MAF1 knockdown induced CDKN1A transcription and chromatin looping concurrently with Pol III recruitment. Simultaneous knockdown of MAF1 with Pol III or BRF1 (subunit of TFIIIB) diminished the activation and looping effect, which indicates that recruiting Pol III was required for activation of Pol II-mediated transcription and chromatin looping. Chromatin-immunoprecipitation analysis after MAF1 knockdown indicated enhanced binding of Pol III and BRF1, as well as of CFP1, p300, and PCAF, which are factors that mediate active histone marks, along with the binding of TATA binding protein (TBP) and POLR2E to the CDKN1A promoter. Simultaneous knockdown with Pol III abolished these regulatory events. Similar results were obtained for GDF15. Our results reveal a novel mechanism by which MAF1 and Pol III regulate the activity of a protein-coding gene transcribed by Pol II.MAF1 represses Pol III-mediated transcription by interfering with TFIIIB and Pol III. Herein, we found that DOI:http://dx.doi.org/10.7554/eLife.06283.001 An organism's genetic material is made of segments of DNA called genes, which contain instructions to make proteins. First, copies of the DNA are made using another molecule called ribonucleic acid (RNA) in a process known as transcription. Then the RNA is used as a template to make a protein. During transcription, enzymes called RNA polymerases move along the DNA to produce the RNA copies.When a cell is actively growing it needs large quantities of new proteins to be made, and so the level of transcription is higher. However, if a cell experiences stress caused by adverse environmental conditions , it can conserve resources by shutting down transcription. For example, one RNA polymerase—called Pol III—makes RNA copies with the help of a protein called BRF1 and several other proteins. However, when a cell is under stress, another protein called MAF1 can interfere with transcription by binding to BRF1, which prevents it from interacting with Pol III.CDKN1A.Previous work has suggested that MAF1 can also inhibit the activity of another RNA polymerase called Pol II, but it was not clear how this could work. Lee et al. studied the effect of MAF1 on transcription in human cells. The experiments show that MAF1 blocks the transcription of many genes that are transcribed by Pol II, including one called CDKN1A is involved in regulating many important processes, including the growth of cells and cell death. Cells that produced lower amounts of MAF1 had higher levels of CDKN1A transcription, and several proteins—including Pol II, Pol III and BRF1—were more able to bind to this gene. However, this effect was not observed in cells that also produced lower levels of Pol III or BRF1, suggesting that Pol III is needed for Pol II to be able to transcribe CDKN1A.CDKN1A, and possibly other genes transcribed by Pol II, by regulating the activity of Pol III. Further research is needed to understand the details of how this works.Taken together, Lee et al.'s findings suggest that MAF1 inhibits the transcription of DOI:http://dx.doi.org/10.7554/eLife.06283.002 Transcription by RNA polymerase III (Pol III) is regulated by MAF1, which is a highly conserved protein in eukaryotes . MAF1 reTBP transcription due to binding of MAF1 to the Elk-1 site on the TBP promoter  indicated that most of these genes are related to cell proliferation. Among them, CDKN1A  was significantly upregulated and the mechanism of induced transcription of this gene after MAF1 knockdown was further investigated.MAF1 has also been proposed to have the potential to repress Pol II-mediated transcription via repression of promoter . Thus, tCDKN1A is a cyclin-dependent kinase inhibitor that inhibits cell cycle progression through interaction with cyclins and cyclin-dependent kinases (CDKs). As a member of the Cip and Kip family of CDK inhibitors, CDKN1A mediates p53-dependent cell-cycle arrest at the G1 phase by inhibiting the activity of CDK2 and CDK1 . In addition, CDKN1A also inhibits the activity of proliferating cell nuclear antigen and blocks DNA synthesis and repair as well as cell-cycle progression. As a result, CDKN1A can regulate many cellular processes, such as proliferation, differentiation, apoptosis, metastasis, cell survival, and stem cell renewal. Expression of CDKN1A can be regulated at the transcriptional level by oncogenes and tumor suppressor proteins that bind various transcription factors to specific elements in response to a variety of intracellular and extracellular signals  and immunoblot using multiple siRNAs  and HCT116p53−/− (p53-null) cell lines to analyze whether the induced CDKN1A expression is dependent on p53. The absence of p53 in HCT11653−/− was confirmed by immunoblot  and a p53 mutant breast cancer cell line (MDA-MB-231)  . TogetheCDKN1A activation after MAF1 knockdown could be due either to interference of binding of transcription factors to the CDKN1A promoter by MAF1 or to the active recruitment or activation of Pol III after MAF1 knockdown. To determine which of these two mechanisms are involved in this process, we carried out simultaneous knockdown of both MAF1 and Pol III. The former mechanism would not be affected by the double knockdown, whereas the latter would be. The effect of Pol III knockdown was analyzed by qRT-PCR and immunoblot using multiple siRNA sequences  content . The hignockdown . We perfnockdown . MAF1 knormation . The R-lnockdown . The resCDKN1A expression is strongly upregulated after MAF1 knockdown, and simultaneous knockdown of MAF1 and Pol III diminished the induced expression. These results indicate that removal of MAF1 may induce recruitment of Pol II and