just prior to addition of galactose to serve as an uninduced control.All 7 cells were collected at the indicated time points. RNA was purified by use of RNeasy , and cDNA was made by SuperScript II RT-PCR  using oligo dT primers /CT Input, and was normalized to the enrichment in a telomeric region (TEL06R). The 0 min time point was set to 1. Sequences of the primers used in the ChIP experiments are listed in ts cells expressing HA tagged TBP by trichloroacetic acid. Proteins were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis, and western blotting was performed with antibodies targeting the HA epitope tag on TBP (Santa Cruz) and anti Mcm2 antibodies (Santa Cruz).ChIP was performed with 2.5x10eviously  with minGAL genes are a group of genes, which is induced when galactose is used as the carbon source in S. cerevisiae. To investigate, how topoisomerase deficiency influences transcription of this group of genes, we have studied transcription of GAL1, GAL2, GAL7 and GAL10 in an S. cerevisiae strain having a deletion of the TOP1 gene and a temperature-sensitive mutation in the TOP2 gene (top10Δtop2ts). A schematic presentation of the individual GAL genes is shown in GAL genes [GAL gene transcription or merely a kinetic delay, we performed an experiment with wild type and top1Δtop2ts cells as outlined in GAL genes were activated by addition of galactose. Cells were collected before transfer to inducible conditions (“0”) and after an induction period of 150 min, and mRNA levels were determined by qPCR. In top1Δtop2ts cells the induction levels of all four GAL genes remained very low even after 150 minutes under inducing conditions relative to the levels obtained in wild type cells  [right) [GAL genes or for continued transcription, we took advantage of the fact that deletion of GAL80 leads to constitutive expression of the GAL genes due to elimination of the need for external stimuli. Wild type, gal80Δ, as well as gal80Δtop1Δtop2ts cells were therefore cultured as shown in GAL gene mRNA levels. In contrast to wild type cells gal80Δ and gal80Δtop1Δtop2ts cells displayed high mRNA levels of the GAL genes under de-repressive conditions at the “0” minutes time points (reflecting the situation at the permissive temperature for top2ts)  , 21. When [left) , 23 or bn [left) , 25. Bin [right) , 26. To top2ts) . Interesgal80Δ and gal80Δtop1Δtop2ts cells we investigated the level of RNA polymerase II in the coding region of the GAL genes in the two strains. This was performed by chromatin immunoprecipitation (ChIP) with antibodies against the C-terminal domain of the RPBI subunit of RNA polymerase II  was performed with antibodies targeting the cMyc epitope tag. Top1 and Top2 were enriched approximately 2 and 3 fold, respectively, in the individual GAL gene promoters  to the TATA box is the first step in the assembly of the preinitiation complex . In ordepe cells . Howevert cells, , this deGAL genes, whereas ongoing transcription can occur in the absence of Top1 and Top2. Furthermore, we demonstrate that topoisomerases are required for TBP binding to the TATA box, or in a step downstream of nucleosome eviction, but upstream of TBP binding. We have previously shown that topoisomerases are required for transcriptional activation of the phosphate regulated PHO5 gene, where the enzymes are necessary for binding of the transcription factor Pho4 to the promoter region [GAL gene induction pathway, as wild type and top1Δtop2ts cells display equal nucleosome promoter clearing (PHO5 gene and the GAL genes are probably reflected in the dissimilarities in the induction pathways. In the case of PHO5, the Pho4 transcription factor has to bind the PHO5 promoter in order for nucleosome remodeling to occur [GAL system, is already bound to the promoter, but its chromatin remodeler recruiting domain is blocked by Gal80 [PHO5 system.Our findings have revealed that topoisomerase activity is required for transcriptional activation of the r region . In the clearing . The disto occur . In contby Gal80 . Thus, tGAL genes in top1Δtop2ts cells during transcriptional activation (Figs GAL genes are activated (gal80∆top1Δtop2ts mutant is deregulated upon transfer to the restrictive temperature. However, as the half-lives of the mRNA’s from GAL1, GAL2, GAL7, and GAL10 are 18, 49, 27, and 20 minutes, respectively [top2ts, we find this explanation unlikely. A more plausible explanation is that after the RNA polymerase has escaped from the preinitiation complex, some of the general transcription factors remain in the promoter region, creating a reinitiation scaffold [GAL genes have been shown to exhibit gene looping [Interestingly, no RNA polymerase II or TBP enrichment is observed in the promoters of the ion Figs  and 5, wctivated . This inectively , and we scaffold , which c looping , 33, wheGAL gene promoters are removed with equal kinetics in both wild type and top1Δtop2ts cells (GAL gene promoters through interaction with Gal4 [in vitro study has demonstrated that negative