PU.1 are rare in human acute myeloid leukaemia (AML), they are common in murine models of radiation-induced AML, and PU.1 downregulation and/or dysfunction has been described in human AML patients carrying the fusion oncogenes RUNX1-ETO and PML-RARA. To study the transcriptional programmes associated with compromised PU.1 activity, we adapted a Pu.1-mutated murine AML cell line with an inducible wild-type PU.1. PU.1 induction caused transition from leukaemia phenotype to monocytic differentiation. Global binding maps for PU.1, CEBPA and the histone mark H3K27Ac with and without PU.1 induction showed that mutant PU.1 retains DNA-binding ability, but the induction of wild-type protein dramatically increases both the number and the height of PU.1-binding peaks. Correlating chromatin immunoprecipitation (ChIP) Seq with gene expression data, we found that PU.1 recruitment coupled with increased histone acetylation induces gene expression and activates a monocyte/macrophage transcriptional programme. PU.1 induction also caused the reorganisation of a subgroup of CEBPA binding peaks. Finally, we show that the PU.1 target gene set defined in our model allows the stratification of primary human AML samples, shedding light on both known and novel AML subtypes that may be driven by PU.1 dysfunction.Transcriptional dysregulation is associated with haematological malignancy. Although mutations of the key haematopoietic transcription factor  Disturbance of these networks can lead to malignant transformation, for example, RUNX1 and its binding partner CBFB control multiple aspects of normal haematopoiesis, but fusion proteins such as RUNX1-ETO or CBFB-MYH11 can cause acute myeloid leukaemia (AML).3 PU.1 was recently shown to contribute to the early establishment of haematopoietic transcriptional programmes,4 and is known to have a key role during foetal haematopoiesis.6 In the adult, PU.1 functions as a major determinant of both myeloid and B-cell lineages8 with its effect often modulated by interactions with other TFs such as CEBPA,9 GATA110 and c-JUN.11 More recently, PU.1 has been identified as a major regulator of cell cycle both in blood stem cells and during myeloid maturation.13PU.1 is an ETS-family TF that has multiple roles in normal haematopoiesis. Structurally, it consists of a transactivating region, a middle PEST region involved in protein–protein interactions particularly with the IRF TFs and a C-terminal DNA-binding domain.Pu.1 cause lethality either in utero5 or soon after birth,14 whereas partial knockdown to levels of 20% by deletion of the Pu.1-14 Upstream Regulatory Element is compatible with foetal life, but causes AML within 3–8 months. This had led to the theory that residual PU.1 activity is required to promote leukaemogenesis.15 However, an alternative model with somatic Pu.1 deletion causing a complete loss of protein also causes AML,16 and Pu.1 hypomorphic mutants are frequently observed in radiation-induced mouse models of AML.20 This suggests that any loss of PU.1 below a certain threshold may be sufficient for leukaemogenesis;21 however, a recent report suggests that some PU.1 function/levels are needed to maintain stem cell function of leukaemic cells.22Loss of PU.1 in murine models can cause AML, but the precise relationship and mechanism remains unclear. Germ-line knockouts of PU.1 and Upstream Regulatory Element mutations have been recorded in human AML, they are much rarer than in mice.27 Better described are mechanisms suppressing PU.1 function, such as disruption of PU.1 transactivation activity by RUNX1-ETO,11 or interference with PU.1 binding (including at its own promoter site) by PML-RARA.28 Importantly, there has as yet been no specific investigation into the transcriptional programmes associated with the loss of PU.1 activity in AML.Although cases of de novo binding of wild-type PU.1 has the capacity to recruit CEBPA to a subset of new sites. Finally, we show that the PU.1 target gene set in our model can be utilised to stratify primary human AML samples, shedding light on both known and novel AML subtypes that may be driven by PU.1 dysfunction.To investigate the effects of restoring wild-type PU.1 to a PU.1 mutant leukaemia model, we developed an inducible PU.1 system, and showed that the induction of wild-type protein causes transit from the leukaemic state to monocytic differentiation. Using chromatin immunoprecipitation (ChIP)-Seq, we produced genome-scale maps of DNA binding by PU.1, the associated TF CEBPA and the H3K27Ac histone mark before and after PU.1 induction, and complemented these with gene expression profiling data. Unexpectedly, mutant PU.1 was bound to a large number of genomic regions, but induction of wild-type PU.1 resulted in a substantial expansion of binding sites, a subset of which was associated with elevated histone H3K27 acetylation, which in turn correlated with increased expression of nearby target genes. Our results also show that M asparagine, 2 mM L-glutamine and 50μM beta-mercaptoethanol. Cells were partially adherent requiring trypsin treatment for passage, and were maintained at 3–10 × 105 cells/ml.X18.1.1 cells were maintained in high glucose RPMI 1640 supplemented with 10% foetal calf serum, 1% penicillin/streptomycin, 300 μGenomic DNA was purified by phenol-chloroform extraction. RNA was extracted using Tri-Reagent  and then treated with DNase I , following the manufacturer's specifications. For DNA sequencing, exonic fragments of endogenous Pu.1 were amplified by PCR and cloned into the PGEM-T Easy vector . Both strands of all five exons were sequenced and compared with the Pu.1 sequence of reference (obtained from Ensembl). Integration of PuER was confirmed by PCR using primers encompassing the Pu.1-ERTM fusion region. All primers used are listed in M.X18.1.1 cells were transduced with PuER or empty vector (EV) control using retrovirus produced with Psi-Eco Retrovirus packaging vector  in 293 T cells. Cells were infected by centrifugation in the presence of polybrene and then selected with 0.5 μg/ml puromycin. Clonal cell lines were obtained by limiting dilution and further expanded. 4-Hydroxytamoxifen (OHT) (Sigma) inductions were carried out at 100 nFlow cytometry was performed on a BD LSRFortessa  cell analyser using the following antibodies: CD11b  and F4/80 . Cell proliferation was assayed by counting live cells following Trypan Blue exclusion.M TriS pH7.4, 150 mM NaCl, 1% NP40, 0.25% Na deoxycholate, 1 mM EDTA) was run on a 12% sodium dodecyl suphate polyacrylamide gel, and transferred to polyvinylidene fluoride membrane by overnight wet blotting. Membranes were probed using primary antibodies against ERα , PU.1  and β-actin .Protein lysate in modified RIPA buffer , CEBPA , H3AcK27  and rabbit IgG . Library construction was performed using the Illumina TruSeq DNA Sample Kit  according to the manufacturer's instructions. Sequencing was performed on the Illumina HiSeq 2000 platform. Reads were mapped to the mm10 mouse reference genome using Bowtie2.30 Mapped reads were converted to density plots and displayed as UCSC genome browser custom tracks, and peaks called using MACS2.31ChIP was performed as previously described32 ChIP-Seq peak coordinates were combined between PU.1− and PU.1+ conditions for each TF ChIP, and peaks overlapping by at least 1 bp were merged. Coverage scores were counted using the intersectBed function for each merged peak region, and then normalised for peak length and total read counts (per 1 million reads). For H3K27Ac, read coverage regions were extended to 800 bp, and then normalised as above.Using BEDTools,lumi package.33 Data were quantile-normalised, and differentially expressed probes identified using the limma package.34 Probes with adjusted P-value⩽0.05, and fold change>1.2 were considered significant.Triplicate samples were amplified using TotalPrep 96-RNA amplification kit  and hybridised to MouseWG-6v2 microarrays (Illumina) by Cambridge Genomic Services. Raw data were filtered in R to remove any probes with detection call ⩾0.01 in all samples, and then transformed by variance-stabilising transformation in the ggplot2.35Normalised ChIP-Seq read counts were combined into one matrix and used for subsequent differential score calculations and correlation analyses, as well as integration of ChIP-Seq and gene expression data. All analysis was performed in R with plots created using De novo motif analysis on the central 80 bp peak regions was performed using Homer.36 Only significant motifs  with ⩾5% peak coverage were reported. Geneset enrichment analyses were performed using Enrichr.37 ChIP-Seq peaks were compared with those of primary macrophages (GSM537983)36 using the Codex webtool.38 PU.1 target genes were used for unsupervised analysis of published expression arrays  in R wiwww.ncbi.nlm.nih.gov/geo) and assigned the identifier GSE63317.The raw and processed data from the ChIP-Seq and microarray experiments reported in this publication have been submitted to the NCBI Gene Expression Omnibus 43 or a control EV within an MSCV-puro retroviral packaging system . Finally, RNA for gene expression microarrays was prepared from the same experiment, to link alterations in TF occupancy and/or histone modification levels with transcriptional changes  (n=1734) defined by (i) ⩾fourfold increase in PU.1 read count, (ii) ⩾fourfold increase in H3K27Ac read count and (iii) H3K27Ac read number ⩾50 per region. For comparison, we looked at peaks with ⩾fourfold increase in PU.1 but unchanged H3K27Ac , and those with no change in PU.1   , indicat<0.0001) . On the  n=2761) . Of noteII peaks .46 our data are consistent with tissue-specific functions of PU.1 being largely mediated by enhancer region binding,36 and shows that these binding events may have an important role in the phenotypic switch to monocytic maturation.Analysis of the genomic distribution of PU.1-binding peaks revealed a marked enrichment of the peaks with increased read counts for intra- and intergenic localisation  compared with those with no read-count change (65.9 for Group III). Even though the association of peaks to genes is complicated by the increased recognition of long range promoter-enhancer interactions,P-value