Bivalent chromatin refers to overlapping regions containing activating histone H3 Lys4 trimethylation (H3K4me3) and inactivating H3K27me3 marks. Existence of such bivalent marks on the same nucleosome has only recently been suggested. Previous genome-wide efforts to characterize bivalent chromatin have focused primarily on individual marks to define overlapping zones of bivalency rather than mapping positions of truly bivalent mononucleosomes.Here, we developed an efficacious sequential ChIP technique for examining global positioning of individual bivalent nucleosomes. Using next generation sequencing approaches we show that although individual H3K4me3 and H3K27me3 marks overlap in broad zones, bivalent nucleosomes are focally enriched in the vicinity of the transcription start site (TSS). These seem to occupy the H2A.Z nucleosome positions previously described as salt-labile nucleosomes, and are correlated with low gene expression. Although the enrichment profiles of bivalent nucleosomes show a clear dependency on CpG island content, they demonstrate a stark anti-correlation with methylation status.We show that regional overlap of H3K4me3 and H3K27me3 chromatin tend to be upstream to the TSS, while bivalent nucleosomes with both marks are mainly promoter proximal near the TSS of CpG island-containing genes with poised/low expression. We discuss the implications of the focal enrichment of bivalent nucleosomes around the TSS on the poised chromatin state of promoters in stem cells.The online version of this article (doi:10.1186/s12920-016-0221-6) contains supplementary material, which is available to authorized users. Local epigenetic control lies in positioning of nucleosomes around transcription start sites (TSS) and control regions, along with post-translational modifications of histone tails and/or the presence of non-canonical histone variants such as H2A.Z and H3.3 , 2. Theshylation –13. TherInitial characterization of bivalent chromatin described a “zonal” phenomenon in which a broad domain of H3K27me3 surrounds a more narrow occupancy of the H3K4me3 mark near the TSS . What haIn the present study, using a modified technique for sequential ChIP, we elucidate the genomic positioning of individual bivalent nucleosomes and their relations to other aspects of epigenetic regulation. We report a marked bias of bivalent nucleosomes immediately flanking the TSS exclusively in genes with proximal promoter CpG islands which are unmethylated. Intriguingly, we observe a relationship between these individual bivalent nucleosomes and previously established salt-labile nucleosomes at transcription start sites known to contain the variant histones H2A.Z and H3.3, which are key to initiation of transcription .A critical aspect of our study is the requirement of highly pure mononucleosomes in large quantities, enough for genome-wide analysis. Traditionally, most maps of histone modifications are created by performing ChIP on formaldehyde-crosslinked chromatin fractionated either by sonication or limited digest with micrococcal nuclease (MNase). While valuable, such maps have reduced resolution, owing to the generation of mixed populations of multinucleosomal fragments, since each modification could be pulled down sequentially on a substrate where these marks reside on neighboring nucleosomes. Therefore the only way to distinguish truly bivalent nucleosomes from heterogeneity within a cell population is to use sequential ChIP analyses of the two different marks on mononucleosome substrates. Hence, we aimed to obtain highly purified mononucleosomes as substrate and modified the sequential ChIP protocol for use in massively parallel sequencing.Traditional purification protocols extract nucleosomes under physiological salt concentrations (100–150 mM NaCl). However, nucleosomes closer to the transcription start sites, containing histone variants H3.3 and H2A.Z are labile at these salt conditions . The monMLH1 promoter, which revealed that amplicons larger than 147 bp were undetectable  cell lines RKO  versus SW480 , as established from previous work [MLH1 is silenced in RKO and associated with both abnormal CpG island promoter DNA methylation as well as some H3K27me3 enrichment. Contrasting this, MLH1 is highly active and marked by H3K4me3 in SW480. Indeed, we observed identical results when both sucrose gradient preparations and mononucleosomes obtained by quick-prep were probed using single ChIPs  antibody was crosslinked to protein A/G beads using disuccinimidyl glutarate (DSG), preventing its elution into the secondary ChIP (2°) step, thus increasing sensitivity and avoiding any ambiguity of analysis  in the same population yielded no enrichment , used as positive controls, but no enrichment in samples subjected to 1° anti-H3K4me3 pull-down followed by 2° anti-H3K27me3 pull-down  are used, revealing a lack of H3K4me3 at the region analyzed. Thus, concluding from the test combinations of 1°H3K4me3 → 2°H3K27me3 antibodies or the reverse (1°H3K27me3 → 2°H3K4me3) pull down in either cell line demonstrated complete lack of enrichment of both marks, indicating that the MLH1 promoter region examined lacks bivalent chromatin in both SW480 and RKO  analyses in RKO and SW480, using the same ary ChIP ° step, tary ChIP ° step, town Fig. . An exacCDO1, SFRP1 and SOX17, genes , to leosomes .To help fortify the above results, we used an additional proteomics approach to examine the pull-down products from sequential ChIP to test if both marks co-exist on the same histone peptide. We purified the core histone H3.1 and its transcriptional variant H3.3 by HPLC  and theno IP and (ii) several batches of anti-H3K4me3 antibodies were sensitive to crosslinking by DSG (unpublished results) introducing ambiguity in pull down activity, and hence can only be used in the 2°IP step (which doesn’t involve crosslinking). This restricted the directionality of seq-ChIP for scale up to genome wide platforms.We used ‘quick-prep’ purified mononucleosomes from NCCIT cells as a substrate for sequential-ChIP, in which nucleosomes isolated by anti-H3K27me3 pull-down were subsequently subjected to secondary ChIP by anti-H3K4me3, followed by massively parallel sequencing to examine genome-wide patterns of H3K4me3, H3K27me3, and bivalency at nucleosome resolution. In addition, we mapped H2A.Z as it is associated with the open chromatin region of active genes . We perfSFRP1 in Fig. To provide initial context for our genome-wide analyses, we first viewed a series of candidate genes, including those analyzed by our local ChIP studies Fig. , for theTo elucidate the general distribution of bivalent nucleosomes around TSS, we classified genes into different categories based on H3K4me3 and H3K27me3 enrichment around the promoter  using SICER for calling significant peaks of enrichment Fig. -c. TSS-rTo get an insight into the genomic position of bivalent nucleosomes, the sequencing reads binned at 10 bp intervals across the −/+5 Kb region around the TSS were plotted after averaging and normalizing to the average input reads for each of the promoter classes. As expected, H3K4me3-exclusive promoters show a general enrichment of this mark on either side of the TSS  with parThe positions of individual bivalent mononucleosomes, mapped by sequential-ChIP sequencing reads, reveal several distinct features compared to the individual marks Fig. , but witst row, Fig. nd row, Fig. nd row, Fig. nd row, Fig. rd panel, Fig. th row, Fig. To further understand the relationship between CpG islands, gene expression, and positions of bivalent nucleosomes, we classified the promoters into quintiles  of increasing CpG densities and contrasted these with genes having no promoter CpG-island. Within each such group based on CpG content, genes were further sub-classified into quintiles by gene expression levels . The chromatin profile of the enhancer regions when divided into subsets based on the presence or absence of a CpG islands however, reveal that enhancers with CpG island (s) have elevated H3K27me3 and bivalent nucleosomes, as well as increased H2A.Z . Thus, like the promoter regions, presence of bivalent nucleosomes is again correlated to presence of CpG islands.In addition to the promoter regions, we further analyzed the presence of bivalent nucleosomes at enhancer elements , which are not only associated with a myriad of chromatin marks , includiGiven the prevalence of bivalent nucleosomes in CpG islands and the known relationship between bivalency in ESC and later DNA hypermethylation in cancer cells, we examined the association of our bivalent nucleosomes with hypermethylated promoters. Previous data from our lab  has showTo deepen our understanding of how seemingly opposing chromatin modifications combine to alter gene expression, we report the first genome-wide positional mapping of nucleosomes with H3K4me3 and H3K27me3 marks. We performed modified sequential-ChIP on purified mononucleosomes followed by next-generation sequencing to map the positions of individual bivalent nucleosomes relative to the distribution of the individual H3K4me3 and H3K27me3 marks that constitute zonal bivalency. Further, we analyzed the relationship of bivalent mononucleosomes with gene expression, promoter CpG content and DNA methylation. The idea that combinatorial marks can coexist on the same nucleosome is important for deciphering the roles of histone modifications and their implications for control of gene expression . The metThe concept of bivalent chromatin consisting of opposing H3K4me3 and H3K27me3 modifications is thought to help maintain genes in a low but poised state of expression in stem cells , 39. VirOur findings stress the need to consider chromatin bivalency in two contexts—namely the zonal patterns first identified , but alsWith regards to true bivalent mononucleosomes, our current findings challenge some previous studies and conclusions, probably due to alterations in the