superhelicity results in a more active transcription of the immunoglobulin heavy chain gene when only TBP, TFIIB and RNA polymerase II are present [Transcriptional activation of a gene is a complicated process involving a plethora of proteins. The DNA superhelical changes occurring in topoisomerase deficient strains will most likely affect the activation process in a step involving protein-DNA interactions. As the nucleosomes bound at the ts cells , but TBPith Gal4 , 35. Theith Gal4 . The steith Gal4 . Rather,ith Gal4 , 15, 36.ith Gal4 –39. Thisith Gal4 . Further present .gal80Δtop1Δtop2ts cells. In these cells, TBP is already bound to the DNA template before cells are exposed to the positive supercoiling, which follows the transfer of cells to the restrictive temperature for top2ts, and although TBP binding may be sensitive to DNA superhelicity, its dissociation may not necessarily be affected. Alternatively, the transient negative supercoiling generated due to polymerase movement during elongation could maintain binding of TBP, as has been suggested earlier [If lack of topoisomerases affects TBP binding, one may speculate, how reinitiation can occur in  earlier .GAL gene activation, being required for TBP binding downstream of nucleosome eviction. This is in contrast to our earlier finding with the PHO5 inducible gene system, where topoisomerase activity was found to be required for binding of the transcription factor Pho4 prior to nucleosome eviction [In conclusion, we show that topoisomerase activity is essential for eviction . Topoiso


Autoimmune diseases occur more often in females, suggesting a key role for the X chromosome. X chromosome inactivation, a major epigenetic feature in female cells that provides dosage compensation of X-linked genes to avoid overexpression, presents special vulnerabilities that can contribute to the disease process. Disruption of X inactivation can result in loss of dosage compensation with expression from previously sequestered genes, imbalance of gene products, and altered endogenous material out of normal epigenetic context. In addition, the human X has significant differences compared to other species and these differences can contribute to the frequency and intensity of the autoimmune disease in humans as well as the types of autoantigens encountered. Here a link is demonstrated between autoimmune diseases, such as systemic lupus erythematosus, and the X chromosome by discussing cases in which typically non-autoimmune disorders complicated with X chromosome abnormalities also present lupus-like symptoms. The discussion is then extended to the reported spatial and temporal associations of the inactive X chromosome with the nucleolus. When frequent episodes of cellular stress occur, the inactive X chromosome may be disrupted and inadvertently become involved in the nucleolar stress response. Development of autoantigens, many of which are at least transiently components of the nucleolus, is then described. Polyamines, which aid in nucleoprotein complex assembly in the nucleolus, increase further during cell stress, and appear to have an important role in the autoimmune disease process. Autoantigenic endogenous material can potentially be stabilized by polyamines. This presents a new paradigm for autoimmune diseases: that many are antigen-driven and the autoantigens originate from altered endogenous material due to episodes of cellular stress that disrupt epigenetic control. This suggests that epigenetics and the X chromosome are important aspects of autoimmune diseases. Epigenetics is the area of biology that connects environmental factors with gene expression patterns in cells. We are still discovering new aspects of epigenetics so a detailed and comprehensive definition of epigenetics is still developing . There i5mC) in eukaryotes, particularly in promoter regions of genes, is associated with suppression of gene expression since the 5mC alters protein binding sites and recruits additional chromatin modifying factors, such as histone deacetylases. This control is supported by binding of proteins, such as the DNA methyl binding protein 2 (MECP2), which add another layer of control, supporting suppression or activation depending on the transcriptional context of the underlying genes. In the case of MECP2, it can bind both 5mC and 5-hydroxymethylcytosine (5hmC). However, DNA methylation can be reversed by a stepwise process that involves the recently discovered conversion of 5mC to 5hmC, followed by conversion to unmethylated cytosine. This process, 5mC to 5hmC to cytosine, is not yet fully understood but reversal of DNA methylation can potentially lead to changes in the expression of the underlying genes , which can be 100s–1000s of bases in length, also have roles in epigenetic control. The lncRNAs can suppress transcription from multiple genes. The X-inactivation specific transcript (XIST) is an example of the lncRNAs. XIST RNA is involved in the silencing of one of the two X chromosomes in