of <0.05 and compared the overall change of expression for each of the three groups previously defined. Not only were many of the genes that changed expression associated with Group I peaks (96 out of 284), but also genes associated to Group I showed the most increased expression following PU.1 induction, compared with Group II and Group III peaks   . This coTo further examine the relationship between PU.1-binding events and gene expression, we took the 96 gene overlap of the 1282 genes mapped from the PU.1 Group I peaks with the 284 genes that showed increased expression following PU.1 induction . Gene on39  and unchanged CEBPA binding  were selected for comparison. The small number of Group II peaks indicates that the vast majority of CEBPA peaks with increased binding after PU.1 induction were associated with H3K27Ac change, and are likely to be functionally relevant for gene transcription , when compared with Group II and Group III (90.4% and 86.5% respectively) . CompariDe novo motif analysis of the PU.1 peak sequences showed CEBPA motifs overrepresented in all three groups of PU.1 peaks defined in the previous section. However, the percentage of regions with CEBPA motifs was higher in Group I PU.1 peaks (36.0%), compared with either Group II (26.4%) or Group III (26.1%) ( (26.1%) . This obCo-binding between peak sets showed that 355/1734 (20.5%) of the Group I PU.1 peaks were at sites of pre-existing CEBPA, a proportion no higher than that of the  Group II and III PU.1 peaks (1331/5056 (26.33%) and 1517/2761 (54.94%), respectively). This does not support a significant role for CEBPA in marking sites for subsequent functional PU.1 binding.We then investigated the alternative possibility that Group I PU.1 peaks bind to new genomic regions and recruit subsequent CEBPA binding. Of the 405 Group I CEBPA peaks identified, 355 (87.7%) were bound at Group I PU.1 peaks; in comparison, none (0.0%) of them were co-bound by PU.1 Group III peaks . This su40  of the t translocations and 20/35 (57.1%) of the t translocations , suggesting a separate class of cases where high PU.1 levels direct a degree of monocytic differentiation, but alternative mechanisms are responsible for the leukaemia phenotype  AML cell line.54It therefore appears that a proportion of normal CEBPA-binding events are impaired by the absence of PU.1 in the leukaemic phase, which may contribute to the differentiation block seen in PU.1-dysregulated leukaemias. Our findings are consistent with a non-leukaemic PuER model of Pu.136 and associated with elevated H3K27 acetylation.55 These observations are therefore consistent with the notion that introducing wild-type PU.1 into this leukaemic model activates a transcriptional programme related to normal cell maturation, overcoming the differentiation block. Our findings not only provide a clearer understanding of the mechanism for the role of PU.1 suppression in AML, but also identify activation of PU.1 as a potential therapeutic target for differentiation therapy in human AML.PuER induction caused both quantitative and qualitative differences in binding between wild-ype and mutant PU.1, and induced PU.1-binding sites with a concomitant increase in histone H3K27 acetylation were primarily localised to enhancers. Cell-type-specific binding of PU.1 in non-leukaemic cells had previously been reported to largely be enhancer-specific,PU.1 mutations are uncommon in human AML,56 upstream mechanisms causing reduced PU.1 expression and function are well described for the fusion genes RUNX1-ETO11 and PML-RARA,57 as well as by FLT3-ITD mutations.59 Analysis of primary AML samples using the set of 96 PU.1 target genes, clustered the majority of RUNX1-ETO and PML-RARA samples together in a group characterised by low PU.1 expression. The presence of other AML cases in this cluster suggests that other as yet unidentified pathways may converge on PU.1 under-expression, giving rise to a common molecular defect.Although 60 it is difficult to see at present how genetic targeting will achieve the efficiency required to target all leukaemic cells within a patient for effective differentiation. As ATRA has proved in PML-RARA-driven AML,61 pharmacological interventions on the other hand represent a potent method of overcoming differentiation block and treating the leukaemia. Interestingly, PU.1 induction has recently been demonstrated by intravenous cytokine activation,62 and it may be that a small molecule-based therapeutic to upregulate PU.1 and alter downstream target expression will have clinical potential to treat PU.1-dysregulated AML.Although genome reprogramming technologies are already showing potential in manipulating TF expression to alter phenotype,


The histone variant H3.3 plays a critical role in maintaining the pluripotency of embryonic stem cells (ESCs) by regulating gene expression programs important for lineage specification. H3.3 is deposited by various chaperones at regulatory sites, gene bodies, and certain heterochromatic sites such as telomeres and centromeres. Using Tet-inhibited expression of epitope-tagged H3.3 combined with ChIP-Seq we undertook genome-wide measurements of H3.3 dissociation rates across the ESC genome and examined the relationship between H3.3-nucleosome turnover and ESC-specific transcription factors, chromatin modifiers, and epigenetic marks.Our comprehensive analysis of H3.3 dissociation rates revealed distinct H3.3 dissociation dynamics at various functional chromatin domains. At transcription start sites, H3.3 dissociates rapidly with the highest rate at nucleosome-depleted regions (NDRs) just upstream of Pol II binding, followed by low H3.3 dissociation rates across gene bodies. H3.3 turnover at transcription start sites, gene bodies, and transcription end sites was positively correlated with transcriptional activity.H3.3 is found decorated with various histone modifications that regulate transcription and maintain chromatin integrity. We find greatly varying H3.3 dissociation rates across various histone modification domains: high dissociation rates at active histone marks and low dissociation rates at heterochromatic marks. Well- defined zones of high H3.3-nucleosome turnover were detected at binding sites of ESC-specific pluripotency factors and chromatin remodelers, suggesting an important role for H3.3 in facilitating protein binding. Among transcription factor binding sites we detected higher H3.3 turnover at distal cis-acting sites compared to proximal genic transcription factor binding sites.Our results imply that fast H3.3 dissociation is a hallmark of interactions between DNA and transcriptional regulators.Our study demonstrates that H3.3 turnover and nucleosome stability vary greatly across the chromatin landscape of embryonic stem cells. The presence of high H3.3 turnover at RNA Pol II binding sites at extragenic regions as well as at transcription start and end sites of genes, suggests a specific role for H3.3 in transcriptional initiation and termination. On the other hand, the presence of well-defined zones of high H3.3 dissociation at transcription factor and chromatin remodeler binding sites point to a broader role in facilitating accessibility.The online version of this article (doi:10.1186/1756-8935-7-38) contains supplementary material, which is available to authorized users. The basic unit of chromatin is the nucleosome, which consists of 147 bp DNA wrapped around histone proteins. Incorporation of histone variants into chromatin critically influences the properties of nucleosomes that play important roles in regulating transcription and epigenetic memory. Among three major histone H3 variants, H3.1, H3.2, and H3.3, H3.3 marks important DNase I hypersensitive sulators . The incsulators , 7.H3.3 plays important regulatory roles in both transcriptional activation and repression. Upon gene activation, H3.3 is incorporated into transcription start sites (TSSs) and coding regions of genes, where it continues to be deposited even when transcription ceases , 9.Histone marks that are associated with gene activation, such as acetylation marks and H3K4me3, are typically found on H3.3 whereas marks associated with gene silencing such as H3K27me3 and H3K9me3 are predominantly found on H3.1 and H3.2 . The preHistone and nucleosome turnover have been studied extensively in yeast, where a single histone variant H3.3 is expressed. More recently, we reported genome-wide histone H3.3 turnover in mouse embryonic fibroblasts and showed that the dynamics of histone turnover appear to be largely conserved among eukaryotes . Our resEmbryonic stem cells (ESCs) are characterized by their pluripotent differentiation capacity that is manifested by a unique chromatin structure. ESCs carry an open chromatin configuration with a large number of bivalent genes that are marked by both active and repressive marks, which resolve upon lineage commitment –16. UnliGenome-wide histone turnover has traditionally been studied using inducible expression systems that include epitope-tagged histones or, alternatively, metabolically labeled histones that are immuno-precipitated by ChIP and subsequently subjected to deep sequencing techniques. TET-inducible histone expression combined with ChIP-Seq typically result in initial increases, followed by decreases in read density over many genomic regions due to read coverage limitations , 18, 19.In this study, we set out to examine histone dynamics by measuring the dissociation rates of previously incorporated histones. To this end, we generated a Tet-OFF ESC line that expresses HA/FLAG-tagged H3.3 and allowed us to track the dissociation of H3.3 upon TET-induced inhibition of H3.3 expression. Using an analytical approach that quantifies the dissociation rates of H3.3 in high resolution, we find that the H3.3 dissociation rate from chromatin varies distinctly across the ESC genome.Our analyses identified distinct dynamics of H3.3 as they relate to transcriptional regulation and heterochromatin integrity. Transcription initiation and end sites are marked by very high dissociation rates of H3.3, that correlate well with gene activity. In contrast, inside coding regions H3.3 equilibrium levels are low