Pol III to activate transcription. To examine this possibility, chromatin-immunoprecipitation (ChIP) analysis followed by quantitative PCR (qPCR) was performed under various knockdown conditions. Efficiency of MAF1 or Pol III knockdown as well as simultaneous knockdown of MAF1 and Pol III was verified by the binding of Pol III to pretRNAArg and pretRNALeu genes. The results showed enhanced binding of Pol III at pretRNAArg and pretRNALeu after MAF1 knockdown, and the binding was diminished after double knockdown  and NM_001220778 (short form), which are 2.25 kb apart  that could be transcribed by Pol III and therefore might represent the target-binding site of MAF1. To test this possibility, we used an in vitro DNA binding reaction . In vitro DNA-protein binding assays performed by the above method or colorimetric assay  both showed that the DNA with deleted or mutated A-box sequences exhibited significantly lower binding of MAF1, which indicates the specificity of MAF1 for the Pol III promoter element  and p300 were shown to act cooperatively to regulate H3K4me3 modification and CDKN1A transcription (MAF1 knockdown-induced transcription of CDKN1A was downregulated after CFP1 (CXXC1) knockdown , after MAF1 knockdown, and this enhanced binding was also abolished when there was simultaneous knockdown of Pol III and MAF1 (Because TBP is an important factor required in both Pol II (part of TFIIB) and Pol III (part of TFIIIB) transcription , we nextand MAF1 . Simultanly MAF1 , which iand MAF1 , indicatCDKN1A forms in the UCSC Genome Database after MAF1 knockdown, we performed 3C analysis to investigate whether chromatin looping occurs between these two regions. Chromatin looping was detected between the regions after MAF1 knockdown, but not in adjacent regions (MAF1 with either Pol III or BRF1 (a subunit of TFIIIB) disrupted the looping formation  in the reporter and examined the effect of mutation on reporter expression. Under regular cell culture conditions, no significant change in GDF15 promoter activity was observed in our results when the A-box was mutated or deleted, in consistent with the results of GDF15 promoter diminished the upregulation of the reporter after MAF1 knockdown  of Pol II and Pol III demonstrate a novel mechanism of activating Pol II genes through Pol III-mediated activation mechanism. Gene expression induced by chromatin remodeling through SINEs has also been described in neuronal genes that undergo acetylation of distal promoter SINEs by p300 to translocate genes to transcription factories . The chromatin looping we observed for CDKN1A and GDF15 after MAF1 knockdown may be mediated through the proximity and interaction between the sets of transcriptional factors recruited in the two 5′ flanking regions after chromatin remodeling as proposed by Histone H3K4me has been suggested to serve as a hallmark of enhancer . Examinar region . HoweverBecause the background expression of Pol II genes slightly decreased when Pol III was knockdown, Pol III may be able to regulate the expression of some minor alleles without MAF1 being bound to the promoter. However, it is difficult to unequivocally validate this possibility without specific technology that can efficiently separate different alleles in cells.MAF1 knockdown. Of these, 76% contained SINEs within the promoter region, which indicates the regulation potential of these genes by MAF1 and Pol III. However, microarray analysis of steady-state mRNA level alone is not sufficient to show whether these genes are directly regulated by MAF1 at the transcriptional level or as the result of downstream secondary effects. Further analysis using in vitro transcription, reporter gene analysis, and nuclear run-on would be required to unambiguously establish how general Pol II genes are regulated by MAF1 and Pol III. In vitro transcription using HeLa cell extracts with depletion or addition of specific transcription regulators could provide very strong support of the involvement of specific factors in transcription regulation.Human genome analysis indicated that 71% of genes contained SINEs in their promoter regions. Microarray analysis showed that 124 genes were upregulated after RPPH1, to which BRF2, Pol III, GTF2B, and Pol II bind, can be transcribed by either Pol II or Pol III  and B-box (−105 to −95) that encompass the Elk-1-binding site of the TBP promoter. In our analysis, MAF1 knockdown only slightly increased TBP expression (1.6-fold) compared with the results reported by Johnson et al. (twofolds). This may be due to the already high expression of TBP in cell lines, and we did not detect binding of MAF1 to this active promoter.Recently, MAF1 was shown to be a negative regulator of transcription of all three polymerases, Pol I, Pol II, and Pol III, through mediating pression . JohnsonMAF1 by knockdown then leads to recruitment of TFIIIB through enhanced binding of TBP; the shared surface of TBP then directs both Pol II and Pol III binding through association with TFIIB and TFIIIB, respectively. Further recruitment of active regulatory factors would then induce transcription by Pol II and Pol III. This model is consistent with those proposed by previous studies, which were based on a component of TBP-associated complexes, p300, interacting with SET1C-coupled histone modifications to activate CDKN1A transcription  and HCT116p53−/− (p53-null) cell lines were originated from Bert Vogelstein (John Hopkins University) and cultured in McCoy's 5A medium , and cultured in RPMI, HuMEC and DMEM medium , respectively. HCT-116A medium . Each meMAF1 , Pol III (POLR3A) , BRF1 (SASI_Hs01_00131187), CFP1 (SASI_Hs02_00322879), and TBP (SASI_Hs01_00122768) was achieved by transfection with Lipofectamine RNAiMax (Invitrogen) according to the manufacturer's protocol for 72 hr. MISSION siRNA Universal Negative Control (Sigma) was used as knockdown control. Cells were transfected in serum-free medium. After 8 hr, the siRNA containing medium was replaced with complete medium.Knockdown assay was performed using siRNA obtained from MISSION RNA . Inhibition of expression of g for 15 min), resolved on a 10% SDS-polyacrylamide gel, and transferred onto a nitrocellulose membrane. The antibody dilutions used were rabbit anti-POLR3A , rabbit anti-MAF1 , rabbit anti-CDKN1A , and mouse anti-tubulin .Cells were lysed at 4°C in RIPA lysis buffer . The lysates were cleared by centrifugation  for three times. Total RNA was extracted using TRIreagent (Invitrogen) protocol. The integrity of the RNA extract was checked by 1.2% (wt/vol) agarose gel electrophoresis and the concentration of RNA was estimated by ultraviolet spectrophotometry.Affymetrix microarray was performed using Human U133 plus 2.0 . Details of the methods for RNA quality, sample labeling, hybridization, and expression analysis were according to the manual of Affymetrix Microarray Kit. All Affymetrix data are MIAME compliant and that the raw data have been deposited in a MIAME compliant database, GEO. The microarray data were deposited at the NCBI GEO website (GEO accession number GSE42239).GAPDH) or 18S to obtain the relative gene expression.Reverse transcription was performed by using superScript III RNase H- Reverse Transcriptase (Invitrogen) and random hexamer according to the manufacturer's protocol. Quantitative PCR was performed using KAPA SYBR FAST  on ABI StepOnePlus Real-Time PCR System (Invitrogen). All reactions were performed in triplicate with KAPA SYBR FAST plus 10 μM of both the forward and reverse primer according to the manufacturer's recommended thermo cycling conditions, and then subjected to melting curve analysis. The calculated quantity of the target gene for each sample was divided by the average sample quantity of the housekeeping genes, glyceraldehydes-3-phosphate dehydrogenase .MCF-7 knockdown cells were collected by trypsinization and washed twice with ice-cold PBS. The cells were resuspended in 0.3 ml of PBS and fixed by slowly adding 3 ml of 70% cold ethanol. Cells were fixed at −20°C for 1 hr. The fixed cells were washed with ice-cold PBS and rehydrated for 15 min. After centrifuging at 200×2, 150 mM sucrose, and 0.5% Nonidet P-40). Nuclei were isolated, and the pellets were resuspended in 1 ml of glycerol buffer . 1 ml of transcription buffer  was added in the nuclei along with 10 μl of biotin-16-UTP or UTP for run-on reaction or negative control, respectively (Roche). After incubation at 29°C for 30 min, the reaction was terminated by the addition of 12 μl of 250 mM CaCl2, and 12 μl of RNase-free DNase I and incubated at 29°C for 10 min. To purify RNA, a TRIreagent extraction, phenol-chloroform extraction, and isopropanol (Sigma) precipitation were then performed. A small aliquot  was saved as input control. Dynabeads M-280 streptavidin (Invitrogen) were mixed with an equal volume of the isolated RNA samples for 20 min at 42°C for 20 min and 2 hr at room temperature. After washing with 15% formamide and 2× SSC, the beads were resuspended in 45 μl of nuclease-free water. Reverse transcription was performed by using superScript III RNase H—Reverse Transcriptase (Invitrogen). Total cDNA was then synthesized by means of random hexamer primed reverse transcription of captured molecules. The gel pictures were quantified with ImageJ (provided by NIH: http://imagej.nih.gov/ij/). The purified run-on products where normalized with internal control (GAPDH) to obtain the relative transcription levels for each gene.Nuclear run-on reactions were performed by supplying biotin-16-UTP to nuclei, and labeled transcripts were bound to streptavidin-coated magnetic beads as described by MAF1 (SASI_Hs01_00135954) was achieved by transfection with Lipofectamine RNAiMax (Invitrogen) according to the manufacturer's protocol for 72 hr. DNA purification and single-stranded R loop foot-printing were carried out as previously described with slight modifications  may be seen possibly due to DNA breathing during the prolonged incubation at 37°C in our data and others (Knockdown assay was performed using siRNA obtained from MISSION RNA (Sigma). Inhibition of expression of Pol III (SASI_Hs01_00046568) and ications . 500 ng d others . Approxid others .3. Proteinase K was added to the samples, and rotated at 65°C for 2 hr followed by 95°C for 10 min and cooled down to room temperature. RNase A was added and samples were incubated at 37°C for 1 hr. After genomic DNA extraction, qPCR was performed. The degree of enrichment is calculated relative to the ratio of signals obtained in the input DNA fraction subtracting IgG-immunoprecipitated DNA.ChIP assay was performed according to the manufacturer's protocol  with slight modifications. Human MCF-7 cells were fixed with 1% of formaldehyde at room temperature for 10 min. The cells were lysed and the chromatin was sonicated to 200–500 bp fragments by Bioruptor sonicator . Chromatin was immunoprecipitated by using Pol III , Pol II , MAF1 , H3K4me3 , H3K27me3 , TBP , H3K9Ace , H3K27Ace , CFP1 , p300 , POLR2E  BRF1 or IgG  antibody, with 10 μg/ml of BSA and 50 μl of Dynabeads Protein A and G (Invitrogen) for overnight at 4°C. The beads were washed once with each washing buffer, including low salt immune complex wash buffer, high salt immune complex wash buffer, and LiCl immune complex wash buffer, and twice with 1× TE