way we extract nucleosomes to best preserve the salt labile fractions of chromatin. Others have shown that PRC2 components are enriched around promoters , 41 withThe observed enrichment of bivalent nucleosomes at active genes could be due to non-specific flow-through of H3K4me3 in the primary ChIP with anti-H3K27me3, and subsequent enrichment in the secondary ChIP with anti-H3K4me3 antibody. However, the ChIP-PCR data in Fig. With respect to individual bivalent nucleosomes, perhaps the most important and potentially functional aspect of our findings is their relationship to the TSS. For both, genes classified as carrying zonal bivalency and those which are predominantly marked by H3K4me3, there exists a strong correlation between bivalent mononucleosomes and the so-called “nucleosome-free region”. Strikingly, this region is known to harbor salt-labile nucleosomes that contain at least two important non-canonical histone variants: H3.3 and H2A.Z, which can be incorporated and evicted from chromatin in a DNA replication-independent manner , 43. TheOur data adds to the growing body of work examining the relationship between localization of bivalency and CpG island/density Fig. , as prevIn summary, to the best of our knowledge, this study is the first attempt to map combinatorial histone modification maps at a genome wide level using mononucleosome substrates. Our findings refine the concept of bivalent chromatin and further classify bivalency into two different yet associated subcategories of zonal bivalency and individual mononucleosome bivalency. In further studies, the relationship between these two types of bivalency and the potential roles of each in gene regulation must be considered. Not only should the techniques developed herein be instrumental in fostering genome-wide mapping of combinatorial marks, but the results open doors to studies of functions for such patterns and particularly those for truly bivalent mononucleosomes in genome positioning and the control of gene transcription.2 at 37 °C to 70–80 % confluence in 150 mm culture dishes. Cells were pre-chilled on ice, harvested by scraping, and washed once with chilled PBS with freshly added 1X complete proteinase inhibitor (Roche) and 1 mM AEBSF. The pellet volume was measured by weight and stored at −80 °C until further use.SW480, HCT116 and DKO cells were grown in McCoy’s 5a (Corning) while RKO cells were grown in MEM (Corning) supplemented respectively with 10 % FBS (Gemini). NCCIT cells were grown in RPMI (Corning) with 15 % FBS. All cells were grown in 5 % CO2, 2 mM CaCl2) and re-suspended in the 3 volumes of the same buffer containing 0.2 % Triton X-100. Samples were treated with 3 U/ml of MNase for 10 min at 37 °C followed by mild sonication to release the digested nucleosomes. Reactions were stopped by addition of 30X stop buffer . Chromatin was cleared by centrifugation at 13,000 × g for 20 min at 4 °C, and the supernatant was crosslinked with 0.15 % formaldehyde (reaction stopped by 50 mM glycine) followed by repetition of the centrifugation step. The supernatant was further fractionated in a 5–25 % sucrose gradient (S.G.) prepared in low salt ChIP buffer  and separated at 40,000 rpm for 16 h in a Beckman SW40 Ti rotor. Pooled mononucleosome peak fractions were exchanged for 1X ChIP buffer  using PD10 desalting columns  and adjusted to 10 μg/ml to be used for ChIP.All preparations were carried out in buffers with freshly added proteinase inhibitor cocktail and 1 mM AEBSF maintained at 4 °C/on ice unless mentioned otherwise. Each single ChIP experiment was performed from 1X volume of nuclei isolated from 6 million cells as previously described , followe2, 4 mM CaCl2, 0.4 % Triton X-100) on ice, followed by treatment with 8 U/ml of MNase at 37 °C for 10 min shaking at 100 rpm. The reaction was stopped with appropriate volume of 30X MNase stop buffer  followed by centrifugation at 13,000 × g for 20 min at 4 °C. Supernatant was diluted in PBS to a chromatin concentration to 200 μg/ml (A260) and crosslinked with 0.15 % formaldehyde for 10 min at RT before stopping the reaction with 50 mM glycine. The preparation was cleared by centrifugation at 13,000 x g for 20 min at 4 °C. The salt concentration from both nucleosome preps was readjusted to 1X ChIP buffer composition using appropriate stock solutions to obtain a chromatin concentration of 50 μg/ml to be used in ChIP.Alternatively, for “quick prep protocol” (Q.P.), nuclear pellets were washed (minus detergent) and resuspended in one volume of 2X MNase reaction buffer  reactions were anti-H3K4me3 , anti-H3K27me3 , anti-H2A.Z  and anti-H3 . All immunoprecipitation buffers were fortified with freshly added proteinase inhibitor cocktail and 1 mM AEBSF. For each reaction of primary ChIP (1°), 5 μg of respective antibodies were bound to 40 μl of pre-blocked protein A + G Dynabeads  overnight at 4 °C, followed by two washes in PBS. Antibody-bound beads (Ab-beads) were crosslinked using 80 μM DSG at RT for 10 min, and the reaction was terminated using 50 mM glycine for 30 min followed by extensive washing of the crosslinked Ab-beads with ChIP buffer. Beads were blocked at 4 °C overnight, washed with ChIP buffer and subsequently incubated with the appropriate immunoprecipitation substrate  overnight at 4 °C with rotation. Samples were washed multiple times in varying conditions and combinations of buffers optimized for specificity with respect to each antibody, namely ChIP buffer, high salt ChIP buffer  and RIPA buffer . The immunoprecipitate was then eluted in ChIP elution buffer  at 65 °C for 10 min with agitation.Alternatively for Sequential ChIP, five 1° ChIP reactions were eluted in 100 μl 0.5 % SDS, 200 mM β-mercaptoethanol at 55 °C for 2 min with agitation. Pooled eluted supernatants were then passed through a G25 spin column  pre-equilibrated with 1X ChIP buffer, followed by dilution of the resulting eluate with 1X ChIP buffer to about 20-fold excess. These eluates were used as substrate for secondary ChIP (2°) with a specific pre-bound antibody, bound and/or crosslinked to protein A + G Dynabeads (as detailed above) and incubated at 4 °C overnight followed by extensive washing with ChIP and RIPA buffer. The immunoprecipitate was eluted in 1X ChIP elution buffer.Crosslinks on the eluted immunoprecipitates were reversed by overnight incubation at 65 °C. The eluted DNA was treated with 100 μg/ml RNase A (Thermo) at 37 °C for 1 h followed by Proteinase K (NEB) treatment at 20 μg/ml in a final concentration of 0.5 % SDS. The resulting DNA was cleaned up using a Qiagen PCR purification kit, and eluted in RNase DNase free 10 mM Tris pH 7.5 for further analysis. DNA obtained by ChIP was analyzed by PCR using primers shown in Additional file g for 20 min at 4 °C and used as substrate for immunoprecipitation.Uncross linked mononucleosomes prepared by the “quick prep” protocol were acid precipitated and used to purify individual histones using an Aquapore RP300 HPLC column with acetonitrile as the medium of flow, as described . Peak frFollowing immunoprecipitation with respective substrates, beads were eluted directly in 1X NuPage LDS sample buffer (Life Technologies) supplemented with 20 mM β-mercaptoethanol. The eluate was then heat denatured, dot blotted onto nitrocellulose membrane  and probed with anti-H3K4me3 antibody. HRP-linked donkey anti-rabbit IgG  was used as secondary antibody. After extensive washing, the blots were developed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific).Immunoprecipitated DNA was subjected to sequencing library preparation and sequenced on Applied Biosystems SOLiD (V3). Reads were aligned to hg18 (NCBI36) using Bioscope 1.2.1, finding all alignments between the first 25 bp of the read (seed) and the reference sequence with up to two mismatches. Each match is extended to the full length of the read, scoring 1 point for matching and −2 points for mismatching bases. The read is trimmed to the length with the highest score. If there is only one alignment or if an alignment scores significantly higher than the others for the same read, it is considered unique and reported.Single end sequencing reads were processed using SAMtools  to removPeaks were called using SICER  to detecCpG island data was downloaded from UCSC (hg18_cpgIslandExt). Promoters with CpG islands were identified as promoters that have a CpG-island within +/−1500 bp from the transcription start site (TSS). Genes with CpG-islands were sub settled into 5 groups  based on increasing CpG-density as defined in the CpG-island data from UCSC. Gene promotes that did not match any CpG-island were called non-CpG-island promoters. Alternate annotated transcripts of a gene, for which any of the other TSS had a CpG-island, were removed from the analysis. Otherwise, alternate transcripts with CpG-islands were considered as individual promoter sites.Combined genome-segmentation data (ChromHMM and Segway software) for human embryonic stem cells was downloaded from ENCODE . This reEnhancers with an overlapping CpG-island were identified as enhancers with CpG-islands. About 30 % of the sampled enhancers have CpG-islands.The Integrative Genomics Viewer (IGV) browser was used to display ChIP-seq data . For thehttp://bioinf.wehi.edu.au/limma/). The log2 intensities of probes were used for plotting.RNA was extracted from the NCCIT cells and processed for hybridization on an Agilent 4 × 44 K array as described previously . The mocP-value > 0.05) were not considered. Promoters with at least one probe with β-Value ≥ 0.75 were considered as methylated promoters while those with < 0.25 were considered as unmethylated.For genome-wide DNA-hypermethylation analysis, the Infinium methylation array (27 k) was used to analyze bisulfite-treated DNA  as per standard protocols . β-ValueAll genomic analysis was done in the R computing platform using Bioconductor packages. Plots were further annotated in Adobe Illustrator.