and display low turnover. H3.3 dissociation rates were found to differ greatly depending on post-translational modifications with higher dissociation rates associated with active marks and lower rates at heterochromatic marks. On the other hand, binding sites of pluripotency factors and chromatin remodelers were marked by invariably high turnover rates. Surprisingly, we found that distant-acting regulatory sites are dynamically regulated by higher rates of H3.3 dissociation than we found at proximal regulatory sites. Therefore we conclude that distinct H3.3 turnover rates provide a molecular mechanism to accommodate chromatin architecture, transcription as well as accessibility of ESC specific transcription factors and chromatin remodelers.out) ), with the expression cassette integrated at the ROSA26 locus [), with tR20), witequ). Addition of DOX resulted in the rapid downregulation of HA-H3.3 after 6 h and HA-H3.3 levels continued to decline over a time course of 48 h  as the change of reads from time point 0 h to time point 6 h. We validated the ChIP-Seq read enrichment and decline of HA-H3.3 at two control regions by ChIP-PCR. ChIP-Seq profiles revealed high enrichment of HA-H3.3 at the 3’UTR of Rps19 and depletion of HA-H3.3 at an intergenic region on chromosome 8. ChIP-PCR experiments confirmed enrichment at Rps19, which declined over the time course of DOX addition    (Pearson’s correlation coefficient (PCC) = 0.712, 0 bp , suggesting that distinct modes of H3.3 deposition and eviction exist at coding regions and transcription start and end sites.H3.3 equilibrium levels and dissociation rates were equal at exon-intron junctions relative to other regions within the coding region and therefore H3.3 exchange may not be involved in splicing processes.Extragenic transcription by RNA Pol II generates various types of short and long non-coding RNAs. Seventy percent of non-coding RNAs are represented by short enhancer-derived RNAs (eRNAs) that are transcribed in both directions , 22. At Within a 10 kbp interval of the genic region, the highest Pol II peaks are detected within a 1 kbp region around TSSs and are positively correlated with gene expression levels. We then compared our measurements of H3.3 dissociation with RNA Pol II enrichment across the direction of transcription. The highest enrichment levels of H3.3 around Pol II binding sites are measured exactly at the binding peak of RNA Pol II. Interestingly, the peak of highest dissociation occurred around 50 to 100 bp upstream of the TSSs, which is typically nucleosome-depleted and located between the -1 and +1 nucleosome  is a complex of ATP-dependent chromatin remodelers, histone methylases, demetylases, and deacetylases . The NuRH3.3 is a carrier of various post-translational histone modifications that influence gene transcription. To test if histone modifications affect nucleosome stability, we examined H3.3 dissociation rates at sites of several histone modification domains.We detected the highest levels of H3.3 deposition at sites associated with active histone marks including H3K4me1, H3K4me3, H3K9ac, and H3K27ac . In all cases of individual transcription factors, satellite repeats in the non-coding region showed a higher (more than two-fold) enrichment than the other repeat elements. Specifically, the number of Klf4, Esrrb, STAT3, Oct4, and Sox2 binding sites are 14.3%, 10.3%, 10.3%, 7.3%, and 7.8%, respectively, and only 2.1%, 4.8%, 4.6%, 2.8%, 3.2%, respectively, in other non-genic repeat elements  are characterized by their pluripotent differentiation capacity that is manifested by a unique hyper-dynamic chromatin architecture . The hiscis and trans factors are thought to determine nucleosome positioning and exchange [Active gene promoters have a highly phased structure of nucleosomes that are dynamically exchanged to allow assembly of the transcriptional machinery . Nucleosexchange , 38. ChrUnexpectedly, we found that non-genic regions of highly dynamic H3.3 within 1 kbp from the nearest Pol II binding site overlap tightly with chromatin remodelers. The presence of H3.3 at these RNA Pol II flanking sites is curious and may mark TESs of non-coding RNAs.H3.3 is a carrier of various histone modifications, but is mostly modified with active histone modifications . In lineOur analysis of binding sites of ESC specific factors, chromatin remodelers, and structural chromatin organizers revealed that, regardless of H3.3 equilibrium levels, H3.3 dissociation is invariably high at protein factor binding sites and drops sharply where these factors are absent. Hence, high H3.3 dissociation is a better predictor of protein factor binding than is H3.3 enrichment. At satellite repeats we detected substantially higher H3.3 dissociation than we did at other repeat elements. The elevated H3.3 dissociation was correlated with a substantially higher proportion of pluripotency factor binding within satellite repeats. Hence, the binding of ESC specific factors appears to be a strong determinant of H3.3-nucleosome turnover in ESCs. Nucleosomes are generally thought to impede transcription factor binding to their DNA motives, although some pioneer factors may bind directly to nucleosomes –43. HencInterestingly, we found higher H3.3 dissociation rates at distal non-genic sites compared to proximal sites around TSSs. At the same time, equilibrium levels of H3.3 were higher at proximal H3.3 sites when compared to distal H3.3 sites.Lower H3.3 turnover at genic regions may partially be explained by gene body H3.3, which is typically exchanged at a low rate. Yet, several marks and protein factor binding sites that are not associated with gene bodies also display higher H3.3-nucleosome turnover at distal sites compared to nucleosomes at proximal sites. Higher distal H3.3 turnover may result from H3.3-H4 tetramer splitting events, which have been reported at higher rates from enhancers compared to non-enhancers .In summary, our analysis of equilibrium levels and dissociation rates in mouse ESCs revealed different modes of H3.3 deposition and turnover that pertain to their pluripotency. We detected differential trends of H3.3-nucleosome turnover as they relate to Pol II-dependent transcription, pluripotency factor binding, and proximity to genes.In this study, we utilized a TET-OFF ESC line to measure dissociation rates of H3.3 nucleosomes upon inhibition of epitope-tagged H3.3 expression. Genome-wide analyses revealed differential H3.3 turnover at functionally distinct genomic regions. High H3.3 turnover was associated with genic and extragenic sites of Pol II binding sites. At genic sites H3.3 turnover was directly correlated with gene activity suggesting that H3.3 displacement and deposition may be direct functions of transcription. Pluripotency factor binding sites as well as binding sites of chromatin enzymes displayed high H3.3 turnover even when present at heterochromatic sites, which typically feature slow H3.3 turnover, suggesting that both histone modifications and protein-DNA interactions are important determinants of nucleosome turnover. Distal regulatory sites are featured by higher turnover than gene proximal sites suggesting that the chromatin of enhancers must be kept in a highly dynamic state in order to facilitate cell type specific gene activation.ES(MC1R(20)) cells were a kind gift from Minoru Ko. The plvx-Tight-Puro-HA/FLAG H3.3 expression construct was generated as described in . LentiviESC cultures were seeded at clonal density and cultured for 5 days in ES medium (+/-2 μg/mL DOX), or directly fixed when cultured on feeder cells. Cells were washed, fixed in 4% paraformaldehyde and tested for alkaline phosphatase (AP) activity using an AP kit (Millipore).Total RNA was isolated using the RNeasy kit (Quiagen) and cDNA was made from 1 μg of total RNA using the SuperScript III First-Strand Synthesis System (Invitrogen). RT-PCR was performed with the following Taqman probes: Gapdh, Mm03302249; Oct4, Mm03053917; Nanog, Mm01617763; Sox2, Mm00488369; AFP, Mm00431715; Sox17, and Mm00488363.Cells were lysed with RIPA buffer, and whole-cell lysates were resolved on SDS-PAGE, transferred onto nitrocellulose membranes, and blotted with anti-HA , anti-FLAG  antibodies at 1:1,000. For anti-H3.3 western blotting, histones were isolated by acid extraction as described in  prior toFor ‘TET-OFF’ experiments, ESCs were cultured over several passages (weeks) on feeder cells in the absence of DOX and were subsequently passaged onto feeder-free plates prior to the inhibition of HA-H3.3 expression. To repress HA/FLAG-H3.3 expression we treated cells with 2 μg/mL doxycycline hyclate before crosslinking with formaldehyde at various time points. ChIP experiments were performed as described previously with an antibody against HA . ChIP-PCIn order to correlate HA/FLAG-H3.3 stability with the presence of histone modifications and/or gene expression levels, we constructed ChIP-Seq libraries for H2A.Z, H3K4me1, H3K4me3, H3K9ac, H3K27me3, and RNA-Seq libraries.For RNA-Seq library construction, poly-adenylated RNA was isolated from 5 ug of total RNA isolated from undifferentiated ESCs cultured on gelatin-coated plates, using the Dynabeads mRNA Direct kit (Invitrogen). Double-stranded cDNA was generated with the Double-stranded cDNA synthesis kit (Invitrogen), sonicated, and subsequently processed exactly like ChIP DNA.ChIP material was blunt-ended and phosphorylated with the End-it-Repair kit (EPICENTRE). Illumina genome sequencing adaptors were ligated with T4 DNA ligase (New England Biolabs) after addition of adenosine nucleotides, using exo-Klenow. Samples were PCR amplified with multiplexed Illumina genomic DNA sequencing primers. PCR products (250 to 450 bp in size) were gel purified and submitted for Illumina deep sequencing.ChIP-Seq reads were mapped to the mouse reference genome (mm9) by perfect and unique matching without allowing any mismatch or gap. The reads were then extended to 150 bp from their 5’ end. We applied a modified average low pass filter for deconvolution of noisy blurred images. The normalized H3.3 level at a 250 bp window is a linear combination of the number of the HA-H3.3 nucleosomes at the site and the number of HA-H3.3 