buffer. Precipitates were eluted with 1% of SDS and 100 mM of NaHCO3C assay was performed according to  with somCDKN1A (with or without MIR3), ACTB, GDF15 (including deleted or mutated A-box) and RPPH1 template DNA was obtained by PCR followed by gel elution  according to the manufacturer's protocol. The deletion of MIR3 was performed as described by PCR-mediated deletion and checked by sequencing (3. Input DNA was prepared as 1 ng of template DNA (5% of 20 ng). Proteinase K was added to the samples, and rotated at 65°C for 2 hr followed by 95°C for 10 min and cooled down to room temperature. DNA isolated from immunoprecipitated protein-DNA complex was subjected to qPCR. The degree of enrichment is calculated relative to the ratio of signals obtained in the input DNA fraction.quencing . The purquencing . The in CDKN1A (5′-AATCAACAACTTTGTATACTTAAGTTCAGTGGACCTCAATTTCCTCATCTGTGAAATAAA-3′) as well as mutated A-box template DNA (5′-AATCAACAACTTTGTATA CTTCCCATCCCAAAACCTCAATTTCCTCATCTGTGAAATAAA-3′) was obtained by oligo synthesis from Genomics . The oligos were annealed and used for in vitro DNA-protein binding assay by the DNA-Protein Binding Assay Kit (Colorimetric) provided by Abcam (ab117139). The assay was performed according to manufacturer's protocol. In brief, 40 ng of biotin-labeled DNA template and 500 ng of MAF1 protein (His tag)  was used for the binding assay. For competition assay, 200 ng of competitor DNA was added to the mixture. Anti-6× His tag antibody  and Goat anti-Mouse IgG2b heavy chain (HRP) antibody  were prepared and added according to manufacturer's protocol. Blank control was performed without the addition of protein as specified by the kit and the degree of enrichment is calculated by subtracting with blank control.Biotin-labeled (labeled at 5′) and non-labeled CDKN1A (−864 to +41 of NM_001220778 for short form and −1249 to +92 of NM_001220777 for long form), GDF15 (−889 to +110), and TAF5 (−998 to +157) genes were cloned into the pGL3-basic Reporter Vector . Knockdown assay was performed as mentioned above for 24 hr before MCF-7 cells were transfected with the plasmids using Lipofectamine LTX (Invitrogen), along with a plasmid expressing β-galactosidase for normalization. The plasmids were transfected for 48 hr, and the cells were lysed and luciferase assay was conducted using the Luciferase Assay System (Promega) using a fluorimetric plate reader.The upstream promoter regions of R Nuclear Extract in vitro Transcription System (Promega Cat. #E3110) according to the manufacturer's protocol with slight modifications. Template DNA was prepared by linearizing the constructed promoter region of CDKN1A, GDF15, and TAF5 as used in Luciferase assay, as well as RPPH1 promoter as used in in vitro MAF1 binding assay. In vitro transcription was performed by incubation with linear form of constructed promoter region with nuclear extract, transcription buffer, magnesium ion, GTP, CTP, ATP, biotin-16-UTP, RNase inhibitor, and 30 μg of yeast tRNA. Negative control was performed by incubation with non-biotin labeled NTPs. 0.2 μg of α-amanitin was added during in vitro transcription for inhibition of Pol II transcription. 3 μg of MAF1 protein (His tag)  used in in vitro MAF1 binding assay was pre-incubated with template DNA before adding nuclear extract to enable binding of MAF1 to the template DNA. Anti-Pol III , anti-Pol II , anti-MAF1 , or anti-IgG  antibody was pre-incubated with nuclear extract for 15 min to deplete the target protein of interest. After incubation at 30°C for 1 hr, the reaction was terminated by the addition of 175 μl of HeLa Extract Stop Solution (Promega Cat. #E3110). A TRIreagent extraction, phenol-chloroform extraction, and isopropanol (Sigma) precipitation were then performed to purify RNA. A small aliquot  was saved as ‘total nuclear RNA’ for each condition. The biotinylated RNA was isolated using streptavidin-coated magnetic beads as described in Run-on assay. Reverse transcription was performed by using superScript III RNase H- Reverse Transcriptase (Invitrogen). Total cDNA was then synthesized by means of random hexamer primed reverse transcription of captured molecules.In vitro transcription was performed by using HeLaScribe review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and four reviewers, one of whom is a member of our Board of Reviewing Editors.Thank you for sending your work entitled “MAF1-regulated-Pol III-mediated activation of The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.The reviewers found the findings to be potentially exciting as they extend and expand thinking about how protein-coding genes are transcribed, in particular a novel role for RNA polymerase III in this process. Overall, this report presents some exciting new findings that are very timely in view of (i) extensive ChIPseq data in the literature demonstrating the localization of components of the Pol III transcription machinery adjacent to Pol II-transcribed genes and (ii) the widespread localization of SINE elements (of unknown function) in the human genome and near promoters. However, the reviewers found several issues that must be addressed in the revised manuscript before it could be recommended for publication:CDKN1A and GDF15, are the two most potently transcribed p53 target genes . However, the authors do not examine the effects that p53 may play in their system. It is entirely possible that most of the effects described upon MAF1 knockdown are driven indirectly by activation of p53 downstream of a cellular stress response caused by deregulated RNAPIII function. The authors should check for p53 induction upon MAF1 knockdown. Key experiments