P=1.54 × 10−38), and residing in a predicted enhancer element. We show this region physically interacts with the transcription start site of ARID5B, that alleles of rs7090445 have differential enhancer activity and influence RUNX3 binding. RUNX3 knock-down reduces ARID5B expression and rs7090445 enhancer activity. Individuals carrying the rs7090445-C risk allele also have reduced ARID5B expression. Finally, the rs7090445-C risk allele is preferentially retained in HD-ALL blasts consistent with inherited genetic variation contributing to arrest of normal lymphocyte development, facilitating leukaemic clonal expansion. These data provide evidence for a biological mechanism underlying hereditary risk of HD-ALL at 10q21.2.Despite high-hyperdiploid acute lymphoblastic leukaemia (HD-ALL) being the most common subgroup of paediatric ALL, its aetiology remains unknown. Genome-wide association studies have demonstrated association at 10q21.2. Here, we sought to determine how this region influences HD-ALL risk. We impute genotypes across the locus, finding the single nucleotide polymorphism rs7090445 highly associated with HD-ALL ( ARID5B.Risk for the paediatric cancer high-hyperdiploid acute lymphoblastic leukaemia (HD-ALL) has been associated with genetic variants at 10q21.2. Here, the authors characterize this region, establishing a single risk variant and showing its role in dysregulated expression of  Twenty to twenty-five per cent of ALL is characterized by high-hyperdiploidy (51–67 chromosomes), making high-hyperdiploid ALL (HD-ALL) one of the major subgroups of paediatric cancerSubsequent clonal expansion is likely to be influenced by both inherited genetic susceptibility, and as yet unidentified environmental risk factors. A number of genetic disorders are associated with an increased risk of ALL including Down’s syndromeARID5B) at 10q21.2  demonstrated robust association at a locus in the gene AT rich interactive domain 5B  driving the 10q21.2 genetic association with ALL susceptibility as a basis for understanding HD-ALL initiation and addiction mechanisms. We identify a potential mechanism contributing to the additional risk to ALL conferred by 10q21.2. Variation at rs7090445 disrupts P<1.0 × 10−35  and an odds ratio of >2.4 at 10q21.2 in HD-ALL (ARID5B and are in strong linkage disequilibrium (LD) defining a single risk haplotype  as plausible functional SNPs based on their putative enhancer characteristics, defined by relevant histone markers , transcription factor binding, DNase I hypersensitivity and sequence conservation , were trisomic for chromosome 10, consistent with previous findingsARID5B expression, with the rs7090445-C and rs7896246-A risk alleles associated with reduced expression  (We next examined if the genotypes of rs7090445 or rs7896246 were associated with ficantly . Followi(P>0.05) , and C10t-test P=0.008 and P=0.028, ARID5B.To measure the effect of rs7090445 and rs7896246 alleles on enhancer activity we performed luciferase reporter assays in MHH-CALL2 and SEM BCP ALL cell lines. Transfection with constructs containing the rs7090445 risk C-risk allele displayed significantly lower normalized luminescence compared to non-risk T-allele constructs  or any other genetic variation 250 bps upstream or downstream of rs7090445, sequencing coverage within this interval was at least 45X. To verify that RUNX3 also binds to rs7090445 in MHH-CALL2 we performed ChIP-qPCR; enrichment for rs7090445 was observed relative to IgG isotype controls, consistent with differential RUNX3 binding driving allele specific enhancer activity  binding for alleles of rs7896246 and rs7090445. Reads mapping to each SNP allele from experiments were mapped and enumerated . Only RUactivity . We exam4.1 ref.  in conju4.1 ref. . To furt complex . Hence wARID5B and the rs7090445 risk region map within a single TAD . To examine the TAD structure of the 10q21 rs7090445 region and identify local chromatin patterns, we analysed publicly accessible Hi-C GM12878 datangle TAD . Furtherd levels , consistLa cells . Collecttwo loci .RUNX3 in ARID5B regulation we analysed tumour gene expression data from three cohorts of paediatric B-ALL totalling 385 cases . While a consistent association was shown between ARID5B and RUNX3 gene expression in HD-ALL the small size of these subsets of the data may have precluded demonstration of a statistically significant relationship. No consistent correlation between ARID5B and RUNX3 was observed in ETV6/RUNX1 positive ALL  experiments. Since transfection efficiency was found to be poor in MHH-CALL2 cells, siRNA RUNX3 knockdown was performed in GM11832 (homozygous for rs7090445 T). We also confirmed that RUNX3 binds to rs7090445 in GM11832 by ChIP-qRT (RUNX3 was accompanied by a significant reduction in ARID5B messenger RNA (mRNA) . Of these 21 had duplicated the risk allele (C) and 10 had duplicated the non-risk allele (T)  of 2,699 individuals in the 1958 British Birth Cohort genotyped using Hap1.2M-Duo Custom array data and 2,501 individuals from the UK Blood Service. The German GWAS comprised 1,155 cases, including 176 with HD-ALL (mean age at diagnosis 6.0 years), genotyped using Illumina Human OmniExpress-12v1.0 arrays. Controls comprised 2,024 healthy individuals from the Heinz Nixdorf Recall studyThe United Kingdom (UK)-GWAS and German-GWAS of B-cell precursor (BCP) ALL have been previously reported11Epigenetic profiles of association signals are composed of chromatin state segmentation (ChromHMM), DNAse I hypersensitivity, transcription factor ChIP-Seq and histone modification data in the lymphoblastoid cell line GM12878 from the ENCODE project2 in either RPMI with 10% FBS , 20% FBS RMPI (MHH-CALL2) or 10% DMEM (HeLa) supplemented with GlutaMAX . REH BCP-ALL cell line used as a model of ETV6:RUNX1 translocation positive ALL. Cell line identity confirmed by translocation specific PCR primers available on request. HeLa cell line used as a non B-cell control. Cell lines were tested for mycoplasma , no positive results were obtained.MHH-CALL2, SEM, REH B-ALL and HeLa cell lines were obtained from the DMSZ , GM12878 and GM11832 lymphoblastoid cell lines (LCL) were obtained through the Coriell Institute . Cell lines were maintained at 37 °C, with 5% CO6 MHH-CALL2, 3 × 106 GM11832 and 2 × 106 SEM cells were transfected with 2.5 μg of pGL3 promoter construct and 50 ng of pRL-SV40 , for normalization in 100 μl of Solution V using either program X-01 (MHH-CALL2 and GM11832) or T-02 (SEM). After 16 h promoter activity was assayed using the Dual-Luciferase reporter assay system (Promega) using a Fluoroskan Ascent FL (Labsystems). Six biological replicates each containing 3 technical replicates, were performed for MHH-CALL2 and SEM.The putative regulatory regions containing rs7090445 and rs7896246 were amplified from genomic DNA from the CEU LCL cell lines NA12004 and NA10851 respectively using primers detailed in http://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/wgEncodeRegTfbsClustered/ wgEncodeRegTfbsClusteredV3). ChIP-seq fastq data for proteins binding to either loci was downloaded from Encode (http://hgdownload.cse.ucsc.edu/goldenPath/hg19/encodeDCC/) and whole genome fastq data for the GM12878 was downloaded from Illumina Platinum Genomes project (AC:ERR194147). Fastq data were aligned to the human reference genome (human_g1k_v37), edited to mask common SNPs in build 141, using Stampy v1.0.28 sequence alignment tool. Allele specificity of ChIP-seq data was assessed using a binomial test of allele specific read counts, assuming an equal distribution. Biological replicates were analysed separately and combined using Fisher’s method.Proteins binding rs7896246 and rs7090445 were inferred from Encode ChIP-Seq data (T/C]GATAGCTTTGAGACCTTCTG-3′) were annealed to generate double-stranded EMSA probes. Each 20 μl binding reaction contained 50 fmol labelled target DNA, 1 × binding buffer . Reactions were incubated in the dark for 30 min at room temperature. Competition assays were performed by adding 200-fold molar excess of unlabelled probes. Super-shifts were performed by adding 2 μg RUNX3 antibody  to the binding reaction and incubating for 15 min before the addition of labelled probe. DNA-protein complexes were resolved by electrophoresis on a 6% DNA retardation gel (Life Technologies) in 0.5 × Tris-borate-EDTA (TBE) at 4 °C. Gels were imaged using the Odyssey Fc Infrared Imaging System (LI-COR Biosciences). An uncropped EMSA gel image is provided in Nuclear protein was extracted from GM12878 cells using NE-PER nuclear and cytoplasmic extraction kits (Thermo Fisher Scientific). Fluorescently labelled (DayLight 682 nM) and unlabelled complementary oligonucleotides (Eurofins Genomics) flanking rs7090445 , with the modification that after mircococcal nuclease digestion of DNA, cells were sonicated in a Biorupter for 10 min at 4 °C, to aid chromatin release. 25 μg of chromatin was incubated with 1 μg of anti-RUNX3 (Santa Cruz sc-376591) or 1 μg IgG2b . After washing qRT-PCR was performed using primer sequences detailed in http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE63525)Hi-C data derived from GM12878, digested with MboI, was retrieved from the NCBI  libraries were generated according to Naumova 20 for siRNA assays. Each biological replicate contained three technical replicates. PCR quantitation was by standard curve method. Five-fold serial dilutions of DNA or complementary DNA (cDNA) were used to control for differential primer efficiency. For siRNA assays target gene expression was normalized to the geometric average of PPIA, TBP, G6PD and LAMIN A/C.qRT-PCR was performed using SYBR Green PCR master mix (ThermoFisher Scientific). Samples were analysed undiluted for ChIP assays or 1/100 diluted in H20 were used for standard curve quantitation.GM11832 homozygous for rs7090445 T-allele was transiently transfected by electroporation using 200 nM of siRNA  for 48 h (siRNA sequences detailed in n=154) and GSE13351 (n=92) and RMA-normalized using a custom array definition file from (http://brainarray.mbni.med.umich.edu/Brainarray/Database/CustomCDF/20.0.0/ensg.asp) using the R packages Affy and limma. RNA-seq data from 139 B-cell ALL was quantified using RSEM as described previouslyP values were calculated in R version 3.2.2.mRNA expression data on paediatric B-cell ALL was retrieved from Gene Expression Omnibus accessions GSE13425 (2(x+1) transformed. To control for ploidy effects only cases with chromosome 10 trisomy for were considered for genotype expression correlations and chromosome duplication bias tests.90 cases of HD-ALL were genotyped using either Illumina Human 1M-duo Infinium BeadChip, HumanOmni1-Quad BeadChip or IlluminaOmni5M BeadChips27Existing GWAS data is available from the authors upon requestHow to cite this article: Studd, J. B. et al. Genetic and regulatory mechanism of susceptibility to high-hyperdiploid acute lymphoblastic leukaemia at 10q21.2. Nat. Commun.8, 14616 doi: 10.1038/ncomms14616 (2017).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Tables, Supplementary Figures, and Supplementary References