nucleosomes in the two adjacent 250 bp-length windows. In the linear combination, the number of HA-H3.3 nucleosomes at the center is weighted 1 and the number of HA-H3.3 nucleosomes in the two neighborhoods at both ends is weighted 0.5.For our analysis of H3.3 dissociation rates and equilibrium levels, we only considered genomic regions that show significant enrichment of HA-H3.3 at 0 h of the ‘TET-OFF’ time course and day 6 of the TET-ON time course.out was calculated following an exponential decay equation:The H3.3 dissociation rate, λ’N(t) is amount of H3.3 in a chromatin at time t in TET-OFF experiment.Where, i , we used the following logistic equation to scale dissociation rates to values from 0 to 1:We estimated the H3.3 dissociation rates for any given locus by counting the number of HA-H3.3 nucleosomes mapped at 0 h and 6 h following inhibition of HA-H3.3 expression. To calculate the relative H3.3 dissociation rates at a locus Ni(0 h) : the number of HA-H3.3 dyads mapped to locus i at 6 h after inhibition of H3.3-HA expression.Ni(6 h) : the number of HA-H3.3 dyads mapped to locus i at 6 h after inhibition of H3.3-HA expression.Equilibrium levels of HA-H3.3 at any given locus was estimated from the enrichment of HA-H3.3 at 0 h of TET-OFF experiments.Ni(0 h) : the number of HA-H3.3 dyads mapped to locus i at 0 h after inhibition of HA-H3.3 expression.Ii(0 h) : the number of nucleosome dyads mapped to locus i from input DNA sequencing at 0 h after inhibition of HA-H3.3 expression.et al. (GSE11431) [et al. (GSE12241) [et al. (GSE21202) [et al. (GSE13084) [et al. [et al. (GSE27844) [et al. [Transcription factor ChIP-Seq for Nanog, Oct4, Sox2, Smad1, E2F1, Tcfcp2I1, CTCF, Zfx, STAT3, KLF4, Esrrb, n-Myc, p300 in mESC were obtained from Chen SE11431) . H3, H4KSE12241) . KDM2A CSE13084) . Med12,  [et al. . HDAC1, SE27844) . LMR/UMR [et al. . DNaseI The raw ChIP-Seq data in SRA format were transformed into fastq files and mapped to the reference genome (mm9). The 30 to 50 bp sequences from the ChIP-Seq data were mapped to the mouse reference genome (mm9) by perfect and unique matching without allowing any mismatch or gap. The reads were then extended to 150 bp from their 5’ end.The RNA-Seq analysis was performed using the Tuxedo software package with default settings. RNA-Seq reads were mapped to the mouse genome (NCBI37/mm9) using Bowtie2. Tophat with default settings was used to detect splice sites. The Cufflinks software package was used to assemble transcripts based on the Refseq mRNA sequence database (mm9). A total of 48,228 transcripts were detected from two RNA-Seq replicate experiments and their mean values were used for further analysis. Transcripts were sorted according to their FPKM (fragments per kilobase of exon model per million mapped fragments). The ‘top 10%’  of transcripts was defined as having expression levels greater than 30 FPKM, the ‘top 20%’  greater than 10 FPKM and less than 30 FPKM, ‘medium’  between 3 and 10 FPKM, ‘low’  between 1 and 3 FPKM. Silenced transcripts were defined as having expression levels between 0 and 1 FPKM.All statistical tests were performed using the R, stat package. To test the significance of Pearson correlation coefficients, we used the cor.test in the R stat package. To test the significance of enrichment and depletion, we performed a Chi-squared test using the chisq.test in the R stat package.Our ChIP-Seq and RNA-Seq data sets have been deposited in the Gene Expression Omnibus data base with accession number GSE63641.(A) HA-H3.3 ESCs were cultured on feeder cells (upper panel) and cultured for 3 days or seeded at clonal density and grown for 5 days (lower two panels) with and without DOX prior to alkaline phosphatase staining. (B) Total RNA was isolated from undifferentiated HA-H3.3 ESCs and from day 8 EBs. Quantitative PCR analysis was performed for analysis of expression of pluripotency factors  as well as differentiation markers . (PDF 3 MB)Additional file 1: Figure S1:(A) ChIP-Seq profiles of two control regions: Rps19 (positive) and Intergenic Chr.8 (negative) over a 48-h time course after inhibition of HA-H3.3 expression. (B) HA-H3.3 ESCs were crosslinked at various time points after DOX addition and ChIP-PCR validation was performed with primers spanning the region highlighted in red. (PDF 1 MB)Additional file 2: Figure S2:


I relate a story of genesis told from the point of view of multi-subunit RNA polymerases (RNAPs) including an Old Testament  and a New Testament  and CTD interactome in eukarya). The Old Testament: at their active site, one class of eukaryotic interfering RNAP and ubiquitous multi-subunit RNAPs each have two-double psi β barrel (DPBB) motifs (a distinct pattern for compact 6-β sheet barrels). Between β sheets 2 and 3 of the β subunit type DPBB of all multi-subunit RNAPs is a sandwich barrel hybrid motif (SBHM) that interacts with conserved initiation and elongation factors required to utilize a DNA template. Analysis of RNAP core protein motifs, therefore, indicates that RNAP evolution can be traced from the RNA-protein world to LUCA  branching to LECA (the last eukaryotic common ancestor) and to the present day, spanning about 4 billion years. The New Testament: in the eukaryotic lineage, I posit that splitting RNAP functions into RNAPs I, II and III and innovations developed around the CTD heptad repeat of RNAP II and the extensive CTD interactome helps to describe how greater structural, cell cycle, epigenetic and signaling complexity co-evolved in eukaryotes relative to eubacteria and archaea. Analysis of RNAP structures shows significant family resemblance between eubacteria and eukarya (see below). Of course, this argument for LUCA and the relatedness of extant life forms could be advanced based on the highly conserved structures of many, many proteins, not just RNAP. LUCA is ancient: approximately 3.5 to 3.8 billion years ago (bya). With respect to many core protein motifs in essential processes, there appears to be surprisingly little evolutionary innovation since LUCA (see below).Multi-subunit RNAPs are found in all cellular life on earth, which has diverged into three domains: eubacteria, archaea and eukarya (including humans). Eubacteria and archaea have a single RNAP. Within the cell nucleus, eukarya have at least three: RNAP I, II and III, allowing for much broader specialization in gene regulation. RNAP I synthesizes rRNA. RNAP II synthesizes mRNA, which specifies protein sequences. RNAP III synthesizes 5S RNA, tRNA and some small RNAs. RNAP IV and V are found in some plants and synthesize regulatory RNAs. That highly homologous multi-subunit RNAPs are found in all cellular life is evidence for a last universal common ancestor (LUCA) from which eubacteria, archaea and eukarya are derived, making -Neurospora crassa.Multi-subunit RNAPs evolved around two DPBBs .,The schematic shown in http://jivarahasya.blogspot.com/).Thermus thermophilus (Tt) RNAP are highly conserved in core motifs, demonstrating their kinship.The sandwich barrel hybrid motif (SBHM) embedded between the 2nd and 3rd β sheets of the β subunit type DPBB provides an interaction surface for initiation and elongation factors that are conserved in the three domains of life .6 In eubhttp://jivarahasya.blogspot.com/). So the ribosome ribozyme function was selected in competition with less capable protein functions. By contrast, proteins are adept at binding positively charged Mg atoms through negatively charged aspartic acid, as in RNA synthesis by multi-subunit RNAPs  were invaded by a protein cofactor including two DPBB motifs.Furthermore, in all cellular life, gene regulation imposes many constraints on evolution of RNAP core protein motifs because changing RNAP activity may alter many dependent and potentially essential processes required for gene expression. In eukaryotes, most evolutionary innovation is expected in RNAP II add-ons rather than within core RNAP motifs. MRNA synthesis by RNAP II specifies protein identity, which gives different cell types unique characteristics. Innovation around RNAP II gene regulation is essential to generate eukaryotic organisms of high complexity, such as animals of highly complex body structure.-At the C-terminal end of its β’ type subunit (named Rpb1), RNAP II has an unusual repeat structure termed the CTD for carboxy terminal domain.What makes eukaryotes distinct from mostly single-celled eubacteria and archaea? Specifically, what evolutionary innovations permit eukaryotes to generate high complexity in DNA genome, cell structure, cell communication and organism plan? A hypothesis of this paper is that immense innovation developed around gene regulation, centered on and interacting with the CTD of RNAP II. Innovation confined to mRNA synthesis was crucial to development of organism plans of increasingly higher complexity, and offered the additional benefit of, and probable requirement for, insulating ribosomal, transfer, 5S and regulatory RNAs from innovations in mRNA synthesis.-Yang and Stiller-,The CTD of RNAP II was a major innovation at the inception of the eukaryote lineage separating the regulation of mRNA synthesis, which in eukaryotes requires highly nuanced regulation, from rRNA synthesis, tRNA synthesis and small regulatory RNA synthesis, which generally require less sophisticated control. Many systems of surprisingly high complexity have evolved around the CTD to modulate RNAP II gene expression including TFIID, SAGA, mediator, TFIIH, COMPASS (Set1 lysine methyltransferase), capping factors, splicing factors, the PAF complex, Set2 complex, 3′-end RNA processing machinery, termination factors, recycling factors and nuclear pore mRNA transport factors .13-15,20-Drosophila) include NELF and utilize promoter-proximal pausing; some animals with less complex body types  lack NELF subunits and do not demonstrate promoter-proximal pausing. From similar logic, innovations such as the notochord in the vertebrate lineage may relate to modifications in P-TEFb recruitment to overcome promoter proximal pausing, such as the Super Elongation Complex (SEC) and/or Brd4, factors implicated in human viral infections (HIV-1→AIDS),,,As a milestone in the eukaryote animal lineage, promoter-proximal pausing of RNAP II is a gene regulation innovation associated with generation of complex body plans and centered on the CTD.