should be repeated in an isogenic system -/+ p53. This could be done with stable p53 knockdown MCF7 cells  or HCT116 p53 -/+ cells .1) The possible role of p53. The two genes examined throughout the manuscript, CDKN1A gene shows that there are two promoter regions (hereafter referred as long and short form)’. This description contrasts with many earlier reports that clearly show a major promoter for CDKN1A with minor alternative start sites. What exactly are the long and short forms? What NM genebank reference numbers do they correspond to? Where are they located relative to the well-characterized p53 enhancers? Importantly, the authors show equivalent Ser5-phospho-RNAPII binding to both promoters, but high H3K4me3 and TBP binding are found only at the downstream promoter encoding the ‘short form’, indicating that this promoter is the functional promoter. Confusingly, although the authors show equivalent binding of Ser5-phospho-RNAPII to both promoters, RBP5, a subunit common to all three polymerases is found preferentially at the downstream promoter. How much RNA is being produced from the ‘long’ and ‘short’ promoters? Some form of quantitative assay should be done to define the relative strength and RNA output of these two ‘promoters’.2) Promoter definition. The authors indicate that ‘Examination of MAF1 knockdown enhanced CDKN1A gene expression from both long and short form promoters through Pol III-mediated promoter looping’. In the next subheading they repeat: ‘MAF1 knockdown enhanced GDF15 gene expression through PolIII-mediated promoter looping’. This is a massive misinterpretation of data, because there is no experiment showing a cause-effect relationship between looping and gene activity, only a correlation. The authors state that ‘Moreover, simultaneous knockdown of MAF1 with either Pol III or BRF1 (subunit of TFIIIB) disrupted the looping formation  Massive misinterpretation of ‘chromatin looping’ data. The authors repeatedly state throughout the manuscript (including the Title) a cause-effect relationship between chromatin looping and gene activation. They state in a subheading of the results that ‘ormation . These oMAF1 may represent a peculiar result of the specific genetic make-up of this single, genetically complex cell type.4) The authors rely on the use of a single cell line, MCF7. As these are breast cancer cells that have sustained multiple genetic alterations, it is unclear whether the findings will hold in normal cells or even in other tumor cell lines. This needs to be addressed as the results with 5) The bulk of the findings rely in the use of RNAi approaches but the authors use only a single targeting sequence for the knockdown of each particular target. Multiple targeting sequences need to be used and the extent of downregulation at both the protein and RNA levels needs to be shown to reduce the possibility of off-target effects and to further validate the conclusions.6) As a general comment both on the cellular knockdown data and the in vitro immuno-depletion data, the authors do not generally indicate the extent of depletion of the factors of interest, and this may well account for some of the modest, albeit significant, effects. Thus it would help to have data/comments on the depletion efficiencies.MAF1 knockdown on CDKN1A expression appears relatively modest  and perhaps improved data?7) In the nuclear run-on assay of CDKN1A expression. However, a close examination the data in CDKN1A is actually reduced about 30% (and that of GDF15 more than 50%). This fits the model that Pol III can positively regulate the expression of CDKN1A and GDF15, but is potentially inconsistent with the authors' failure to see any Pol III occupancy prior to MAF1 knockdown. Is this just due to the lower sensitivity and higher background in the ChIP analysis?8) The authors state that knocking down Pol III alone did not affect MAF1 ChIP signal is only 3-fold above background. Because this data provides important evidence of direct regulation, and even though it is supported by the in vitro demonstration of direct MAF1 binding to the SINE element, the authors might consider ways to modify the ChIP protocol to increase the signal/background ratio.9) In MAF1 binding to the p21 SINE element 10) While the in vitro data for the direct  element  seems coMAF1 mechanism involving direct binding to promoter elements, the reader is advised (in the Introduction) of prior reports indicating MAF1 binding to BRF1 and/or Pol III as well as Pol III/MAF1 co-occupancy on target genes, and thus anticipates that MAF1 may be repressing Pol III function by a related mechanism on the target genes analyzed here. This, of course, appears not to be the mechanism from the ChIP analyses that follow the functional studies, but I think the rationale and results could be presented with more clarity and with clear indications that, perhaps surprisingly, they do not fit the original models but suggest an alternate mechanism that is pursued (by the direct binding studies).11) In relation to the interesting  new [Editors’ note: further concerns were raised before acceptance.]CDKN1A and Chromatin Looping” for further consideration by eLife. Your revised submission has been evaluated by the original Reviewing Editor and Senior Editor.Thank you for resubmitting your article entitled “MAF1-Regulated-Pol III-Mediated Activation of eLife. The authors have performed additional experiments in response to the reviewers suggestions, mostly to include missing controls and replicates, but the manuscript still fails to produce a clear picture of how exactly MAF1 and POLIII regulate protein-coding genes such as CDKN1A