female cells. Since most X-linked genes are used at equivalent levels in both male and female cells, only one X chromosome is needed. Early in embryonic development each human female cell randomly chooses one of its two X chromosomes to be inactivated, leaving only one active X chromosome. This establishes X-linked gene dosage compensation such that female and male cells have equivalent expression and product levels for most X-linked genes. The X chromosome selected for inactivation expresses multiple copies of XIST which bind to contiguous chromatin along the X chromosome and recruit other epigenetic suppressing factors, such as DNA methyltransferases (DNMTs). X inactivation results from synergy of XIST RNA, DNA methylation, histone deacetylation as well as many other factors . The resDXZ4, XIST, loc550643, and FIRRE). Each of these lncRNAs may have a role in maintenance of the X inactivation state in their vicinity. The two domains observed in the inactive X chromosome lay on either side of the DXZ4 gene, which is in the middle of the X long arm, Xq  so that their expression can be more efficiently regulated. Epigenetic suppression and compacting of chromatin also stores potential DNA supercoiling stress that, when released, can disrupt and alter chromatin over hundreds even thousands of bps, rapidly unraveling heterochromatin into more accessible extended loops. Each nucleosome stores supercoiling stress that, when released, can flux through the chromatin causing twisting and disruption of the chromatin structure as the chromatin adjusts to accommodate the stress . The relFigure 1A) in each cell while male cells have only one X chromosome , this suspicion of the X is supported by the observed rates of SLE in Klinefelter’s syndrome in which  males with an extra X chromosome have a rate of SLE 14x greater than  males . Previously it was thought that rates of autoimmune diseases in Turner’s syndrome patients would be similar to rates observed in  males, in effect, a rate lower than seen in  females. In reality, there are no published reports of classic  Turner’s syndrome with SLE, possibly due to a lack of specific studies to address this question , 3:1 in rheumatoid arthritis (RA), and 9:1 to 10:1 in systemic lupus erythematosus (SLE), Sjögren’s syndrome (SjS) and autoimmune thyroiditis . This ovquestion . Howeverquestion . There iquestion . For othThis general approach of analyzing the infrequent occurrences of autoimmune symptoms in what might otherwise be considered non-autoimmune disorders can be very beneficial in expanding our understanding of autoimmune diseases. But we must be open to broad explanations that can involve genetics and/or epigenetics, and can involve the innate immune response, the adaptive immune response, and even events preceding any immune involvement.Figure 2). In some cases, the gene in question may have genetic mutations, insertions, deletions, or duplications that alter the gene product function and/or the levels of gene expression. In other cases there might be epigenetic changes that alter the level of gene expression without changes in the underlying DNA sequence. The methyl CpG binding protein (MECP2), which suppresses transcription by capping methylated DNA sites and recruiting histone deacetylases, shows decreased MECP2 mRNA in association with its contributing risk for lupus (MECP2 mRNA expression appears to result from single nucleotide polymorphisms (SNPs) in the vicinity of the MECP2 gene, SNPs that alter the gene expression but not the functioning of the protein. The MECP2 gene is located at Xq28, i.e., toward the end of the long arm of the X chromosome.In relation to the X chromosome, a few specific X-linked genes have demonstrated an association with autoimmune diseases (or lupus . This deIRAK1) which is also located at Xq28 near the MECP2 gene. IRAK1 interacts with the interleukin-1 receptor to up regulate a transcription factor, nuclear factor κB activating protein (NKAP), which activates the innate immune response. Several SNPs have been identified in the DNA sequence in and around IRAK1. One mutation in the gene leads to an S196F change in the IRAK1 protein which leads to enhanced up regulation of NKAP and, thereby, contributes to an increased risk for lupus (Another gene that has been linked to autoimmune diseases is the interleukin-1 receptor-associated kinase 1 gene (or lupus .CD40LG (a.k.a. CD154), located at Xq24, which codes for a membrane protein expressed at the surface of activated CD4 T cells , also believed to have involvement in some autoimmune diseases, is a key transcription factor controlling activation of regulatory T cells (Treg cells). The methylation status of the gene receptor .HERV-Fc1, with only one copy in the genome located at Xq21.33, has shown increased expression in cases of MS .Human endogenous retrovirus are suspected of roles, possibly reverse transcription activity, in autoimmune diseases . Among tes of MS . The meaAnother source of potential reverse transcription