The maintenance of the correct cellular information goes beyond the simple transmission of an intact genetic code from one generation to the next. Epigenetic changes, topological cues and correct protein-protein interactions need to be re-established after each cell division to allow the next cell cycle to resume in the correct regulated manner. This process begins with mitotic exit and re-sets all the changes that occurred during mitosis thus restoring a functional G1 nucleus in preparation for the next cell cycle. Mitotic exit is triggered by inactivation of mitotic kinases and the reversal of their phosphorylation activities on many cellular components, from nuclear lamina to transcription factors and chromatin itself. To reverse all these phosphorylations, phosphatases act during mitotic exit in a timely and spatially controlled manner directing the events that lead to a functional G1 nucleus. In this review, we will summarise the recent developments on the control of phosphatases and their known substrates during mitotic exit, and the key steps that control the restoration of chromatin status, nuclear envelope reassembly and nuclear body re-organisation. Although pivotal work has been conducted in this area in yeast, due to differences between the mitotic exit network between yeast and vertebrates, we will mainly concentrate on the vertebrate system. CDK1-cyclin B activity is crucial for mitotic entry, and its inhibition promotes mitotic exit. The APC/C-Cdc20 complex timely degrades the mitotic cyclins and promotes mitotic exit through CDK down-regulation. Although this represents a crucial event for mitotic exit, dephosphorylation of CDK1 substrates is an essential step, and phosphatases take control of the transition progression , and all these isoforms appear to have specific roles in the cell cycle , a scaffolding subunit (A) and most of the complexes also contain a variable subunit (B) that acts as a substrate specifier. The B subunits are B55, B56 and PR72, and they have different isoforms Hunt .Studies on the identification of phosphatases that control mitotic exit have suggested not only that both PP1 , LBR, emerin and other lamina-associated proteins (LAP1/LAP2). It is at the inner nuclear membrane (INM) that chromatin binding proteins such as BAF and HP1 (heterochromatin protein 1) provide a bridge between the lamina and chromatin. The outer lamina extends to the endoplasmic reticulum (ER) and into the cytoplasm. Intercalating open spaces in the nuclear envelope (NE) are the nuclear pore complexes (NPC). These provide an open structure for rapid transport of proteins and transcripts into and out of the nucleus. The central core of the NPC is the Nup107–160 complex; the nucleoporins Nup153, Nup50 or ELYS are located on the chromatin side, whereas the cytoplasmic NPC is characterised by the presence of Nup214, Nup88 and Nup358 ), which allows separation of sister chromatids after chromosomes have condensed. This process is reversed shortly afterwards (within 30/90 min after NEBD), and the new G1 nucleus re-organises to its original state.Several kinases play a major role in NEBD. Dissociation of chromatin from the nuclear envelope occurs through phosphorylation events on lamins (A/C and B) and lamina-chromatin-associated proteins. The first V-type filaments that disappear from the rim are the A-type lamins in early prophase, a target of CDK1  requires the re-association of all the components that were disassembled during mitosis in a sequential and timely fashion, and therefore, it is also a highly regulated mechanism. Protein phosphatase 1 is recruited to the periphery of chromosomes at telophase directing dephosphorylation of lamin B and promoting its polymerisation  or by the position of the segregating chromatin within the anaphase cell or a combination of both of the above. A topological control of the process appears to occur in drosophila where the position of the chromatin seems to allow or prevent reformation of the NE during mitotic exit , histone modifications, transcriptional machinery interactions and nuclear bodies are all required to ensure proper gene expression programmes. Here, we will discuss how these processes are controlled during the passage throughout mitosis.S. cerevisiae that H3S10ph also leads to deacetylation of H4 thus enhancing the condensed chromatin status  . Mitotic TBP complexes seem to contain phosphatase activity (PP2A) necessary to prevent the condensation of chromatin via the local dephosphorylation of condensin. However, as a general theme, dissociation of transcription activators from chromatin sites is the most common situation, and, although it remains unknown why some genes are kept under the regulation of transcription factors, it is tempting to suggest that this allows genes to be in a potentially active state ready for transcription upon mitotic exit.BRD4 is another transcription-associated protein that recruits the positive transcription elongation factor b (pTEFb) complex for elongation of RNAPII through phosphorylation past the transcription start site (TSS) and onto the coding region of a gene. BRD4 has been shown to associate with mitotic chromatin in some  but not all cell lines (HeLa)  ∼35 % global translational repression of the bulk of mRNAs and (2) ∼200 of mRNAs that show large gene-specific changes in their translation efficiency during mitosis. This latter group encompasses mRNAs in which translation is paused during mitotic entry and resumed upon mitotic exit. An example is that of Emi1, a gene involved in inactivating the APC, which is reduced to very low levels during mitosis in order to allow the activation of APC protein and progression of the cell cycle. When cells exit mitosis, translation of Emi1 is quickly activated. The advantage of this mechanism is the ability of regulating protein levels in a very short period of time compared to transcription, and its rapid reversibility enables protein synthesis to restart quickly when cells exit from mitosis and enter G1 . In early G1, when RNAPII transcription is re-activated, pre-mRNA splicing machinery starts accumulating in the nucleus, including the migration of MIGs  accumulate small nuclear ribonucleoproteins (snRNP), SMN and coilin. They remain associated in mitosis in so-called mitotic cajal bodies (MCB). Upon reformation of the nuclei, MCBs disintegrate, and the components, first coilin and then SMN, start accumulating in the nucleoplasm for the new formation of CB  bodies are thought to regulate post translational processes as they partner with SUMO and ubiquitin ligases. They have also been associated with translocations in cancer. PML bodies remain aggregated during mitosis in mitotic accumulations of the PML protein (MAPPs). Reformation of this nuclear body includes transition from the cytoplasm of its main components, namely SP100 and DAXX  divide compartments into nuclear subdomain containing clusters of multiple regulatory elements tethered by long-range interactions  a protein that exists in the de-phospho (D) and phospho-state (P), (2) a kinase that converts the protein from D to P and (3) a phosphatase that converts the protein from P to D. If either the kinase or the phosphatase is bound to cellular structures, then a gradient of phosphorylation can be established within the cell. This system works both for freely diffusible substrates and for substrates where the diffusion coefficient is dictated by other cellular movements e.g., the movement of the chromatids towards the poles. This model has been shown to exist for Aurora B during mitotic exit where the kinase phosphorylates chromosome substrates that are dephosphorylated by PP1, a chromosome-associated phosphatase at anaphase   and spatially (molecular gradients) regulated. However, just the simple observation of a mammalian cell dividing prompts us to consider that the mechanics of the process may play important roles as well. The movement towards the poles, the invagination and cleavage of the furrow, the spreading of the cells all generate local tensions. Moreover, the reformation of the nuclear membrane and intranuclear structures may well exert a mechanical role in the establishment of the chromosome territories and chromatin organisation in a few hours window after mitosis.This aspect is not yet well studied but there are indications that mechanical forces are important players to be considered in the process. Recent work from Funabiki’s laboratory has shown that drastically changing microtubule dynamics during pronuclear reassembly in Xenopus egg extracts causes the appearance of distorted and irregularly shaped nuclei. The chromatin-associated protein Dppa2 (development pluripotency associated 2) appears to be the regulator of this process. The importance of this mechanical clue in the formation of the G1 nucleus is revealed also by the fact that these nuclei present a delayed and disorganised DNA replication  might be involved in the condensation and silencing of mitotic chromatin. Similarly, ejection of chromatin binding proteins through phosphorylation events might just be the tip of the iceberg with regards to modifications that will need to be reverted for re-establishment of chromatin environments in G1. Reassembly of the nuclear envelope implicates a consortium of synchronised events to ensure the rim formation, nuclear pore assembly and import functions. The physics behind the pushing and pulling forces of a cell undergoing mitosis also remains far from understood. How kinases/phosphatases timely regulate the breakdown of the nuclear envelope, movement of chromatin to distal parts, and even the organisation of chromatin upon the formation of the G1 nucleus remains to be elucidated.The cooperation between the development of new imaging technologies, single cell analyses and mathematical modelling will be essential to understand in the four-dimensional space the pivotal factors which regulate the formation of a structure required for efficient regulation of the nucleus after mitosis is over.


Haloferax volcanii have highly divergent putative TFEβ homologues with unknown properties. Here, we demonstrate that Haloferax TFEβ lacks the prototypical iron-sulphur cluster yet still forms a stable complex with TFEα. A second metal cluster contained in the zinc ribbon domain in TFEα is highly degenerate but retains low binding affinity for zinc, which contributes to protein folding and stability. The deletion of the tfeB gene in H. volcanii results in the aberrant expression of approximately one third of all genes, consistent with its function as a basal transcription initiation factor. Interestingly, tfeB deletion particularly affects foreign genes including a prophage region. Our results reveal the loss of metal centres in Hvo transcription factors, and confirm the dual function of TFE as basal factor and regulator of transcription.The basal transcription factor TFE enhances transcription initiation by catalysing DNA strand-separation, a process that varies with temperature and ionic strength. Canonical TFE forms a heterodimeric complex whose integrity and function critically relies on a cubane iron-sulphur cluster residing in the TFEβ subunit. Halophilic archaea such as  DNA melting and template loading (The RNA polymerase (RNAP), auxiliary factors and molecular mechanisms of transcription are conserved between archaea and eukaryotes. Many Archaea inhabit extreme environments and their enzymes have adapted to cope with conditions of high temperature or salinity, while the underlying changes of protein chemistry enabling this tolerance remain poorly understood. The molecular engineering challenges of initiating transcription in a start site-specific fashion are independent of the type of RNAP and the domain of life. RNAPs need to engage with the promoter region of the double-stranded DNA template, the two DNA strands need to be separated, the template strand loaded into the RNAP active site, and individual NTP substrates bound in a geometry conducive to catalysis. All cellular RNAPs depend on additional basal transcription factors to facilitate this process. In archaea three basal transcription factors  assist RNAP during initiation. TBP and TFB bind to the TATA and BRE motifs and are necessary and sufficient for RNA polymerase recruitment to the promoter and transcription initiation. TFE is not strictly required for transcription  