and GDF15.Despite a commendable effort by the authors to try to address the concerns raised by the reviewers, the revised manuscript fails to meet the high standards of scholarship expected for publication in MAF1 and POLIII likely affecting the activity of the enhancer region at the CDKN1A locus, and the manuscript should be rewritten in light of this fact.The major issue is that, in response to a request for clarification from the reviewers, it is now evident that the genomic region that the authors have called the ‘p21 long promoter’ (p21L) is indeed the enhancer regulatory region of this gene, known to be bound by p53 and many other transcription factors. This region, located 2.25kb upstream of the canonical p21 promoter, produces enhancer-derived RNAs (eRNAs) and may engage with the promoter through a chromatin loop. In view of this, the entire dataset must be reinterpreted as Finally, the revised manuscript failed to correct some points raised by reviewers. As an example of a major oversight, the authors tested for ‘p53 activation’ by Q-RT-PCR, when it is well established that p53 activation upon cellular stress occurs at the protein level, and thus should have been tested by Western blot. 1) The possible role of p53. The two genes examined throughout the manuscript, CDKN1A and GDF15, are the two most potently transcribed p53 target genes . However, the authors do not examine the effects that p53 may play in their system. It is entirely possible that most of the effects described upon MAF1 knockdown are driven indirectly by activation of p53 downstream of a cellular stress response caused by deregulated RNAPIII function. The authors should check for p53 induction upon MAF1 knockdown. Key experiments should be repeated in an isogenic system -/+ p53. This could be done with stable p53 knockdown MCF7 cells  or HCT116 p53 -/+ cells .CDKN1A and GDF15 are p53 target genes . Since CDKN1A expression is induced in both of these cell lines, the induction is independent of p53  Promoter definition. The authors indicate that ‘Examination of CDKN1A gene shows that there are two promoter regions (hereafter referred as long and short form)’. This description contrasts with many earlier reports that clearly show a major promoter for CDKN1A with minor alternative start sites. What exactly are the long and short forms? What NM genebank reference numbers do they correspond to? Where are they located relative to the well-characterized p53 enhancers? Importantly, the authors show equivalent Ser5-phospho-RNAPII binding to both promoters, but high H3K4me3 and TBP binding are found only at the downstream promoter encoding the ‘short form’, indicating that this promoter is the functional promoter. Confusingly, although the authors show equivalent binding of Ser5-phospho-RNAPII to both promoters, RBP5, a subunit common to all three polymerases is found preferentially at the downstream promoter. How much RNA is being produced from the ‘long’ and ‘short’ promoters? Some form of quantitative assay should be done to define the relative strength and RNA output of these two ‘promoters’.CDKN1A with minor alternative start sites. Indeed, most published papers refer to the transcription start site (TSS) of short form promoter, whereas the well-characterized p53 enhancers are located 1.4 kb and 2.3 kb upstream from TSS, which is near long form promoter . In our manuscript, the long form promoter refers to the upstream region of NM_001220777, whereas the short form promoter refers to the upstream region of NM_001220778 according to NM genebank reference numbers (CDKN1A (“L-Ex-1” for long form and “Ex1” for short form) with respect to the identified p53 response elements and ChIP amplicons  is depicted in We thank you for highlighting that many earlier reports clearly show a major promoter for  numbers . The relCDKN1A gene in the UCSC Genome Database shows that there are two transcription start sites, NM_001220777 (long form) and NM_001220778 (short form), which are 2.25 kb apart” (CDKN1A promoter”). Although the expression of both forms is induced after MAF1 knockdown, the short form has a higher expression level and stronger promoter activity in MCF-7 cell line  Massive misinterpretation of ‘chromatin looping’ data. The authors repeatedly state throughout the manuscript (including the Title) a cause-effect relationship between chromatin looping and gene activation. They state in a subheading of the results that ‘MAF1 knockdown enhanced CDKN1A gene expression from both long and short form promoters through Pol III-mediated promoter looping’. In the next subheading they repeat: ‘MAF1 knockdown enhanced GDF15 gene expression through PolIII-mediated promoter looping’. This is a massive misinterpretation of data, because there is no experiment showing a cause-effect relationship between looping and gene activity, only a correlation. The authors state that ‘Moreover, simultaneous knockdown of MAF1 with either Pol III or BRF1 (subunit of TFIIIB) disrupted the looping formation . These observations suggest chromatin looping between the long and short form promoter regions is important for the activation of CDKN1A transcription from both promoters’, and ‘The above results demonstrate that MAF1 knockdown can activate CDKN1A expression through promoter looping induced by recruitment of Pol III and Pol II along with histone-modifying factors’. Later on, they state ‘These results further demonstrate unambiguously that MAF1 protein represses the promoter activity of CDKN1A and GDF15, and that recruitment of Pol III after MAF1 depletion is crucial for the activation of transcription of these genes through promoter