activity is the long interspersed element, LINE1 (L1). L1 elements originated from a functional gene that codes for reverse transcriptase activity (RNA to DNA) and endonuclease activity that aids in inserting reverse transcribed DNA into the genome at new sites. L1 elements comprise 17% of the human genome and there are estimated to be more than 500,000 L1 element copies scattered throughout the human genome . A definIn vivo, most L1 elements are sufficiently methylated to keep them inactive. However, recent reports have demonstrated that, in lupus patients, some L1 elements are hypomethylated in CD4+ and CD8+ T cells, B cells, and neutrophils  is the last chromatin replicated in S phase which adds to the difficulties in maintaining the properly silenced epigenetic state.LINE1 elements are proposed to serve an additional function with regards to the X chromosome besides their retrotransposition activities, their potential as alternate transcription start sites, and their potential as anti-sense interfering RNAs. There is an overall twofold enrichment of L1 elements on the X chromosome (34%) compared to the genome average (17%) but the enrichment is in a gradient, higher in the X chromosome long arm (Xq) but it becomes lower in the short arm Xp; . The L1 6 bps and contains approximately 100 genes including the SRY sex-determining gene. The X chromosome has actually gained genes increasing to 153 × 106 bps and approximately 1,100 genes. Much of the newer material is located in the Xp  gene and the spermidine/spermine-N1-acetyltransferase (SAT1) gene at Xp22.1. The enzymes from these genes are involved in the polyamine pathway: SMS in biosynthesis of spermine from spermidine and SAT1 in recycling of spermine to spermidine and spermidine to putrescine. These two genes can, in effect, work against each other leading to wasteful cycling through polyamine synthesis and recycling. Since polyamine synthesis uses SAM, this could adversely impact the availability of SAM needed for DNA methylation. Normally SMS and SAT1 are silenced on the Xi (SAT1 is particularly interesting in that it can undergo superinduction (rapid increase in expression of 100x or more) in response to cellular stress and SAT1 can acetylate spermidine which can then be oxidized to putrescine (S-adenosylmethionine decarboxylase (AMD1) are elevated in RA synovial fibroblasts, along with putrescine which stabilizes AMD1 and is a precursor for polyamine synthesis (SAT1 and SMS could rapidly deplete SAM by futile polyamine synthesis and recycling.Another set of genes on the X chromosome that have bearing on autoimmune diseases are the spermine synthase (n the Xi  but repetrescine . Recentlynthesis . The polTLR7), located at Xp22.2, has recently been determined to be associated with increased risk for lupus in males in Chinese and Japanese populations when a SNP is found in the 5′ untranslated region (UTR) of the gene  and rare (found in 5% or less of population). So far in the human genome 30 rare fragile sites and 89 common fragile sites have been identified . FragileFMR1); FRAXE ; and FRAXF  and three common fragile sites have been identified: FRAXB (Xp22.31); FRAXC (Xq22.1); and FRAXD . However, the most significant vulnerabilities appear to be from Xp21 through PAR1, in effect, the distal portion of the short arm of the X chromosome. Within this section are: the FRAXB fragile site that can delay replication or sustain breaks; polyamine genes that can impact SAM levels; ‘hot’ L1 genes that have functional reverse transcriptase activity; and an abundance of CpG rich Alu elements. The high content of Alu elements in this region requires extensive amounts of methylation and protein suppressors to avoid their inappropriate expression by RNA pol III and potential reverse transcription by L1 or other reverse transcriptases. In the case of the inactive X chromosome, there would be extensive packaging of the DNA to silence genes and this packaging would contain ample supercoiling stress that can add to the dynamics in chromatin disruption . In this patient’s situation there are two available SAT1 alleles whereas normally with X inactivation, one would be silenced ] karyotype who has SLE ] karyotype . NADPH oxidase is needed in phagocytes to generate oxygen radicals to destroy phagocytized pathogens. The activated NADPH oxidase consists of several subunits including a cytochrome b component which includes a gp91-phox subunit coded by the CYBB gene at Xp21.1 . Figure 5B). These reports suggest difficulties in the females in establishing and maintaining X-linked dosage compensation. Other reports are available presenting X-CGD with lupus and lupus-like symptoms (Another situation arises in X-linked chronic granulomatous disease (X-CGD), a rare immunodeficiency disease (t Xp21.1 . The gp9symptoms .6 bps) and the mouse X (161 × 106 bps) have 95% of their genes in common . The human X chromosome is submetacentric, meaning that there is a centromere between the short arm (Xp) and the long