loading .Sulfolobus solfataricus (Sso) TFE is a heterodimer consisting of TFEα and TFEβ subunits (tfeB). Canonical tfeB genes are conserved in all cren- and thaumarchaeal genomes , while the gene is absent altogether from other euryarchaea like Methanocaldococcus jannaschii and Pyrococcus furiosus  Figure . In S. ciability ,11. Impoiability . In addi RNAPIII . Thus, i2+ .Functional studies have provided detailed insights into the structural and mechanistic basis of archaeal TFE function during OC formation ,2,7,8,142+ . The effntration . Similar+ concentrations, which is thought to have caused global changes in the haloarchaeal proteomes such as low isoelectric points . Thi. Thi+ cotfeB gene from Haloferax volcanii (Hvo) does encode a bona fide TFEβ subunit, which does not encompass an FeS cluster and yet still forms a stable complex with TFEα. In addition, the second metal centre of TFE, the TFEα zinc ribbon that is crucial for RNAP binding, is highly degenerate with only a single cysteine residue contributing to zinc binding. This results in a very low affinity for zinc, yet collision induced unfolding experiments demonstrate that zinc binding stabilizes the protein structure. In contrast to Sulfolobus, the Haloferax tfeB gene is not essential for cell viability. The tfeB deletion causes a slow growth phenotype and leads to the transcriptional misregulation of one third of all genes. This substantiates the role of TFE as global regulator in the archaeal domain.Here, we show that the 6-tag fusion, respectively. HVO_1090 coding for Hvo TFEβ and its variant TFEβ Δ1–72 were cloned likewise via NdeI and XhoI restriction sites into vector pET-21a(+) (Merck). Plasmids were transformed into Escherichia coli Rosetta2 (DE3) (Merck) and heterologous expression was carried out in enriched growth medium for 2 h after induction at 37°C. Cells were resuspended in N(500) buffer , disrupted by sonication, cleared by centrifugation and Hvo TFE was isolated by Ni-affinity chromatography.The gene HVO_1174 coding for Hvo TFEα was PCR-amplified and cloned into a modified version of vector pRSF-1b  via NcoITEV was expressed and purified as described above. Ni-affinity elution fractions were dialyzed against 20 mM Tris/HCl pH 8.0, 300 mM NaCl, 5 mM 2-mercaptoethanol in the presence of His-tagged TEV protease overnight. TEV protease was removed by Ni-affinity chromatography and successful cleavage was verified by SDS-PAGE.A TEV-protease cleavage site was introduced between Hvo TFEα and the C-terminal His-tag by the Phusion site directed mutagenesis method (ThermoFisher Scientific). The resulting protein TFEαSize exclusion chromatography was carried out in N(500) buffer with a flow rate of 0.5 ml/min on a Superose 12 hr 10/300 column (GE Lifesciences) with 0.5 ml fractions collected.H. volcanii, isolation of genomic DNA and construction of deletion mutants was carried out as described previously  were resuspended in 250 μl of unbuffered spheroplast solution   on an Agilent 2100 Bioanalyzer. Total RNA concentration was measured using Qubit RNA BR assay kit . 1 μg of total RNA was used for rRNA depletion using Ribo-Zero rRNA Removal Kit (Illumina). Libraries were prepared using the dUTP method  . 750 μl http://www.bioinformatics.babraham.ac.uk/projects/fastqc). Reads were mapped to the Hvo genome using TopHat (Padj < 0.01) (Sequencing reads were trimmed to remove the adaptor parts using Trimmomatic  and the g TopHat  using pa < 0.01) .In order to calculate transcript abundance, we combined an available transcriptome map of Hvo  with recH. volcanii DS2 genome was employed. Aligned promoter sequences were depicted using WebLogo3 with correction for the genome composition (http:/www.R-project.org) with the LSD package v3.0.The same transcription start site mapping data were used for promoter analysis. DNA sequences were extracted using BedTools getfasta  for posiposition . Synonymposition . AT% of position . Data we4 acetate by ultrafiltration. Zn(II)-acetate was added to the samples at 2:1 molar ratio followed by 1 hr incubation at room temperature. All samples were analyzed on a first generation Synapt mass spectrometer  (in-house prepared gold-coated capillaries (borosilicate glass capillaries with dimensions 1.0 mm × 0.78 mm from Harvard apparatus) created using a needle-puller  and coated with gold using a sputter-coater , as described previously (For nESI mass spectrometry (MS) proteins were buffer changed to 0.5 M NHter, UK) . Sampleseviously . The maseviously .TEV in minimal (M9) medium using as nitrogen source 15NH4Cl (CK Isotopes). The protein was purified as described above by Ni-affinity chromatography, TEV protease cleavage and size exclusion chromatography, but the last step was carried out in an NMR compatible buffer .Isotopically labeled samples for NMR studies were prepared by growing BL21 (DE3) cells for heterologous expression of protein TFEα2H2O. Zn2+ was added using a ZnSO4 stock solution diluted to a final concentration of 165 and 190 mM corresponding to ∼1:1 molar ratio for TFEαTEV and TFEαTEV C138S, respectively. 2D 15N–1H HSQC spectra were acquired for each Zn2+ concentration. Data were processed using the NMRPipe/NMRDraw  /NMRDraw .H. volcanii (Hvo) harbours ORFs annotated in the arCOG database as TFEα and β subunit-encoding genes  (tfeA (HVO_1174) and tfeB (HVO_1090) into compatible plasmids for coexpression in E. coli. Either of the two proteins was fused to a C-terminal His-tag in order to facilitate purification using Ni-affinity chromatography. SDS PAGE analysis of the elution fractions revealed that both TFEα and β subunits co-eluted irrespective of which subunit was tagged, suggesting that they form a stable complex. The fractions containing the co-eluting proteins were furthermore subjected to gel filtration  . In orden Figure . The Sson Figure .Halobacterium salinarum have TFEα variants with all four cysteines conserved, others such as Haloquadratum walsbyi TFEα have the same substitutions as in H. volcanii with a single conserved cysteine.FeS clusters are commonly coordinated by cysteine residues, but alternatively arginine, histidine or aspartate residues can substitute for single cysteine residues ,42. In tHvo TFEα/TFEβ, we carried out native nano electrospray-ionization (nESI) mass spectrometry. The native mass spectrum of TFEα/TFEβ-His confirms the formation of a dimeric complex  that allowed the removal of the affinity tag following purification. To prevent zinc loss due to the additional purification steps, we re-incubated the protein with 1 mM ZnSO4 before the buffer exchange into NH4 acetate compatible with nESI MS. The native mass spectrum of TFEαTEV showed two peaks of similar intensity with a calculated mass of 21 425.5 (± 0.4) Da and 21 490.6 (± 0.0) Da corresponding very well with the theoretical mass of TFEαTEV (21 425.65 Da) and an additional zinc ion  experiments. CIU combines activation of proteins by voltage to induce protein unfolding coupled with ion mobility mass spectrometry to detect the folding status  corresponding to the native folded protein, and a slower travelling population (∼25 ms) equivalent to a more unfolded state triggered by a higher voltage  using the quadrupole mass analyzer, and subjected it to CIU using a voltage ramp in the trap collision cell prior to ion mobility separation. Interestingly a significantly higher voltage (∼25 V) was required to convert zinc-bound TFEαTEV into the more unfolded state, indicating that zinc binding indeed exerted a stabilizing effect on TFEα. To gain further insight into the possible structural differences between WT TFEαTEV and the C138S variant we recorded two-dimensional (2D) 15N–1H Heteronuclear Single Quantum Coherence (HSQC) spectra. The HSQC spectra of TFEαTEV and the C138S variant showed good dispersion and superimposed well on each other with a single crosspeak shift between the two spectra that likely corresponds to C138  that the C138S mutation does not cause larger structural perturbation of TFEαTEV.In order to assess whether zinc binding increased the stability of TFEαg status . Ligand-TEV  and therefore its transcriptome will be more comparable to that of ΔtfeB::trpA+ strain H2644 (650 = 0.2) and subjected it to next generation RNA sequencing (RNA-seq). Consistent with the predicted role of TFEβ as a basal transcription factor, the deletion of tfeB led to changes in relative mRNA abundance for 38% of all genes  . Generally, slow growth is associated with reduced ribosome biosynthesis in pro- and eukaryotes (Padj < 0.01). The majority of these genes show increased relative abundance (32 out of 49)  .The Hvo Δin H2644 . RNA iso < 0.01) . Approxikaryotes . This ho) Figure . Therefof arCOGs  in the s) Figure . InterestfeB strain, while none of the remaining genes showed increased relative abundance. In order to test the effect of tfeB deletion on non-native genes more systematically, we plotted the rare codon frequency of genes (defined as codons with less than 10% synonymous codon usage genome-wide) against the log2-fold change in relative transcript abundance  , with pHV4 stably integrated into the chromosome ,47. Intee Figure . Many gee Figure . The sysh cases) .tfeB deletion globally affects the transcriptome, we calculated estimated transcript levels for the Hvo transcription units . In summary, our results suggest that TFEβ preferentially stimulates transcription of laterally transferred (non-native) genes.To get further insight into how s Figure . DownregHalobacterium TfbA, TfbB/D/F, TfbC/G and TfbE)  . Hvo posE motifs . A motifn Figure .tfeB deletion significantly alters the global transcriptome, particularly affecting non-native genes.Overall, our data suggest that Euryarchaeota, several lineages such as the Thermococcales and Methanococcales have lost the tfeB gene and this loss appears to coincide with a monomeric TFEα variant that seems to be functional on its own, as deducted from in vitro experiments  and TFIIEβ, respectively. Similar to TFEβ, the RNA polymerase subunit D in halophilic archaea appears to have lost the structural FeS cluster  in line with the idea that its WH domain interacts with the non-template strand via electrostatic interactions on the RNAP surface during OC formation (I = 4.33). Under the physiological conditions of high salinity found in the cytoplasm of H. volcanii, electrostatic interactions with DNA are disfavoured. In addition, high salinity directly stabilizes dsDNA by counteracting repellent forces within the phosphate backbone of the two DNA strands. While the extreme natural environments of haloarchaea are likely to have driven the adaptation of haloarchaeal TFEβ, different selective pressures might have led to the loss of the Fe-S cluster in its eukaryotic counterparts. This includes the emergence of different regulatory mechanisms for the RNAPII and III transcription machineries early in the evolution of eukaryotes. Another adaptation occurred in the Hvo TFEα subunit where the canonical ZR evolved into a degenerate form retaining only low affinity for zinc. This adaptation is specific to Haloferax and closely related species while the majority of haloarchaeal TFEα retained the four conserved cysteine residues forming canonical ZR domains. Nevertheless, it may be part of a general trend of reduced cysteine content in haloarchaeal proteomes  and functionally interacts with TFE  and the termination factor Rho to suppress transcription of foreign DNA (tfeB strain did not provide evidence for a specific DNA sequence bias in either up- nor downregulated promoters (Bacteria including eign DNA . A rigorromoters , congrueThe accession number for the RNA-seq data reported in this paper is NCBI GEO: GSE101134.Supplementary DataClick here for additional data file.