-Arabidopsis, mutations that alter the timing of blooms in response to cold and also the hardening of seeds after flowering are found in multiple genes encoding subunits of the PAF complex, which interacts with the CTD and participates in the initiation, elongation and termination phases of the RNAP II transcription cycle.The PAF complex is a central feature of the RNAP II CTD interactome from yeast to plants to humans. The human PAF complex is implicated in diseaseComplex eukaryotic DNA genomes were partly initiated during endosymbiosis of a pro-mitochondrial proteobacterium into an archaeal host.-,Nuanced gene regulation in the eukaryotic lineage has been developed as a series of seemingly endless add-ons and adaptations: repression, anti-repression and anti-anti-repression, etc. Notably, many or most gene activation mechanisms in eukarya appear to be anti-repression mechanisms. Many or most key evolutionary RNAP II add-ons integrate with the CTD, which can be viewed as somewhat similar to, but somewhat more than, a global positioning system to monitor, locate and regulate progression of RNAP II through and beyond the transcription cycle of pre-initiation complex assembly, initiation, promoter escape, capping, promoter-proximal pausing, elongation, splicing, termination, RNAP II recycling, mRNA folding and decoration, and mRNA export and licensing for translation .13-15,20Mostly single-celled eubacteria and generally more complex eukarya have characteristic gene regulatory network strategies that likely reflect and license organism complexity . My opin-,Because only some evolutionary processes can be observed experimentally, “proving” arguments regarding ancient evolution is challenging, but science is much more suited to detailed, accurate and useful description and prediction than “proof.” Also, evolution via natural selection is not nice, and LUCA is strongly suspected of eating the RNA-protein world, limiting the RNA-protein world's availability for current study, just as more modern organisms subsequently may have feasted on or out competed LUCA . DespiteBecause evolution has become a political football, this complicates teaching. Scientists remain adamant about presenting evolution, without which biology would make no sense, a suspected goal of anti-evolutionists. For one thing, without a conceptual framework, biology seems overwhelmingly complex, and, after more than 100 y of critical and sophisticated challenges to Darwinian philosophy, the most powerful organizing principle in biology remains evolution via natural selection. The current work indicates, however, that underlying the remarkable complexity in biological systems are deeper concepts: that although biological systems naturally generate huge variation and complexity, there may not be very many identifiably distinct “strategies” or milestones in evolution. The splitting of RNAP functions into RNAP I, II and III and extension of the CTD heptad repeat on RNAP II, at the dawn of the eukaryotic lineage, represent major “strategies” and milestones. The RNAP II CTD then became a scaffold for co-evolution of the extensive CTD interactome. Surprisingly, the eukaryotic cell cycle and RNAP II transcription cycle, which is centered on the CTD, resemble one another,,Another concept is that it may be most useful to present evolution based on conservation of protein motifs and protein sequences rather than by concentrating on the description of intact organisms , 2, 3, 4I do not mean to imply that the evolution of RNAPs explains all of biology. RNAP, however, provides a rich and coherent lesson because RNAPs are central components in genetic coding and because the evolutionary history of RNAPs extends into the RNA-protein world to LUCA branching to LECA and to the present day, relating a credible story of genesis. Also, gene expression is a core feature of evolutionary advancements, and eukaryotic gene regulation appears to center on the CTD of RNAP II, indicating that the gene regulatory networks of eukaryotes should be understood in significant part through analysis of the extensive CTD interactome . Because


CYP2B6 gene in human liver. However, there are dramatic interindividual variabilities in CYP2B6 expression that cannot be fully explained by the CAR/PXR-based modulation alone. Here, we show that expression level of CYP2B6 was correlated with that of hepatocyte nuclear factor 3β (HNF3β) in human primary hepatocytes prepared from 35 liver donors. Utilizing recombinant virus-mediated overexpression or knockdown of HNF3β in HepG2 cells, as well as constructs containing serial deletion and site-directed mutation of HNF3β binding motifs in CYP2B6 luciferase reporter assays, we demonstrated that the presence or lack of HNF3β expression markedly correlated with CYP2B6 gene expression and its promoter activity. Novel enhancer modules of HNF3β located upstream of the CYP2B6 gene transcription start site were identified and functionally validated as key elements governing HNF3β-mediated CYP2B6 expression. Chromatin immunoprecipitation assays in human primary hepatocytes and surface plasmon resonance binding affinity experiments confirmed the essential role of these enhancers in the recruitment of HNF3β to the promoter of CYP2B6 gene. Overall, these findings indicate that HNF3β represents a new liver enriched transcription factor that is involved in the transcription of CYP2B6 gene and contributes to the large interindividual variations of CYP2B6 expression in human population.CYP2B6 plays an increasingly important role in xenobiotic metabolism and detoxification. The constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) have been established as predominant regulators for the inductive expression of  CYP2B6 gene expression is evident.Although traditionally thought to be of minor significance in pharmacology and toxicology , the cliCYP2B6 gene transcription. Ectopic expression of CAR or PXR alone failed to fully restore the basal and inductive expression of CYP2B6 in non-hepatic cells or hepatoma cell lines that express extremely low levels of liver enriched transcription factors (LETFs) thth3]thiaz5’- GGGGTACCTAGACATACATATACCCAC-3’, 5’- GGGGTACCCATACAGGGATGCAAGCAG-3’, 5’- GGGGTACCGGGATTACAGGTGTGAGC-3’, 5’- GGGGTACCTCAGCATCTGCAGGCTTC-3’, 5’- GGGGTACCACACACCTGGAGCTCAAG-3’, 5’- GGGGTACCGGACAATGTAGCCCCAACCC-3’ and the same reverse primer: 5’- AGTCTACTCGAGCTGCACCCTGCTGCAGCCT-3’. The PCR products were sub-cloned into the KpnI and XhoI sites of pGL3-basic vector, resulting in constructs termed 2B6-300bp, 2B6-387bp, 2B6-600bp, 2B6-1kb, 2B6-1.4kb and 2B6-2kb, and the correct orientation was verified by sequencing. The pCR3-hCAR, pMEX-C/EBPα, pcDNA3.1/HNF3β expression vectors and the CYP2B6 reporter constructs (2B6-1.6kb and 2B6-1.8kb) were obtained or generated as described previously [Luciferase reporter plasmids containing -300bp, -387bp, -600bp, -1kb, -1.4kb and -2kb fragments of the CYP2B6 promoter region were PCR-amplified by using forward primers: eviously , 17, 20.CCGCCTGAGTTACTGTGTGCACTAAGAGTGTA, the HNF3β-b-mut: CCAGTCATATAAGCACATACCCCCTTTACAT GTA, and the HNF3β-c-mut: CCAGAGTGCA AGCTCACCCAAAGCTAAGTA. The constructs were sequenced to confirm the presence of the mutation(s).Site-directed mutagenesis was performed by PCR, using the 2B6-2kb construct as the template, based on the protocol of the QuickChange Multi Site-Directed Mutagenesis Kit from Stratagene . Mutated nucleotides in the HNF3β binding sites are underlined for the HNF3β-a-mut: 6 cells/well in 12-well biocoat plates in DMEM supplemented with 5% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 4 μg/ml insulin, and 1 μM dexamethasone. After attachment at 37°C in a humidified atmosphere of 5% CO2, hepatocytes were cultured in complete William’s Medium E (WME) and overlaid with Matrigel (0.25mg/ml). Cell culture medium was replaced on a daily basis.Human hepatocytes were isolated as described previously  from hum5’-AGACGCCTTCAATCCTGACC-3’ and 5’-CCTTCACCAAGACAAATCCGC-3’; CAR, 5’- GAGCTGAGGAACTGTGTGGTA-3’ and 5’-CTTTTGCTGACTGTTCTCCTGAA-3’; HNF3β, 5’- GGAGCAGCTACTATGCAGAGC-3’ and 5’-CGTGTTCATGCCGTTCATCC-3’; C/EBPα, 5’-TCGGTGGACAAGAACAGCAA-3’ and 5’-TTTCAGGAGGCACCGGAATCT-3’; HNF4α, 5’-CGAAGGTCAAGCTATGAGGACA-3’ and 5’- ATCTGCGATGCTGGCAATCT-3’; and GAPDH,5’-CCCATCACCATCTTCCAG GAG-3’ and 5’-GTTGTCATGGATGACCTTGGC-3’. Fold induction values were calculated according to the equation 2ΔΔCt, where ΔCt represents the differences in cycle threshold numbers between the target gene and GAPDH, and ΔΔCt represents the relative change in these differences between control and treatment groups.Total RNA from human primary hepatocytes was isolated using the RNeasy Mini Kit  and reverse transcribed using High Capacity cDNA Archive kit  following the manufacturers’ instructions. Real-time PCR assays were performed in 96-well optical plates on an ABI StepOnePlus Real-Time PCR system (Applied Biosystems) with SYBR Green PCR Master Mix (Qiagen). Primer sequences for the CYP2B6, CAR, HNF3β, C/EBPα, HNF4α, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are: CYP2B6, HepG2 cells obtained from American Type Culture Collection  were transfected with different CYP2B6 reporter constructs in the presence of HNF3β, with or without hCAR and C/EBPα expression vectors using X-tremeGENE 9 DNA Transfection Reagent . Twenty-four hours after transfection, cells were treated with solvent (0.1% DMSO), PB (1mM), or CITCO (1μM) for 24 h. Subsequently, cell lysates were assayed for firefly luciferase activities normalized against the activities of co-transfected Renilla luciferase using Dual-Luciferase Kit . Data were represented as mean ± S.D. of three individual transfections.GATCAAGAACATGTCGTCGTACGTGCTCGAGCACGTACGACGACATGTTCTTTTTTTG) [Twenty-four hours after seeding, HepG2 cells were infected with negative control adenovirus or increasing concentrations of adenovirus expressing HNF3β purchased from Vigene Biosciences  for 48 h. For HNF3β-RNAi lentivirus, DNA oligonucleotides encoding short hairpin HNF3β RNA (TTTTTTG)  was inseTTTTTTG) . In HNF3Cell