arm (Xq). The mouse X chromosome is telocentric, meaning that there is just one long arm with a centromere at one end  males compared to  males, strongly suggest that the X chromosome is involved. Furthermore, cases of missing XICs and/or duplication of genetic material of the Xp, particularly from Xp21 to the terminus, suggest the X short arm, Xp, has a key role in autoimmune diseases. Unraveling the contributions of the X chromosome in autoimmune diseases is complicated by the natural phenomenon of X inactivation to achieve dosage compensation of X-linked genes. Studying X inactivation and loss of dosage compensation (a.k.a. reactivation or escape from X inactivation) has proven difficult. Much of our studies on autoimmune diseases have used mice as subjects. Genetically, the mouse has been a good model since the human X (153 × 10n common . However one end . The X i one end . TherefoThe inactive X chromosome (Xi) is typically observed as a dense heterochromatic structure in a perinuclear location, as if it has been pushed aside by the more active chromatin. This can help explain the late replication of the Xi relative to other chromosomes since it is less accessible but requires more effort in unpacking, repairing, and replicating DNA, and then repacking into heterochromatin. It places greater demands on methylation for DNA, histones, and translocation of other chromatin proteins, such as SAF-A. This is further complicated by the fragile sites with alternate DNA conformations that must be resolved but the transient release of stored negative supercoiling stress from nucleosomes during replication can add to the formation of alternate DNA conformations, such as Z-DNA .Figure 6, Inset). We can begin to imagine the consequences of this Xi-nucleolus association that might occur if a nucleolus increases its synthetic activities to produce ribosomes, tRNAs, SRPs, splicing components, and other macromolecular structures. The nucleolus increases in size, pushing aside most DNA in order to provide space for the folding of nascent RNA transcripts followed by their association with nascent proteins into ribonucleoprotein complexes. As the nucleolus increases in size, it could engulf part or all of the Xi. Since the nucleolar synthesis uses RNA polymerases I and III (RNA pol I and III) to produce many of the specialized non-coding RNA transcripts, the Xi could then be exposed to RNA pol III that could transcribe many of the Alu elements, including in PAR1. RNA pol III transcribes genes for non-coding RNAs such as the 7SL RNA of the SRP, the U6 RNA, the 5S rRNA of the ribosome, tRNAs, and SINEs  but the spermidine is the more relevant counterion for the in vivo setting , the authors identified more than 40 sites in the 23S rRNA and more than 135 sites in the 50S subunit where the polyamines could potentially interact with the RNA. This suggests that the polyamines could help with the initial folding of the 2,094 bases of the 23S rRNA and be involved in the final ribonucleoprotein assembly.Polyamines are important for RNA folding but they are not necessarily a component of the final RNA or ribonucleoprotein moiety. As shown in tructure . On the tructure . In thistructure . Using aAlthough the polyamines are important in RNA folding and assembly of macromolecular complexes and the polyamine levels increase during stress to facilitate increased production of ribosomes, tRNAs, SRPs, and splicing components, there can be consequences if the polyamine levels are not controlled. An increase in putrescine can support the formation of NAPs which can stabilize infrequent conformations in DNA, RNA, and proteins . At the ODC, SMS and SDS, thereby increasing polyamine synthesis and consumption of SAM . With the increased polyamines and RNA transcripts, including Alu RNA, there can be misfolding of RNA and potential stabilization of this abnormal RNA by polyamines and NAPs. For example, Alu RNA can form cruciforms that could be stabilized by polyamines and NAPs into autoantigens. These RNA transcripts can incorporate the Ro protein into autoantigenic complexes since the function of Ro is to identify misfolded RNA  have identified genes that have statistical significance in specific autoimmune diseases but that still leaves a vague picture of the ‘cause and effect’ in explaining triggering events and progression. Epigenetics as a factor in autoimmune diseases has been gaining interest in the past decade but many researchers still have a genetics-based perspective of epigenetics, that screening for the methylation state of genes will uncover the hypomethylated or hypermethylated ‘lupus’ gene. But epigenetics also involves sections of chromatin between genes such that potential problems can initiate without specific genes being involved. For example, release of DNA supercoiling stress stored in nucleosomes can flux through the chromatin and disrupt sites 1000s of bps away, opening loops of chromatin with genes and pseudogenes that were previously suppressed but