Shigella is a highly adapted human pathogen, mainly found in the developing world and causing a severe enteric syndrome. The highly sophisticated infectious strategy of Shigella banks on the capacity to invade the intestinal epithelial barrier and cause its inflammatory destruction. The cellular pathogenesis and clinical presentation of shigellosis are the sum of the complex action of a large number of bacterial virulence factors mainly located on a large virulence plasmid (pINV). The expression of pINV genes is controlled by multiple environmental stimuli through a regulatory cascade involving proteins and sRNAs encoded by both the pINV and the chromosome. The primary regulator of the virulence phenotype is VirF, a DNA-binding protein belonging to the AraC family of transcriptional regulators. The virF gene, located on the pINV, is expressed only within the host, mainly in response to the temperature transition occurring when the bacterium transits from the outer environment to the intestinal milieu. VirF then acts as anti-H-NS protein and directly activates the icsA and virB genes, triggering the full expression of the invasion program of Shigella. In this review we will focus on the structure of VirF, on its sophisticated regulation, and on its role as major player in the path leading from the non-invasive to the invasive phenotype of Shigella. We will address also the involvement of VirF in mechanisms aimed at withstanding adverse conditions inside the host, indicating that this protein is emerging as a global regulator whose action is not limited to virulence systems. Finally, we will discuss recent observations conferring VirF the potential of a novel antibacterial target for shigellosis. E. coli, including Shigella, are able to live in complex environments and have evolved intricate control systems. The expression of their pathogenic phenotype is the result of a multifactorial process which requires the synthesis of a large set of virulence determinants that may not be simultaneously needed during all stages of the infection process. These determinants are controlled by global regulatory networks, integrating specific regulators with conserved housekeeping processes.Bacterial pathogens must often survive within fundamentally diverse habitats. Dynamic adaptation to the surroundings depends on the ability to sense environmental variations and to respond in an appropriate manner. This involves drastic changes in the transcriptional program of the cell. The capability to swiftly modulate gene expression requires investment by the bacterium in numerous gene functions that not only allow adaptation to different milieus but also enable the cell to co-ordinately rework its response to mutable conditions  is the synthesis of VirF which acts as an antisilencer, relieving H-NS mediated repression at the virB , for its early effectors and their chaperones (Ipa and Ipg proteins), and for MxiE, the last regulator in the cascade  associated with the expression of virulence  encompassing the RNA polymerase binding site . This temperature-dependence relies on the interaction of H-NS with two binding sites within the virF promoter, spaced by a DNA linker region. The accessibility of the target DNA to H-NS varies significantly with temperature and H-NS is able to recognize its binding boxes only at lower temperature: with increasing temperature H-NS binding decreases, and at a temperature higher than 30°C the virF promoter becomes insensitive to H-NS repression. The position of the two H-NS binding sites on the virF promoter has been investigated in detail  and −1 (binding site I) with respect to the virF transcription start site. Site I overlaps the canonical −35 and −10 regulatory elements, suggesting that this region is involved in the transcriptional repression of the virF gene by H-NS: the interaction of H-NS with DNA may prevent the −35 and −10 boxes to be accessed by RNA polymerase  endowed with decreasing intrinsic DNA curvature. The results of footprinting and in vivo transcription assays using these templates highlight a strict correlation between intrinsic DNA curvature and the capability of H-NS to bind to its target sites and repress virF transcription. Moreover, the reciprocal rotational position of the two H-NS sites appears crucial for the thermoregulation of virF: moving the sites by about a half helix turn further apart produces templates which, despite a comparable overall bending level, are almost insensitive to H-NS repression, likely because the protein can no longer form a tight, transcription-blocking nucleoprotein complex. Temperature does not only affect the amplitude of the DNA bend within the virF promoter but also the position of the bending center, identified by means of circular permutation assays , centered around positions +55 (site I), −1 (site II), −130 , and −200 (site IV), have been identified in the virF promoter region . The synthesis of these nucleosides depends on the products of the tgt  and miaA  genes, respectively. The intracellular concentration of VirF decreases in tgt mutants and in miaA mutants, inducing a low virulence phenotype and an avirulent phenotype, respectively  suggests that some of them may encode factors perturbing the invasive process which likely have been silenced during evolution in order to optimize bacterial fitness within the host. Most of these genes encode yet unknown products. In this respect the speG gene, encoding the enzyme spermidine acetyl transferase (SAT), is an exception. In E. coli, SAT prevents the accumulation of spermidine (and the consequent toxic effects) by catalyzing the conversion of this polyamine into its physiologically inert form, acetylspermidine , whose induction by VirF has been observed only at 37°C , was able to severely affect intercellular spreading and induce a significant inhibition of cell invasion , Sapienza Università di Roma, Consiglio Nazionale delle Ricerche, and Institut Pasteur (PTR-24-16).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


SET gene expression. More studies are required to define the in vivo significance of this mechanism, and specifically, its implication for several benign and malignant diseases related to SET dysregulation.SET (SE Translocation) protein carries out multiple functions including those for protein phosphatase 2A (PP2A) inhibition, histone modification, DNA repair, and gene regulation. SET overexpression has been detected in brain neurons of patients suffering Alzheimer’s disease, follicle theca cells of Polycystic Ovary Syndrome (PCOS) patients, and ovarian cancer cells, indicating that SET may play a pathological role for these disorders. SET transcript 2, produced by a specific promoter, represents a major transcript variant in different cell types. In this study, we characterized the transcriptional activation of human SET transcript 2 promoter in HeLa cells. Promoter deletion experiments and co-transfection assays indicated that ZFX, the Zinc finger and X-linked transcription factor, was able to transactivate the SET promoter. A proximal promoter region containing four ZFX-binding sites was found to be critical for the ZFX-mediated transactivation. Mutagenesis study indicated that the ZFX-binding site located the closest to the transcription start site accounted for most of the ZFX-mediated transactivity. Manipulation of ZFX levels by overexpression or siRNA knockdown confirmed the significance and specificity of the ZFX-mediated SET promoter activation. Chromatin immunoprecipitation results verified the binding of ZFX to its cognate sites in the SET promoter. These findings have led to identification of ZFX as an upstream factor regulating  SET (SE Translocation), also known as Template Activating Factor-1β (TAF-1β) and protein phosphatase 2A inhibitor (I2PP2A), was originally identified as a part of SET-CAN fusion protein generated by chromosomal translocation in the patient SE with acute undifferentiated leukemia . In the Studies have shown that SET is a multitasking protein involved in different physiological processes ,4. ThrouAccumulated data have shown that, through PP2A-dependant and PP2A-independent pathways, SET plays a critical role in the cell cycle regulation and contributes to the development of hematological malignancies and solid tumors, especially gynecologic cancers ,14. In bSET gene regulation is not well understood. Through the use of alternative promoters, SET gene produces four mRNA variants with distinct exon 1 of divergent lengths  of  lengths , leadinghttp://www.cbs.dtu.dk/services/Promoter/). Analysis of SET sequences using the SwitchGear Genomics  pointed to the same TSS as predicted by Promoter 2.0 software. Following PCR amplification, the 994 bp fragment containing the 855 bp promoter and 139 bp 5′ UTR was cloned into the pGL3-Basic luciferase reporter vector. In order to define the proximal sequence essential for the transcriptional control of human SET gene, seven sequentially truncated promoters were examined in transfection assay. As shown in SET gene transcription might be regulated in a cell-specific manner. On the other hand, in both cell lines, further deletion  of the promoter led to a stepwise reduction of promoter activities. A significant reduction was noted to occur between P5 and P8, which corresponds to the region −157/+47. Thus, the 204 bp region harbors strong positive cis-active elements indispensable for the promoter activity of the human SET gene.The transcription start site (TSS) and putative promoter were identified with the Promoter 2.0 Prediction Program  and JASPAR database  were used to predict transcription factor binding sites. Several highly scored binding sites were identified within the 204 bp (−157/+47) region, including those for Sp1, E2F1, E2F3, E2F4, EGR1, and ZFX (p < 0.05). Since E2F3a displayed a similar inhibitory activity in P5, P6, and P7 truncated promoters (p < 0.05), respectively. We elected to focus on ZFX, the factor appeared to account for a large portion of the SET promoter activity.The Genomatix software  were found to be located between −157/−45, which corresponded to the region deleted from P5 to P7. Therefore, co-transfection was performed with increasing amounts of ZFX-expressing plasmid and P5 (pGL3-157) or P7 (pGL3-45) to determine the significance of these ZFX-binding sites. ZFX overexpression following cell transfection was verified on mRNA and protein levels with the use of real-time PCR and Western blotting, respectively A. While To determine the significance of each ZFX-binding site, site-directed mutagenesis was performed . Four plTo confirm the above observation, the mutant promoters were examined in the presence of ZFX overexpression. Results of co-transfection experiments with reporters containing mutant promoters and ZFX expression vector indicated that mutation of the fourth site (Site4) led to a blockade of the ZFX-mediated transactivation C. Taken To determine the ZFX binding to its cognate sequences of Site4 (CAGGCCAATGGCGC), we performed a transient chromatin immunoprecipitation (ChIP) assay following cell transfection with P5 or M4 reporter plasmid. As described under Materials and Methods, PCR primers were designed to encompass the immediate upstream of Site4 and downstream of the luciferase-coding sequence. As shown in # accomplishing 55%–65% reduction of ZFX mRNA and 70%–80% reduction of ZFX protein, and Si-ZFX3# accomplishing 40%–50% knockdown of mRNA and 55%–65% protein (# or Si-ZFX3# readily led to a significant reduction of wild type (P5) promoter activity. However, removal of the ZFX-binding Site4 either by mutation (M4) or deletion (P7) rendered insensitivity to changes of ZFX level. Thus, Site4 appears to play a dominant role for the ZFX-mediated upregulation of SET promoter.To further confirm the key function of ZFX-binding Site4, endogenous ZFX expression was knocked down with specific siRNAs. Four siRNA sequences were tested for ZFX inhibition. Results of real-time PCR and Western blotting demonstrated that two siRNAs effectively reduced the ZFX expression, with Si-ZFX2 protein A. As shoChIP assays were performed to investigate if ZFX was able to bind to the native SET promoter in HeLa cells. The precipitated DNA was subjected to PCR amplification with primers flanking the genomic region (−147/+26) containing the four ZFX-binding sites. As shown in #, a decrease in the DNA band representing ZFX binding to SET promoter was observed. In a control experiment, transfection with a scramble siRNA sequence did not significantly affect the DNA band density when compared to the non-transfection group. Correspondently, an increase in the DNA band was observed when ZFX was overexpressed  cells HL-60 and HEL ,22. SuchZFX contains a large acidic transcriptional activation domain, a nuclear localization sequence, and a C-terminal DNA-binding domain consisting of zinc fingers ,26. As aIn summary, SET transcript 2 is a major transcription variant found in many cell types. We have characterized the SET transcript 2 promoter and demonstrated that the Zinc finger and X-linked factor ZFX transactivates the promoter through binding to a site closely located to the transcription start site. The pathophysiological significance of ZFX-mediated SET activation remains to be investigated. Similarly, the upstream signal that controls ZFX expression needs to be identified. Given extensive involvements of SET in Alzheimer's disease, PCOS, and cancers, identification of ZFX as a potent SET regulator is a meaningful step, and further investigations into the ZFX-SET signaling network may provide useful information for the development of new therapeutic modality against human diseases.2 atmosphere.HeLa and human embryonic kidney (HEK) 293 cells were purchased from the American Type Culture Collection (ATCC). Cell cultures were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum . The medium was supplemented with penicillin and streptomycin (100 units/mL for each antibiotics). Cell cultures were incubated in a humidified incubator conditioned to 37 °C and 5% CO® HS  using the primers F1 and R, which were fixed with the NheI and XhoI sites, respectively  was PCR-amplified with PrimeSTAREectively . The ampectively  with a sectively , and subectively . SequencFour ZFX-specific siRNAs and a RISC-free control siRNA were designed and synthesized by Dharmacon . The efficiency of ZFX-specific siRNAs for ZFX knockdown was assessed with real-time PCR and Western blotting. The designation and target sequences of ZFX-specific siRNAs are documented in Transient transfection with plasmid DNA or siRNAs was carried out in HEK 293 and HeLa cells using the Effectene transfection kit  and the HiPerFect transfection kit , respectively. Cells were seeded in 96-well plates with 100 μL of antibiotics-free medium 24 h before transfection. Following 7 h exposure to transfection reagents, the medium was changed to a regular one. At 24 h post-transfection, cells were lysed and luciferase activity was measured using the Dual Reporter assay system  and the LB960 microplate luminometer. The pGL3-Basic vector was used as a control, while the pRL-TK  plasmid expressing the renilla luciferase was used as an internal reference to standardize the luciferase activity. For dose-response experiment, 0–100 ng of ZFX expressing plasmid (pEF1-ZFX) and 100 ng of promoter reporter plasmid/2.5 ng of pRL-TK plasmid were co-transfected into HeLa cells. Transfection experiments were performed in triplicates. At least three independent experiments were repeated.7 cells were fixed with 1% formaldehyde and sonicated on ice with 15 s bursts that were repeated 15 times with 15 s intervals. Optimization experiments were performed in order to obtain an optimal length of chromatin fragments with 200–1000 bp of genomic DNA . Briefly, approximately 1 × 10omic DNA . Target omic DNA . To asseCt-ΔΔ method. The threshold cycles were determined in triplicates with the use of 7900 Real-Time PCR System . HeLa cells were transfected with either ZFX siRNA or siRNA control using the RNeasy Plus Mini Kit . At 48 h post-transfection, total RNA was isolated from cells and quantified with NanoDrop 1000 spectrophotometry . 