homogenate proteins harvested from Ad-HNF3β overexpressing HepG2 cells were resolved on SDS—polyacrylamide gels (12%) and electrophoretically transferred onto blotting membranes. Subsequently, membranes were incubated with antibodies against HNF3β (diluted 1:200), CYP2B6 (diluted 1:200), or β-actin . Blots were washed and incubated with horseradish peroxidase secondary antibodies, and developed using enhanced chemiluminescence Western blotting detection reagent from GE Healthcare .CYP2B6 metabolic activity assay was performed in adenovirus HNF3β infected HepG2 cells on 24-well plate using P450-Glo ™ CYP2B6 Assay kit  following the manufacturer’s instruction. In brief, 48 h after HNF3β adenovirus infection, culture medium was completely removed and cells were washed twice with Krebs-Henseleit buffer , followed by incubation at 37°C for 2 h in 300 μL Krebs-Henseleit buffer containing 3 μM of Luciferin-2B6 (CYP2B6 specific substrate) and 3 mM of salicylamide. Equal volume of Luciferin Detection Reagent was added into each well, and mixed at room temperature for 20 min before luminescence detection . Data were represented as mean ± S.D. of three individual infections.6 cultured human primary hepatocytes were cross-linked with 1% formaldehyde for 10 min at 37°C, washed with ice-cold phosphate buffered saline containing a protease inhibitor cocktail. Nuclear extracts were sonicated. Immunoprecipitation was performed overnight at 4°C using 5μg of anti-HNF3β antibody (Santa Cruz Biotechnology) or normal rabbit IgG  followed by precipitation using protein A coupled to agarose beads. After de-crosslinking and protease digestion, DNA fragments were recovered by QIAquick PCR purification kit (QIAGEN). CYP2B6 promoters containing HNF3β-a , HNF3β-c , and the region between -1.4/-1.6k were amplified by PCR using primers: 5’-TGGACAATGTAGCCCCAACCC-3’ and 5’-GATTGGGTGCTCATTGCAGCC-3’; 5’-CTCATACACATGCAAGGATAC-3’ and 5’-GAGCAAGTGAATGTGTGGG TG-3’; and 5’-GTCAGGCGTAGGATGAGACAG-3’and 5’-TCTTGAGCTCCAGGTGTGTGC-3’. A proximal promoter region of SULT1E1 gene was used as a negative control as reported previously [Experiments were performed using a ChIP assay kit according to the manufacturer’s protocol . In brief, 1 × 10eviously .AGAGTGTAAAGACTGAG), HNF3β-b (TACATGTAAAAATCATA) and HNF3β-c (GCTAAGTAAAAAAGTGC) were used as analytes. The binding assay was performed by injecting 60 μl each of the oligonucleotides at 10 μM in 10 mM Hepes, pH 7.4, containing 150 mM NaCl, 3 mM EDTA, and 0.005% P-20 at the flow rate of 30 μl per minute at 25°C. The association and dissociation between analytes and HNF3β protein were recorded respectively by SPR with a Biacore 3000  following the manufacturer’s instructions. Sensorgrams of the interaction generated by the instrument were analyzed by the software BIAeval 3.2.Recombinant HNF3β protein purchased from Abcam  was covalently linked to the surface of a BIAcore CM5 sensor chip  by direct immobilization as described previously . Oligonup < 0.05 or p < 0.01. Linear regression was analyzed using Pearson’s Correlation Coefficient .All data represent at least three independent experiments and were expressed as mean ± S.D. Statistical significance was determined using one-way analysis of variance followed by post-hoc Dunnett’s test or Student’s t test where appropriate. Statistical significance was set at p < 0.01), C/EBPα , and HNF4α , respectively (p < 0.01)  . These rTo further investigate the effects of HNF3β on CYP2B6 expression, adenovirus-driven HNF3β was used to ectopically over-express HNF3β in HepG2 cells. As shown in It is well-known that expression of CYP2B6 is predominantly regulated at the transcriptional level. Thus, we next investigated whether HNF3β could alter the transcriptional activity of CYP2B6 via a putative luciferase construct containing the first 2 kb of the CYP2B6 5’-flanking region in a pGL3-Basic reporter vector. This region contains the PBREM regulatory module that responds to the nuclear receptor CAR. Consistent with the notion that CAR is constitutively activated in immortalized cell lines , transfein silico analysis of the first 2 kb upstream sequence of the CYP2B6 promoter using the MatInspector release professional program [in silico analysis failed to predict a consensus HNF3β binding site between -1.6 kb and -1.4 kb, our luciferase reporter assay showed that deletion of this 200 bp sequence significantly reduced HNF3β-mediated activation of CYP2B6 promoter  in comparison to that of HNF3β-c (4.82 × 103 1/Ms) though both shared similar dissociation rate constants . In consequence, HNF3β-a exhibited a higher binding affinity (KD = 8.45 ×10−12 M) than HNF3β-c (KD = 7.34 ×10−10 M) . As demonstrated in 10−10 M) . TogetheCYP2B6 gene expression by recognizing and interacting with multiple enhancer modules including two consensus HNF3β binding sites located at -1887/-1871bp and -347/-331bp upstream of the CYP2B6 transcriptional start site. Site-directed mutation of these responsive elements resulted in significant reduction of the CYP2B6 promoter activity. Moreover, HNF3β protein was efficiently recruited to the CYP2B6 promoter through direct interaction with these enhancer modules.CYP2B6, an inducible cytochrome P450 isoform predominantly present in the liver, exhibits large intra- and inter-individual variations in human populations , 35. ExpCYP2B6 gene transcription in HepG2 cells [To date, human CAR and PXR have been recognized as the major transcription factors mediating drug-induced CYP2B6 expression. However, optimal expression of CYP2B6 was only achieved in HPH that maintain physiologically relevant expression of most LETFs. Hepatocyte nuclear factors are a heterogeneous group of evolutionarily conserved transcription factors that are pivotal for the development and maintenance of liver specific features . PositivG2 cells . Using Hin silico analysis in the -1.6k to -1.4k region of the CYP2B6 promoter, deletion of this region unexpectedly resulted in a significant reduction of CYP2B6 luciferase activity, indicating the presence of an unidentified HNF3β binding site which warrants more detailed investigation in the future.Regarded as one of the master regulators of hepatocyte differentiation and maturation, HNF3β regulates the expression of numerous hepatic genes by interacting with respective cis-acting binding elements in the promoters of these genes, including the gluconeogenic phosphoenolpyruvate carboxykinase, insulin-like growth factor-binding protein 1, and tyrosine aminotransferase –43. UnliCYP2B6 gene. It is worth noting that HNF3β was also co-precipitated with the DNA fragment covering the -1.6/-1.4bp region, where an unidentified HNF3β binding motif was speculated. However, given the proximity between the -1.6/-1.4bp and the HNF3β-a containing (-1887/-1871bp) areas, such recruitment may only represent a HNF3β-a dependent phenomenon. Quantitative kinetics for binding affinities between HNF3β and the three predicted binding sites were further estimated by a SPR approach. Consistent with results of the luciferase reporter and ChIP assays, our SPR affinity assays revealed that the HNF3β-a and HNF3β-c elements were efficiently recruited to the HNF3β protein, while the HNF3β-b containing region appeared not to be directly associated with HNF3β.In characterizing the recruitment of HNF3β to the promoter of CYP2B6 in a more physiologically relevant system, results from our ChIP assays in HPH confirmed the interaction between HNF3β protein and chromatin regions harboring the HNF3β-a and HNF3β-c motifs of CYP2B6 gene, in addition to HNF4α and C/EBPα. Ectopic expression of HNF3β in HepG2 cells was associated with increased CYP2B6 transactivation and endogenous expression. Such enhancement can be at least partly explained by the identification and functional characterization of two consensus HNF3β binding sites located in the 5’-flank of CYP2B6 upstream. These findings reveal additional mechanistic bases in our understanding of CYP2B6 gene transcription, and suggest that expression of CYP2B6 is governed by a complex regulatory network including genes such as, CAR, PXR, HNF4α, C/EBPα, and HNF3β. It is worth noting that multiple cis-acting responsive elements for C/EBPα and HNF4α have also been localized within the first 2 kb of the CYP2B6 promoter [In conclusion, our data show that HNF3β represents a novel LETF that regulates the transcription of promoter , 21. Fut


SUMOylation is associated with epigenetic regulation of chromatin structure and transcription. Epigenetic modifications of herpesviral genomes accompany the transcriptional switch of latent and lytic genes during the virus life cycle. Here, we report a genome-wide comparison of SUMO paralog modification on the KSHV genome. Using chromatin immunoprecipitation in conjunction with high-throughput sequencing, our study revealed highly distinct landscape changes of SUMO paralog genomic modifications associated with KSHV reactivation. A rapid and widespread deposition of SUMO-2/3, compared with SUMO-1, modification across the KSHV genome upon reactivation was observed. Interestingly, SUMO-2/3 enrichment was inversely correlated with H3K9me3 mark after reactivation, indicating that SUMO-2/3 may be responsible for regulating the expression of viral genes located in low heterochromatin regions during viral reactivation. RNA-sequencing analysis showed that the SUMO-2/3 enrichment pattern positively correlated with KSHV gene expression profiles. Activation of KSHV lytic genes located in regions with high SUMO-2/3 enrichment was enhanced by SUMO-2/3 knockdown. These findings suggest that SUMO-2/3 viral chromatin modification contributes to the diminution of viral gene expression during reactivation. Our previous study identified a SUMO-2/3-specific viral E3 ligase, K-bZIP, suggesting a potential role of this enzyme in regulating SUMO-2/3 enrichment and viral gene repression. Consistent with this prediction, higher K-bZIP binding on SUMO-2/3 enrichment region during reactivation was observed. Moreover, a K-bZIP SUMO E3 ligase dead mutant, K-bZIP-L75A, in the viral context, showed no SUMO-2/3 enrichment on viral chromatin and higher expression of viral genes located in SUMO-2/3 enriched