now have exposed promoter regions. If we think of Alu elements with all their copies as being genes, albeit small non-coding genes, then there is not one single ‘lupus’ gene but more than a million. That still leaves us short of an explanation for the triggering and progression of autoimmune diseases. We have to think of the higher levels of epigenetics that can be involved, such as X inactivation and its potential for disruption with subsequent overexpression or loss of X-linked genes. And we have to think of epigenetic control as stored potential, not just for individual gene expression, but for DNA stress that can have far reaching impact on gene packaging and DNA conformations. Epigenetic changes can also involve subtle events, such as transient appearance and possible stabilization of alternate DNA conformations in a fragile site that then delay the completion of DNA repair or replication, placing the entire chromosome in jeopardy of degradation or misallocation during cell division so that daughter cells inherit different amounts of chromatin. Among the many components in epigenetic control  the polyamines are particularly interesting due to their versatility, their unique combination of charge and length, and their involvement in RNA folding, nucleoprotein assembly and episodes of cellular stress. In addition, their ability to form aggregates, NAPs, when sufficient putrescine, spermidine, and spermine are available can be potentially even more detrimental by stabilizing autoantigenic conformations of nucleoprotein complexes. The importance of polyamines in nucleolar activities places the polyamines in a critical position during cellular stress when the most inactive component in the nucleus, the inactive X chromosome with all its vulnerabilities, could be engulfed by the most active and dynamic component of the nucleus, the nucleolus. Looking at a list of autoantigen types in lupus, the mystery clears somewhat as we realize that most can be grouped as components of chromatin  or components of the nucleolus . Still, we must keep in mind that there are other possibilities. For example, the DNA may be reverse transcribed DNA in the cytosol and the histones may be nascent histones in the cytosol that bind the reverse transcribed DNA.SAT1 in mice leads to hair loss  to the drinking water . DFMO isair loss . This coWe expect that, as the autoimmune disorder research community gains more understanding of epigenetics and its potential, there will be more projects that are based on hypotheses of epigenetics involvement and less emphasis on finding specific genes and mutations. In addition, while mouse models are of value in understanding the adaptive immune response, the mouse models must be reappraised for their value in studying the innate immune response and early events that precede the autoimmune reaction. Projects targeting the adaptive immune response are of value in determining how to reduce the autoimmune reaction however our hypotheses suggest that projects that increase our understanding of the innate response, cellular and nucleolar stress responses and the stability of the inactive X chromosome, may well prove to be more beneficial in preventing and curing autoimmune disorders. Drug discovery targeting PAD enzymes to reduce NETosis, targeting pathogens, such as EBV, targeting other enzymes involved in epigenetic control and targeting polyamine synthesis and recycling enzymes should prove of greater value for eventual clinical use for prevention and control of autoimmune disorders.We have described the vulnerabilities of the inactive X chromosome which include its need for extensive heterochromatic assembly and maintenance, its perinuclear localization, its proximity to the nucleolus, and the potential detrimental actions that can arise from Alu elements, L1 elements, fragile sites, polyamine genes, miRNAs, and HERVs. The nucleolar stress response, such as the impact of pathogen activity exemplified by EBV, can lead to engulfment of the Xi by the nucleolus and exposure of the Xi to the high concentrations of polyamines and RNA pol III. Abnormal expression of previously sequestered alleles on the Xi, such as miRNAs and Alu and L1 elements, can alter the nucleolar activity and generate polyamine-stabilized (and possibly NAP-stabilized) alternate (misfolded) conformations of RNA and DNA, proteins and nucleoprotein complexes that are potentially autoantigenic. Constant epitopes, such as Ro, La, or Ku proteins, within these abnormal nucleoprotein complexes could also become autoantigenic targets. These autoantigens can then be exposed extracellularly where they elicit an autoimmune reaction. The extracellular exposure of these autoantigens can occur through apoptosis, NETosis (neutrophil extracellular trap release), ETosis, or cell surface presentation. This hypothesis: ‘X chromosome-nucleolus nexus’ fits well with the previously published ‘NAPs in NETs’ hypothesis . TogetheThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.