1 μg of total RNA was reverse-transcribed in a 20 µL reaction using the High Capacity RNA-to-cDNA Kit  following the manufacturer’s instructions. The resulting cDNA was subsequently amplified using primers P1 to P4  and the 3VO4), and 1 mM phenylmethylsulfonyl fluoride (PMSF), supplemented with 1% Protease Inhibitor Cocktail . Protein concentration was measured with the Bradford Assay. 50 µg of protein was loaded and separated in an 8% polyacrylamide SDS-gel and electrotransferred onto polyvinylidene fluoride membranes. The membranes were incubated with primary antibodies, followed by incubation with matching HRP-conjugated secondary antibodies. Color development was carried out using the ECL reagents . GAPDH was detected and the results were used as protein loading controls. The primary antibodies used were: anti-ZFX  and anti-GAPDH .At 72 h post-transfection, cellular proteins were extracted from HeLa cells with RIPA buffer  containing 5 mM sodium fluoride (NaF), 1 mM sodium vanadate  were calculated for each experimental group. All statistical analyses were performed with the use of SPSS 13.0 Software . One-way analysis of variance (ANOVA) was used to evaluate quantitative data containing more than two groups. Data that passed ANOVA test were further analyzed using the student’s


Retinoblastoma protein (pRB) interacts with E2F and other protein factors to play a pivotal role in regulating the expression of target genes that induce cell cycle arrest, apoptosis, and differentiation. pRB controls the local promoter activity and has the ability to change the structure of nucleosomes and/or chromosomes via histone modification, epigenetic changes, chromatin remodeling, and chromosome organization. Functional inactivation of pRB perturbs these cellular events and causes dysregulated cell growth and chromosome instability, which are hallmarks of cancer cells. The role of pRB in regulation of nucleosome/chromatin structures has been shown to link to tumor suppression. This review focuses on the ability of pRB to control nucleosome/chromatin structures via physical interactions with histone modifiers and chromatin factors and describes cancer therapies based on targeting these protein factors. Retinoblastoma protein (pRB) was the first identified tumor suppressor that negatively regulates the G0/G1 to S phase transition of the cell cycle . The mos MAX gene transcription, MAX-dependent prodifferentiation gene expression, and the subsequent suppression of lung cancer development [ MAX enhancer/promoter regions. It is also known that some SW1/SNF complexes containing Brm and/or BRG1 bind to pRB and repress transcription. The ATPases of SWI/SNF/Brm/BRG1 are involved in chromatin remodeling and the pRB-mediated inhibition of cell proliferation. pRB was reported to recruit Brm or BRG1 through their LXCXE domains, thereby repressing gene expression and effectively inducing cell cycle arrest [The SWI/SNF is a chromatin remodeling protein complex that participates in ATP-dependent histone exchange or removal of histones from DNA, thereby altering nucleosome structure and mobilizing higher-order formation of chromatin . SWI/SNFelopment . In thise arrest , 14. Alte arrest . The cele arrest , 36. Brme arrest , these AHistone deacetylase 1 (HDAC1) is also an important pRB binding protein for the inhibition of gene expression. In addition to the direct inhibition of E2F-mediated transactivation, pRB also recruits HDAC1 to the DNA strands near the promoter region of the E2F-target gene . A canon cyclin E promoter, are reduced when cyclin E is silenced; furthermore, the HDAC inhibitor trichostatin A inhibited the pRB-mediated inactivation of cyclin E expression [Histone acetylation opens the chromatin structure so that transcriptional activators can access the target promoter region and stimulate transcription. On the other hand, HDACs catalyze the removal of an acetyl group from lysine residues in histones and nonhistone target proteins. By reducing acetylation, HDACs facilitate the inactivation of gene expression, including pRB-mediated repression of E2F-target gene expression . A previpression .These studies suggest that pRB regulates the local chromatin structure by recruiting HDAC1 to modulate the balance of histone acetylation levels, and HDAC inhibitors may compromise the tumor suppressive pRB-E2F axis. However, a number of studies showed that HDACs are overexpressed in many human cancer cells , 42. Indα, HP1β, and HP1γ) and each HP1 subtype plays common and also distinct roles in human cells. HP1α is mainly located in heterochromatin, while HP1β and HP1γ are associated with both heterochromatin and euchromatin [β- or HP1γ-bound pRB may repress the euchromatic local promoter region of cyclin E by recruiting Suv39h1. This induces heterochromatin formation by recruiting additional Suv39h1 molecules to methylate the neighboring nucleosomes and produce a tightly packed and inactivated promoter region. Consistently, HP1β was found at E2F-responsive promoter regions when pRB was activated to repress these promoters [γ interaction mediates silencing of E2F-target gene expression and heterochromatin formation during senescence [γ-H3K9me3 is also involved in gene silencing in adult cardiac myocytes, which permanently exit the cell cycle [α to repress the expression of E2F-target genes because HP1α was found to be preferentially phosphorylated in the G2/M phase and to bind to histone H3 modified with both K9me3 and phosphorylated serine 10 in mitotic chromosomes [Histone methylation and demethylation are important modifications of nucleosome/chromatin modifications induced by pRB. pRB interacts with the histone methyltransferase, Suv39h1, which is mainly responsible for trimethylation of H3K9 (H3K9me3), although it can also catalyze dimethylation of H3K9 (H3K9me2) , 46. H3Khromatin , 52. HP1hromatin , 54. In romoters . Furthernescence . pRB-HP1ll cycle . Howeveromosomes . HDAC-meomosomes  and Suv3omosomes , 53, 54.omosomes , 60. ThuLSD1 , 61 and At the onset of cell differentiation, cell cycle-driving gene expression is silenced for the exit from the cell cycle. The active histone marks are removed, while the repressive marks are introduced in target nucleosomes near the cell cycle-driving genes. The H3K4me3 demethylase activity of RBP2 has also been demonstrated to contribute to regulating cell differentiation . Studies in vivo [ RB promoter is highly methylated in many types of human cancer cells [In addition to histone methyltransferase and demethylases, pRB binds to DNA methyl transferase 1 (DNMT1), which associates with HDAC in vivo . pRB for in vivo , 65. Bas in vivo , 66–68. er cells , 69, 70.In addition to the regulation of local nucleosome structures at the pRB-E2F-target promoter region, pRB plays a pivotal role in maintaining whole chromosome dynamics, such as heterochromatin formation and mitotic chromosome segregation. Cells expressing mutant pRB that lacks the LXCXE-interacting domain show abnormal chromatin structures, including decondensed chromatin and display butterfly chromosomes . These apRB participates in the formation and maintenance of heterochromatin structure , 72. As L/ΔLΔ) showed diminished methylation of H4K20 at pericentromeric DNA [The H4K20me3 is enriched at pericentromeric heterochromatin, whereas pRB-deficient mouse fibroblasts show reduced levels of H4K20me3 at pericentromeric heterochromatin . Similareric DNA . Furthereric DNA . Intereseric DNA . Notablyeric DNA . These e Hox genes. PcG proteins regulate the Hox expression pattern required for development [Polycomb group (PcG) proteins were originally identified as repressor complexes forelopment , 79. Recelopment , 81. Twoelopment  to modulelopment . In addielopment . pRB is elopment . A recenelopment . Thus, ppRB facilitates cell cycle arrest and thus influences differentiation and senescence , 86 Fig. Since d RB1L/ΔLΔ MEFs, which are unable to maintain senescence [ RB1L/ΔLΔ MEFs also showed that the pRB binds to promyelocytic leukemia (PML) protein, and the LXCXE-interacting domain in pRB was important for PML-pRB binding to establish constitutive heterochromatin H3K9me3 at E2F-target genes [Cellular senescence can be triggered by repetitive replication (replicative senescence), activation of oncogenic genes (oncogenic senescence), telomere shortening, and genotoxic stresses . Senescenescence . The stuet genes –98. Impoet genes . This reet genes –101. Simet genes . pRB asset genes . Taken tEarly studies demonstrated that pRB is a component of nuclear matrix, which consists of highly compartmentalized and insoluble nonchromatin structures . The nucRecently, pRB was discovered to bind to nuclear matrix apparatus protein NuMA , a mitot Rbf mutant showed abnormal and dispersed chromatin during prophase and prometaphase [ RB1L/ΔLΔ cells displayed an inefficient localization of condensin II on chromosomes, delayed progression to metaphase, and lagging chromosomes in anaphase [ RB1−/− cells and RB1L/ΔLΔ cells correlated with an increase in aberrant replication, mitotic errors, and aneuploidy [ RB1 can produce the same phenotype, suggesting that pRB plays a pivotal role in the maintenance of the chromosome structure and stability via physical interactions with chromatin-related proteins.Condensin II complex is another important factor that highlights the role of pRB in mitotic chromosome dynamics and stability . An initetaphase . Additioanaphase . Moreoveeuploidy . SurprispRB acts as a central tumor suppressor mainly by inhibiting the cell cycle progression driven by E2F-target genes. In this context, the involvement of pRB directs antitumorigenesis via a conformational change in the local promoter region with or without epigenetic marks. In many types of human cancer cells, the levels of pRB and pRB binding nucleosome/chromatin-related proteins that act cooperatively with pRB, such as HDACs , PML 9898, and BL/ΔLΔ, a mutant pRB lacking the LXCXE-binding cleft, enhances tumorigenesis and genomic instability in mouse tumor models [The increase in chromosomal instability due to dysfunctional pRB binding may be related to cancer development in the light of the normal pRB role in maintaining the global nucleosome structure and chromosome organization. Indeed, NuMA is overexpressed in colorectal and breast cancer , 115–118r models . All of  RB1 gene expression is repressed via promoter DNA methylation. The proteasome-dependent degradation of pRB promoted by ubiquitin ligase Mdm2, which was the first identified ubiquitin ligase for p53 [Overall, this review focuses on the physical contribution of pRB, which controls local nucleosome structure and whole chromosome organization. pRB inactivation results in dysregulated cell proliferation, apoptosis, differentiation, and senescence, and all those defects can lead to tumorigenesis and cancer progression . Phospho for p53 , 120, is for p53 –126. The for p53  and Nutl for p53 –130. The for p53 . A recen for p53 . These rTargeting enzyme activities related to nucleosome histone modification may be a potent strategy for cancer therapy. Despite its repression of E2F-target gene expression, HDAC is overexpressed in many human cancers, and a number of HDAC inhibitors, including trichostatin A and vorinostat ), are antitumor agents . SAHA wa in vitro inhibitory potency against Suv39h. LSD1 inhibitors, including the small molecules GSK2879552 and ORY1001, have been developed [Chaetocin was the first discovered inhibitor for drosophila histone methyltransferase Su(var)3-9, and it selectively inhibits human Suv39h1 . BIX0129eveloped . A screeeveloped . Studieseveloped . Compouneveloped .Thus, the continued development of inhibitors of CDK4/6 and histone modifiers aims to eradicate cancer cells. Several agents showed sufficient potency in clinical trials. However, selective inhibitors or activators that target the interaction between pRB and its binding proteins during nucleosome/chromatin organization have not been identified. It is understandable that development of such agents, such as LXCXE-binding inhibitors, is difficult because pRB and its LXCXE-dependent interactions have central and diverse functions in living cells. To increase antitumor effectiveness, treatment with a combination of CDK4/6 inhibitors and inhibitors of histone modifiers could inhibit cell cycle progression and induce apoptosis via structural changes in the nucleosome/chromosome. A greater understanding of the direct role of pRB role in chromatin remodeling or chromosome organization will facilitate the development of antitumor agents and therapeutics for pRB-inactivated human cancers.