regions during reactivation. Importantly, virus production significantly increased in both SUMO-2/3 knockdown and KSHV K-bZIP-L75A mutant cells. These results indicate that SUMO-2/3 modification of viral chromatin may function to counteract KSHV reactivation. As induction of herpesvirus reactivation may activate cellular antiviral regimes, our results suggest that development of viral SUMO E3 ligase specific inhibitors may be an avenue for anti-virus therapy. Establishment of KSHV persistent infection requires a dynamic balance between latency, a phase where most viral genes are silenced, and lytic cycle, a phase when nearly all viral genes are expressed. Disruption of this balance may augment virus clearance. During the latent-to-lytic switch, KSHV genomes are subjected to profound epigenetic changes. SUMOylation promotes targeting of proteins to different DNA sites, thereby helping to create specific epigenetic patterns that switch genes between active and inactive stages. It comes as no surprise that SUMOylation may be involved in chromatin remodeling of the KSHV genome during the latent-to-lytic switch and SUMOylation inhibition may disrupt the balance between KSHV latent and lytic cycle. In this study, we identified a profound SUMO-2/3 enrichment in KSHV genome euchromatin regions upon reactivation. SUMO-2/3 modification is responsible for diminishing viral gene expression after reactivation. KSHV SUMO-2/3-specific E3 ligase K-bZIP mediates the SUMO-2/3 enrichment during reactivation. Loss of E3 ligase activity of K-bZIP in the viral context increases viral lytic gene expression and virus production. Our findings demonstrate, for the first time, a SUMO-2/3-specific modification affecting transcription which regulates viral lytic gene expression, and uncovers a novel therapeutic strategy to disrupt persistent infection. DNA blots were hybridized overnight at 65°C with rotation. After washing, the blots were imaged using X-ray film .KpnI and HindIII cleaved BAC16 DNA was separated on 1% agarose gel and visualized by ethidium bromide staining. The DNA in the gel was transferred to NC membrane using downward alkaline transfer. A K-bZIP probe was radiolabeled with [α-iSLK-Puro cells were transfected with 2 μg of BAC16 DNA using FuGENE HD (Roche). Forty-eight hours after transfection, the cells were expanded to a 15 cm Petri dish and selected by hygromycin (600 μg/ml). After three weeks of selection, the hygromycin-resistant and GFP positive colonies were picked and pooled to establish the iSLK-Puro-BAC16 K-bZIP-WT, -WT rev and -L75A cell lines. iSLK-Puro-BAC16 cell lines were maintained as described for iSLK-Puro cells and supplemented with 300 μg/ml hygromycin.To assess viral production, supernatants from control and Dox-induced TREx-F3H3-K-Rta BCBL-1 and SUMO-2/3 knockdown TREx-F3H3-K-Rta-shSUMO-2/3 BCBL-1 cells were collected before and after 48 hours treatment. KSHV virion DNA was prepared using QIAamp MinElute Virus Spin kits as described previously . QuantifTo distinguish the genome-wide distribution of SUMO paralog conjugation on KSHV chromatin and determine their changes during viral reactivation, we conducted chromatin immunoprecipitation (ChIP) assays in combination with high-throughput next generation sequencing (ChIP-seq), a gold-standard method for identifying the genome-wide sites of epigenetic marks. For this, we used the well-characterized KSHV-infected primary effusion lymphoma (PEL) cell line, TREx-F3H3-K-Rta BCBL-1, that expressed a Dox inducible K-Rta protein whose expression switches KSHV from latent to lytic phase . The indAfter Dox treatment for 12 hours, the global expression of SUMO-1 and SUMO-2/3 and successful induction of K-Rta were first confirmed by immunoblotting . The ChISurprisingly, when compared with our previous result of H3K9me3 modification on the KSHV latent genome (0 hpi) , we notiFor further confirmation of the ChIP-seq results, we selected four genes from each of two viral genomic regions, one representing the region of high SUMO-2/3 enrichment with low H3K9me3 mark  , red boxThe discovery of the negative correlation between SUMO-2/3 and H3K9me3 prompted us to further explore the association of SUMO-1 and SUMO-2/3 with different histone marks. We compared the binding pattern of SUMO-1 and SUMO-2/3 before and after viral reactivation from this study with the previously published binding profiles of various chromatin marks  Table 1Table 1. Consistent with our ChIP-on-chip data, SUMO-2/3 showed medium negative correlation to H3K9me3 (r = -0.3) at 12 hpi. Interestingly, no correlation was found between SUMO-2/3 enrichment with any other histone marks after KSHV reactivation. Moreover, no statistically significant correlation was found in SUMO-1 with histone marks on both latent and lytic KSHV genomes. To further explore if the negative correlation of SUMO-2/3 enrichment with heterochromatin mark H3K9me3 is also true on viral promoter regions, a number of promoters with high SUMO-2/3 enrichment at 12 hpi from this study were again compared with the previous study . Among tIn order to gain insight into transcriptional regulation by SUMO-2/3 during KSHV reactivation, an RNA-Seq assay was performed using total RNA purified from TREx-F3H3-K-Rta BCBL-1 cells before and after K-Rta induction. Several viral lytic genes, such as K7 and PAN, showed high expression in control cells. This may due to the spontaneous reactivation of KSHV in a small population of BCBL-1 cells. Viral gene expression changes revealed by this assay show that SUMO-2/3 enrichment was preferentially located at gene regions that show a higher level of transactivation during KSHV reactivation , red boxAs mentioned earlier, the positive correlation between SUMO-2/3 enrichment and viral gene transactivation suggests that SUMOylation on the viral genome may be required for gene shut-off after viral lytic reactivation. To explore this idea, a SUMO-2/3 inducible knockdown TREx-F3H3-K-Rta-shSUMO-2/3 BCBL-1 cell line was used . A time To confirm whether global SUMO-2/3 knockdown results in a corresponding decline in SUMO-2/3 enrichment on the KSHV genome during reactivation, another ChIP assay was performed using control (TREx-F3H3-K-Rta BCBL-1) and SUMO-2/3 knockdown TREx-F3H3-K-Rta-shSUMO-2/3 BCBL-1 cells. ChIP-qPCR result showed that SUMO-2/3 knockdown abolishes SUMO-2/3 enrichment on the high SUMO-2/3 enrichment region of KSHV genome, but has little effect on the high H3K9me3 mark region . This reEnhancement of viral lytic gene expression by SUMO-2/3 knockdown may lead to an increase in viral replication and elevate levels of virus production. To examine the level of KSHV virion production in SUMO-2/3 knockdown cells, the control (TREx-F3H3-K-Rta BCBL-1) and the inducible SUMO-2/3 knockdown TREx-F3H3-K-Rta-shSUMO-2/3 BCBL-1 cells were treated with 0.2 μg/ml Dox for 48 hours, a condition that we can consistently detect relatively low (~ 2- to 3-fold virion induction) but significant induction of KSHV virion production in control TREx-F3H3-K-Rta BCBL-1 cells. The successful induction of K-Rta, expression of K-bZIP and knockdown of SUMO-2/3 was first assessed by immunoblotting . The supet al. 2009 stated that the K-bZIP promoter was the most enriched target of K-bZIP following its overexpression [Our findings strongly suggest that SUMO-2/3 may play a major and critical role in regulating viral gene silencing after transactivation. The next question is how KSHV controls the SUMO-2/3 modification on its genome during lytic reactivation. Interestingly, our recent report demonstrated a unique mechanism by which KSHV modulates SUMOylation via expressing a viral lytic SUMO E3 ligase, K-bZIP . This vipression . Thus, tTo uncover the potential functional role of the SUMO E3 ligase activity of K-bZIP in regulating KSHV gene expression during reactivation in a virus context, we generated a SUMO E3 ligase dead mutant of K-bZIP in BAC16, a KSHV BAC clone generated from Dr. Jung’s laboratory . The leuTo evaluate the potential role of K-bZIP SUMO E3 ligase, iSLK cells harboring different recombinant KSHV-BACmids were induced with Dox for 24 and 48 hours for gene expression and virus production analysis, respectively. Total protein lysates were collected for immunoblotting analysis to assess the successful induction of K-Rta and expression of K-bZIP . Next, wTo determine if the K-bZIP SIM plays a role in its binding on the viral genome, ChIP assays of K-bZIP by using chromatin prepared from non-induced and Dox-induced iSLK-Puro-BAC16 K-bZIP-WT rev and -L75A mutant were performed. Consistent with our ChIP result of K-bZIP occupancy from TREx-F3H3-K-Rta BCBL-1 cells , a signiSince KSHV lytic reactivation is accompanied by transcriptional reprogramming, we extended the observations to virus production. Supernatants from non-induced and Dox-induced (48 or 72 hours) iSLK-Puro-BAC16 K-bZIP-WT rev and -L75A cells were collected and used for detection of virion-associated DNA using real-time TaqMan qPCR amplification or to infect 293T cells, respectively. In agreement with our previous findings, K-bZIP-L75A mutant showed significantly higher viral production . ConsistOne distinct feature of herpesviruses is that there are two phases, latent and lytic, in their lifecycle. Establishment of latency is a common property for herpesvirus to evade host immune responses and establish life-long infection. In addition to viral propagation, lytic reactivation has also been found to be required for maintaining herpesviral persistence . This isde novo infection, a quick transition from euchromatin mark to heterochromatin mark was detected in KSHV genomes [In latent phase, KSHV genome persists as a transcriptionally silent extrachromosomal episome resembling heterochromatin. During lytic phase, many regions of the viral genome adopt a state of euchromatin organization and almost all viral genes are transcribed in a temporally ordered manner. Epigenetic modifications of herpesvirus chromatin very likely play key roles in regulation viral gene expression as well as controlling the switch between latency and lytic replication. This notion is supported by the fact that viral lytic reactivation can be induced by