looping’. The looping observed may as well be a consequence, rather than a driver, of gene activation, or just as well a totally inconsequential event. The manuscript should be rewritten to remove all indication of cause-effect relationship between looping and gene activation.CDKN1A and Chromatin Looping”. The subheadings were also modified to “Pol III is required for transcriptional activation and chromatin looping of CDKN1A/GDF15 after MAF1 knockdown” (in the Results section).Indeed, we have no evidence that chromatin looping plays a causal role in the gene expression of the two genes we studied. Therefore, we have rewritten the manuscript to remove the indications of cause-effect relationship between looping and gene activation. We have changed the title to “MAF1-Regulated-Pol III-Mediated Activation of 4) The authors rely on the use of a single cell line, MCF7. As these are breast cancer cells that have sustained multiple genetic alterations, it is unclear whether the findings will hold in normal cells or even in other tumor cell lines. This needs to be addressed as the results with MAF1 may represent a peculiar result of the specific genetic make-up of this single, genetically complex cell type.p53+/+ (wild-type) and HCT116p53-/- (p53-null), to repeat our experiments. We performed quantitative RT-PCR to analysis the effect of MAF1 knockdown and simultaneous knockdown of MAF1 and POLR3A on the expression of CDKN1A and GDF15 in these four cell lines. Consistent with the MCF-7 results, the expression of CDKN1A and GDF15 is induced after MAF1 knockdown, and simultaneous knockdown of MAF1 and POLR3A abolished the induced expression (MAF1 knockdown strongly upregulated CDKN1A expression”). Together, these results demonstrate that MAF1 regulates CDKN1A expression in non-tumorigenic (MCF-10A), as well as breast (MCF-7 and MDA-MB-231) and colon cancer (HCT116p53+/+ and HCT116p53-/-) cells, and is independent of p53.To address this question, we choose four other cell lines, MCF-10A (non-tumorigenic), MDA-MB-231, HCT1165) The bulk of the findings rely in the use of RNAi approaches but the authors use only a single targeting sequence for the knockdown of each particular target. Multiple targeting sequences need to be used and the extent of downregulation at both the protein and RNA levels needs to be shown to reduce the possibility of off-target effects and to further validate the conclusions.MAF1 and POLR3A. Quantitative RT-PCR and western blot were performed to analyze the knockdown effect of MAF1 and POLR3A using three different siRNAs, including the original one. The results showed that the three siRNAs for MAF1 depleted MAF1 and up-regulated tRNA genes, as well as CDKN1A and GDF15 (MAF1 knockdown strongly upregulated CDKN1A expression”). However, probably due to slow turnover time of MAF1 protein, western blot analysis showed only 45% of reduction for the MAF1 protein in cells treated with siRNA  with the strongest affect. Nevertheless, the reduction percentage for MAF1 is consistent with the up-regulation of CDKN1A in mRNA and protein level, indicating that depletion of MAF1 induces CDKN1A expression . The siRNAs with the strongest knockdown affect for MAF1  and POLR3A  were used to perform knockdown in ChIP, 3C and reporter assays.To address this question, we ordered two more siRNA targeting sequences for 6) As a general comment both on the cellular knockdown data and the in vitro immuno-depletion data, the authors do not generally indicate the extent of depletion of the factors of interest, and this may well account for some of the modest, albeit significant, effects. Thus it would help to have data/comments on the depletion efficiencies.MAF1 and POLR3A are reduced about 45% and 60% in protein level, respectively  (MAF1 and Pol III). We also used anti-Pol III antibody on Pol III transcribed-RPPH1 to show that the reduction is 79%±1%  In the nuclear run-on assay of, the effect of the MAF1 knockdown on CDKN1A expression appears relatively modest . Could the authors provide quantitation of the data for CDKN1A  and perhaps improved data?MAF1 knockdown strongly upregulated CDKN1A expression”). The gel pictures were quantified with ImageJ (provided by NIH: http://imagej.nih.gov/ij/). The purified run-on products where normalized with internal control (GAPDH) to obtain the relative transcription levels for each gene. MAF1 knockdown induced the expression of CDKN1A and GDF15 for 2.9±0.8 and 88.8±12.2 folds, respectively, in run-on assay. This shows that MAF1 knockdown induces newly transcribed CDKN1A and GDF15 mRNA, and the up-regulation is not due to post-transcriptional processing or RNA turnover rates. The run-on results were moved from We repeated run-on assay to provide quantification results (n=3) and a more improved gel picture in 8) The authors state that knocking down Pol III alone did not affect CDKN1A expression. However, a close examination the data insuggest that expression of CDKN1A is actually reduced about 30% (and that of GDF15 more than 50%). This fits the model that Pol III can positively regulate the expression of CDKN1A and GDF15, but is potentially inconsistent with the authors' failure to see any Pol III occupancy prior to MAF1 knockdown. Is this just due to the lower sensitivity and higher background in the ChIP analysis?CDKN1A and GDF15 is indeed reduced about 30% and 50%, respectively, in Pol III knockdown, which fits the model that Pol III can positively regulate the expression of CDKN1A and GDF15. However, the occupancy of Pol III prior to MAF1 knockdown is relatively very weak. Thus, we speculate that since the down-regulation of CDKN1A and GDF15 is not