The interest of scientists for the transcription of the eukaryotic genome started four decades ago with the purification of the three RNA polymerase (Pol) enzymes. Pol I, II and III transcribe different species of RNA with the help of several general/basal transcription factors (TFs). Messenger RNA (mRNA) transcription by Pol II has been intensely studied owing first to the protein-coding function of mRNA and also to the apparent complexity of this system compared to the transcription of ribosomal RNAs by Pol I or small RNAs by Pol III. Indeed, it was shown very early that mRNA transcription required, apart from Pol II, additional enzymatic activities that didn’t seem to be needed in the Pol I or III systems. Surprisingly enough, all these activities reside in TFIIH, a multi-subunit transcription/repair factor that is involved, through its CDK7 and XPB subunits, both in the phosphorylation of the carboxyl-terminal domain of the largest Pol II subunit and in the ATP-dependent opening of the promoter around the transcription start site, respectively . Consequin vitro helicase activity of XPB appeared very low [Recent results contradicted the above Pol II model and have deeply modified our view of transcription initiation in eukaryotes. First of all, in contrast to its ATPase activity that is robust, the very low . Rather,very low .Moreover, technical advances in our capacity to localize transcription factors inside the preinitiation complex (PIC) have revealed that XPB was binding to the promoter DNA downstream from the transcription start site, a location that was not compatible with its role in promoter opening . In additripterygium wilfordii, which binds XPB and inhibits its ATPase activity [in vivo. Through the inhibition of the ATPase activity of XPB, Triptolide and Minnelide probably inhibit the translocation of XPB downstream of the transcription start site thereby disrupting the release of the transcriptional block imposed by XPB itself. Similar observations concerning the involvement of XPB in basal transcription have been made in the last months [The new regulatory step described above depends on the ATPase activity of XPB and is probably the target of the very promising anti-cancer compound Minnelide. This small molecule is a synthetic prodrug of Triptolide, a diterpenoid epoxide endogenously produced by the thunder god vine, activity . Minneliactivity . Severalactivity , validatt months , 6. They


Small Auxin-Upregulated RNA (SAUR) genes encode growth regulators that induce cell elongation. Arabidopsis contains more than 70 SAUR genes, of which the growth-promoting function has been unveiled in seedlings, while their role in other tissues remained largely unknown. Here, we focus on the regulatory regions of Arabidopsis SAUR genes, to predict the processes in which they play a role, and understand the dynamics of plant growth.SAUR10-clade: SAUR8, SAUR9, SAUR10, SAUR12, SAUR16, SAUR50, SAUR51 and SAUR54. Overexpression analysis revealed that the different proteins fulfil similar functions, while the SAUR expression patterns were highly diverse, showing expression throughout plant development in a variety of tissues. In addition, the response to application of different hormones largely varied between the different genes. These tissue-specific and hormone-specific responses could be linked to transcription factor binding sites using in silico analyses. These analyses also supported the existence of two groups of SAURs in Arabidopsis: Class I genes can be induced by combinatorial action of ARF-BZR-PIF transcription factors, while Class II genes are not regulated by auxin.In this study, we characterized in detail the entire SAUR10-clade genes generally induce cell-elongation, but exhibit diverse expression patterns and responses to hormones. Our experimental and in silico analyses suggest that transcription factors involved in plant development determine the tissue specific expression of the different SAUR genes, whereas the amplitude of this expression can often be controlled by hormone response transcription factors. This allows the plant to fine tune growth in a variety of tissues in response to internal and external signals.The online version of this article (10.1186/s12870-017-1210-4) contains supplementary material, which is available to authorized users. SAURs revealed their general capacity to induce cell elongation and growth, whereas knock-out studies yielded little results, probably due to a high level of redundancy amongst the 79 Arabidopsis SAUR genes 11–13 denotes the number of nucleotides in between. We tested this motif with the three different spacing variants, and found a distinct over-representation of this motif in the Class I (auxin-induced) promoters. The fraction of Class I promoters that contained the motif was 0.22 , while this fraction was 0 for Class II promoters (0 out of 20) and 0.07 for the control group. Both the difference between Class I and Class II promoters (p~0.02) and between Class I and control group promoters (p~0.02) was significant. Variations on this combined consensus motif have been published  and only in 2 out of 20 Class II SAURs; the latter being similar to the occurrence of 56 in 500 randomly chosen promoters. The difference between regulated and not-regulated sequences is significant (p = 0.0003). Hence, the PIF5-like motif CAxxxxCATGTG is clearly more often present in the Class I SAURs than in Class II SAURs and control promoters. The alternative CA[AC]xxAxGTG  [SAUR10-clade SAURs are regulated by PIFs. Moreover, in a study by Hornitschek et al. [SAUR10 and SAUR16 are the only SAURs that are significantly down-regulated in pif4pif5 seedlings, and only SAUR10 is down-regulated in pif5 single mutant seedlings. It would be interesting to study the role of the CAxxxxCATGTG motif in the SAUR10 promoter for its PIF5-dependent regulation. The identification of a longer motif here may point to the binding of a second transcription factor in close proximity to the PIF. Our in silico analysis underlines the importance of the PIFs for SAUR regulation in general, but also reveals differences between the group of auxin-induced SAURs and the group of non-auxin regulated SAURs. Except for some enrichment of A/T-rich stretches in the SAUR promoter sequences, no other binding sites were identified that were significantly over-represented in the SAUR promoters compared to the control promoters.All PIFs bind preferentially to G-boxes (5′-CACGTG-3′) , and PIFd SAUR51 , as welld SAUR51   is only present in the promoters of SAUR10, SAUR16 and SAUR50, while SAUR12 lacks an ARF binding site , but whether these concern really functional binding sites and whether this spacing is important remains to be investigated.That reported , 19, andreported  showed tet genes , 41. Theh et al. , suggestSAURs are not only expressed in seedlings, but have specific expression patterns throughout the plant and appear to a large extent developmentally regulated. Furthermore, many SAUR genes respond dynamically to different hormones. In addition to auxin and BR, SAURs can positively or negatively respond to ethylene [SAUR transcripts and SAUR proteins  and cytroteins ; R:FR ratio = 30.1). Reduced R:FR conditions were achieved by supplemental FR (730 nm) irradiation, resulting in a PAR of 83.5 μmol m−2 s−1, and a R:FR ratio of 1.15. Blue light reduction was achieved by filtering the light through a layer of LEE Medium Yellow 010 filter, resulting in a PAR of 70.4 μmol m−2 s−1, and containing 1.06789E-02 W/(sqm*nm) irradiance in the blue light spectrum (400–500 nm), compared to 3.84107E-01 W/(sqm*nm) under control conditions.The overexpression and reporter lines were generated in the Col-0 background. SALK  and JIC SAUR8, SAUR10 and SAUR16 were amplified from genomic DNA  medium, 10% sucrose, 0.9% agar) and incubated in liquid 2.2 g/l MS medium with hormones or in mock (control) medium for 4 h. The following hormone concentrations were used: 5 μM IAA . Class I (SAURs induced by auxin) and Class II (SAURs not regulated by auxin) genes were selected based on Additional file SAURs, a background promoter set was used. This consisted of 500 randomly selected Arabidopsis promoter sequences (excluding the SAUR promoters).Motif searches were performed with 3 kb upstream promoter sequences obtained using the Bulk Data Retrieval tool from TAIR, based on TAIR10 . These were converted to MAST format and motif hits were obtained in the sequence datasets using the MAST tool from the MEME package. Searches for consensus patterns were performed using ps_scan [In addition to the de novo motif search, a set of potentially relevant existing motifs were obtained from literature , 51 and  ps_scan . To test ps_scan .Additional file 1: Figure S1.UPGMA tree of the Arabidopsis SAUR proteins. (PDF 1406 kb)Additional file 2: Table S1.SAURs to auxin and BR. Table S2. Classes of SAUR genes based on Additional file 2: Table S1. Table S3. Primer sequences. (XLSX 38 kb)Overview of published data showing the response of the individual Arabidopsis Additional file 3: Figure S2.Analysis of the T-DNA insertion lines. (PDF 209 kb)Additional file 4: Figure S3.pSAUR:GUS lines. (PDF 869 kb)Expression patterns of the Additional file 5: Figure S4.Dynamics of IAA/BR induction (PDF 132 kb)Additional file 6: Figure S5.SAUR10-clade genes to GA application. (PDF 196 kb)Response of Additional file 7: Figure S6.The location of GUS expression does not change after IAA-BR treatment. (PDF 373 kb)