inhibitors of DNA methyltransferases (DNMTs)  and HDAC genomes . In late genomes . During  genomes . Togetheet al showed that the KSHV latent protein LANA also contains a SUMO-2 specific SIM that is essential for the recruitment of the SUMOylated chromatin remodeling protein KAP-1 which aids in the maintenance of viral latency [SUMO modification is a post-translational modification that not only modulates the function of many transcription factors but also the chromatin organization by recruiting chromatin remodeling enzymes, including DNA and histone modification enzymes, to regulate gene expression. Global analysis of SUMOylation in epigenetic regulation has been done in different eukaryotic cells and indicates that SUMOylation can either restrain ,12 or pr latency . These s latency –12, thesHistone marks have long been studied and believed to be essential in maintaining chromatin structure and therefore manipulating gene expression. Though global SUMO modifications in epigenetic regulation have begun to be studied in eukaryotic cells, the correlation between histone marks and SUMO modifications has not yet been elucidated. Due to the relatively low complexity of the viral genome and the availability of genome-wide histone modification landscapes from KSHV, we were able to compare the genome-wide distribution of SUMO modification with different histone marks  on KSHV et al. noted that the repressive H3K9me3 mark was restricted to two regions of the KSHV genome that mainly encode late viral genes [A previous report from Toth al genes . The absal genes . The sigal genes . Howeveral genes . TogetheSUMO E3 ligases occasionally display paralog-specificity toward certain targets, however, none of the cellular SUMO E3 ligases, including protein inhibitor of activated STAT (PIAS) family, Ran-binding protein 2 (RanBP2), and Pc2, have been demonstrated to have selectivity towards a specific SUMO paralog. Therefore, despite many studies intent on analyzing paralog-specific SUMO modifications of epigenetic and transcription regulators, the distinctive functional specificity of SUMO isoforms in global epigenetic regulation in relationship to gene expression remains largely unknown. This indicates that a specific stimulus that can induce SUMO paralog-specific modification is required to elucidate the SUMO paralog-specific epigenetic regulatory function. By encoding a viral lytic SUMO-2/3 specific E3 ligase, K-bZIP, KSHV possesses as an ideal tool to distinguish the functional specificity of SUMO-2/3 in epigenetic regulation . To studThe identity of the protein(s) modified by SUMO and responsible for global SUMO-2/3 enrichment on the viral genome during reactivation is an important unanswered question. All K-bZIP interacting chromatin binding proteins could be potential candidates, such as the H3K9me3 demethylase JMJD2A . AnotherSurprisingly, during reactivation in K-bZIP-L75A mutated rKSHV, the viral genes in the high H3K9me3 mark region where there is little SUMO-2/3 enrichment, showed defective transcription activity. This implies that K-bZIP may participate in transactivation of viral genes located in high H3K9me3 regions in a SUMO E3 ligase-independent manner. Although K-bZIP is widely accepted as a transcriptional repressor, K-bZIP has also been found to activate gene transcription through direct DNA binding . Direct In addition to transcription regulation, the role of K-bZIP in KSHV replication is also controversial. Several reports have showed that K-bZIP is able to regulate KSHV replication through interacting and regulating Ori-Lyt-binding proteins, such as LANA  or HDAC Although SUMOylation of individual transcription factors is responsible for transcription repression, global SUMO modification of chromatin was found to be essential for either activation of genes  or shut-S1 FigImmunoblotting was performed using TCLs prepared from TREx-F3H3-K-Rta BCBL-1 cells; untreated, or treated with 20 ng/ml TPA and 0.2 μg/ml Dox for the time period shown. Blots were probed with anti-Flag and anti-K-bZIP antibodies. Anti-GAPDH antibody was used for loading control.(TIFF)Click here for additional data file.S2 Fig(A) The expression of SUMO-1 and SUMO-2/3 before and after Dox induction for viral reactivation was analyzed using anti-SUMO-1 and anti-SUMO-2/3 antibody, respectively. Ratio for each blot is the signal/α-tubulin observed at 12 hours Dox treatment setting 0 hour Dox as 1.0. Immunoblotting using anti-K-Rta antibody to confirm the induction of K-Rta in TREx-F3H3-K-Rta BCBL-1 cells after 0.2 μg/ml Dox treatment for 12 hours. Anti-α-tubulin antibody was used for loading control. (B) ChIP assay for SUMO paralogs was performed using chromatin prepared from non-induced (0 hour) and 0.2 μg/ml Dox-treated (12 hours) TREx-F3H3-K-Rta BCBL-1 cells using anti-SUMO-1 or SUMO-2/3 antibody. SUMO-1 and SUMO-2/3 binding to promoters in SUMO-2/3 enrichment and H3K9me3-rich regions were analyzed by real-time qPCR. ChIP enrichment was computed by comparing values obtained from Dox-treated samples to their non-induced controls. Rabbit IgG was used as negative antibody control and IgG enrichment is very low or not visible in both ChIP assays.(TIFF)Click here for additional data file.S3 Fig(A) TCLs from non-induced (0 hour) and 0.2 μg/ml Dox-treated (12 hours) TREx-MH-K-Rta BCBL-1 and TREx-MH-K-Rta-Flag-SUMO-1 and -Flag-SUMO-2 BCBL-1 cells were analyzed by immunoblotting using anti-Flag and anti-K-Rta antibodies. Anti-α-tubulin antibody was used for loading control. Ratio for each construct is the Flag/α-tubulin signal observed for Dox treatment at 12 hour using Dox at 0 hour set as 1.0. (B) ChIP was performed using chromatin prepared from cells treated as described in (A) using anti-Flag antibody. Rabbit IgG was used as negative antibody control and enrichment is not visible in most cases. SUMO-1 and SUMO-2 binding to SUMO-2/3 enrichment and H3K9me3-rich regions were analyzed by real-time qPCR using primer pairs as described in (TIFF)Click here for additional data file.S4 Fig18 primer. The expression level of SUMO-2, SUMO-3 (B), and four viral genes representing SUMO-2/3 enrichment region as described in (A) pLenti4-shSUMO-2 and -3 were introduced into TREx-BCBL-1 cells by lentiviral transduction. 0.2 μg/ml of Dox was added to cells 48 hours after transduction to induce SUMO-2/3 knockdown. TCLs were collected 24 hours after Dox treatment and analyzed by immunoblotting using anti-SUMO-2/3 antibody. Anti-α-tubulin antibody was used for loading control. (B and C) Total RNA isolated from cells treated as described in (A) was reverse transcribed using oligo-d(T)(TIFF)Click here for additional data file.S5 FigTCLs isolated from non-induced (0 hour) and 0.2 μg/ml Dox-induced  TREx-F3H3-K-Rta BCBL-1 and TREx-F3H3-K-Rta-shSUMO-2/3 BCBL-1 cells were subjected to immunoblotting analysis using anti-SUMO-2/3 antibody. Induction of K-Rta and expression of K-bZIP was confirmed by using anti-K-Rta and anti-K-bZIP antibodies. Anti-GAPDH antibody was used for loading control. Ratio for each cell line is the SUMO-2/3/GAPDH signal observed for Dox treatment at 0 (for shSUMO-2/3), 12, 24, and 48 hour using TREx-F3H3-K-Rta BCBL-1 cells at 0 hour set as 1.0.(TIFF)Click here for additional data file.S6 Fig(A) The expression level of three viral lytic genes representing the SUMO-2/3 enrichment and high H3K9me3 region were quantified by real-time qPCR using cDNA from (TIFF)Click here for additional data file.S7 Fig(A) TREx-F3H3-K-Rta BCBL-1 and TREx-F3H3-K-Rta-shSUMO-2/3 BCBL-1 cells were treated as described in (TIFF)Click here for additional data file.S8 Fig(A) Western blot analysis of LANA expression in iSLK-Puro-BAC16 K-bZIP-WT rev and -L75A cells. GAPDH was probed as control. Ratio for each cell line is the LANA/GAPDH signal observed for Dox treatment at 24 and 48 hour using Dox at 0 hour set as 1.0. (B) Genomic DNA from iSLK-Puro-BAC16 K-bZIP-WT rev and -L75A cells was prepared using phenol/chloroform extraction and KSHV genome copy was determined by real-time qPCR using orf19 and orf20 specific primer pairs. A primer pair specific for the promoter region of cellular gene RTP4 was used as control. The fold was computed by comparing K-bZIP-L75A copy number values to K-bZIP-WT rev.(TIFF)Click here for additional data file.S9 FigChIP for K-bZIP was performed using chromatin prepared from non-induced (0 hour) and 1 μg/ml Dox-treated (24 hours) iSLK-Puro-BAC16 K-bZIP-WT rev and -L75A cells using anti-K-bZIP antibody or rabbit IgG. K-bZIP binding to promoters in SUMO-2/3 enrichment and H3K9me3-rich regions was analyzed by real-time qPCR. Enrichment using rabbit IgG negative antibody control is not visible in some plots.(TIFF)Click here for additional data file.S10 Fig(A) iSLK-Puro-BAC16 K-bZIP-WT rev and -L75A cells were treated with 1 μg/ml Dox for 48 hours. Non-induced (-Dox) and Dox-treated (+Dox) cells were fixed by 4% paraformaldehyde and stained using anti-K-Rta and anti-K-bZIP antibodies. Representative immunofluorescence assay (IFA) stained images showing K-bZIP positive cells in K-Rta expressing cells. (B) The K-bZIP positive cells in K-Rta expressing cells were quantified from >20 microscopic fields. Number of K-bZIP positive cells (pink) among K-Rta expressing cells (red) was calculated as % of K-Rta positive cells. *; P<0.05(TIFF)Click here for additional data file.S11 Fig(A) The expression of K-Rta and K-bZIP in iSLK-Puro harboring parental BAC16 clone, iSLK-Puro-BAC16 K-bZIP-WT, and iSLK-Puro-BAC16 K-bZIP-WT rev and -L75A cells before and after Dox induction for 24 hours was analyzed by immunoblotting. GAPDH was probed as control. (B) Supernatants harvested from iSLK-Puro-BAC16 K-bZIP-WT, -WT rev and -L75A cells treated with or without Dox for 72 hours were filtered and used to infect 293T cells. GFP positive cells were analyzed by fluorescence microscopy  48 hours after infection. (C) The GFP positive cells were quantified using the average from >20 microscopic fields. ***; P<0.001. NS; non-significant.(TIFF)Click here for additional data file.S12 Fig(A) TCLs were collected from iSLK-Puro-BAC16 K-bZIP-WT rev and -L75A cells treated as described in (TIFF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.