as efficient as tRNA genes after Pol III knockdown, the amount of Pol III binding at CDKN1A and GDF15 prior to MAF1 knockdown may be too low to be differentiated from background (MAF1/Pol III knockdown). Thus, although knockdown Pol III showed a decrease in Pol III binding at tRNA genes, the amount of Pol III is relatively the same as the CDKN1A and GDF15 knockdown control and MAF1/Pol III knockdown. It is possible that there is a minor subpopulation of alleles with Pol III bound at the promoter, and they are responsible for the low level background transcription. Knockdown of Pol III will thus reduce the background expression level from this subset of alleles. However, the low level of binding does not allow us to show that unequivocally. Therefore, we modify the manuscript in the Discussion to state that Pol III may be able to regulate expression of CDKN1A and GDF15, before MAF1 knockdown, but the low level of Pol III binding prior to MAF1 knockdown does not allow us to validate this possibility unequivocally (Discussion).The expression of 9) In, the MAF1 ChIP signal is only 3-fold above background. Because this data provides important evidence of direct regulation, and even though it is supported by the in vitro demonstration of direct MAF1 binding to the SINE element, the authors might consider ways to modify the ChIP protocol to increase the signal/background ratio.MAF1 ChIP is also relatively higher than other published MAF1 ChIP data, which showed a 2-fold signal/background ratio at Pol II and Pol III promoter in yeast and mammalian cell lines  during complex binding, as well as using various washing conditions . The condition used (see Methods) is already our optimized condition after testing various conditions. Our 3-fold signal/background ratio for l., 2011 ; Johnsonl., 2011 ; Palian l., 2011 ). Whethe10) While the in vitro data for the direct MAF1 binding to the p21 SINE element  seems convincing, especially with the SINE deletion control, it would be very interesting to have more detailed data on the interaction site in light of the novelty of this observation. Have the authors any further information from DNase footprinting, gel shifts with point mutations in the Pol III promoter elements etc. on this point, especially in relation to their suggestion in the Discussion that “MAF1 and Pol III/TFIIIB may recognize similar DNA elements”? CDKN1A promoter. We performed in vitro binding assay using the A-box-mutated SINE element as described before (MAF1 binds to the short interspersed element (SINE) of CDKN1A”). In addition, we also performed in vitro DNA-protein binding assay by using a colorimetric assay kit provided by Abcam (ab117139) to further validate that MAF1 binds to the Pol III promoter element, but not the mutant form  In relation to the interesting  new MAF1 mechanism involving direct binding to promoter elements, the reader is advised (in the Introduction) of prior reports indicating MAF1 binding to BRF1 and/or Pol III as well as Pol III/MAF1 co-occupancy on target genes, and thus anticipates that MAF1 may be repressing Pol III function by a related mechanism on the target genes analyzed here. This, of course, appears not to be the mechanism from the ChIP analyses that follow the functional studies, but I think the rationale and results could be presented with more clarity and with clear indications that, perhaps surprisingly, they do not fit the original models but suggest an alternate mechanism that is pursued (by the direct binding studies).We thank you for highlighting that we should modify the manuscript to present the rationale and results in more clarity and indications that our model does not fit the original models. Therefore, we have rewritten the last paragraph of the Introduction and modified the first paragraph of the Discussion to suggest an alternate mechanism as shown by the direct binding studies.[Editors’ note: a second round of author responses follow.]Despite a commendable effort by the authors to try to address the concerns raised by the reviewers, the revised manuscript fails to meet the high standards of scholarship expected for publication in eLife. The authors have performed additional experiments in response to the reviewers suggestions, mostly to include missing controls and replicates, but the manuscript still fails to produce a clear picture of how exactly MAF1 and POLIII regulate protein-coding genes such as CDKN1A and GDF15.Overall, this report presents some exciting new findings that are very timely in view of (i) extensive ChIPseq data in the literature demonstrating the localization of components of the Pol III transcription machinery adjacent to Pol II-transcribed genes and (ii) the widespread localization of SINE elements (of unknown function) in the human genome and near promoters”. The decision is based mainly on three criticisms, which we would like to respond to here.not PCNA as stated in the rejection letter. PCAF is lysine acetyltransferase 2B , also known as P/CAF, or KAT2B; and it has been shown to be associated with p300 for chromatin modification. We performed ChIP of PCAF to show that this epigenetic factor becomes associated with CDKN1A promoter after MAF1 knockdown  is indeed the enhancer regulatory region of this gene, known to be bound by p53 and many other transcription factors. This region, located 2.25kb upstream of the canonical p21 promoter, produces enhancer-derived RNAs (eRNAs) and may engage with the promoter through a chromatin loop. In view of this, the entire dataset must be reinterpreted as MAF1 and POLIII likely affecting the activity of the enhancer region at the CDKN1A locus, and the manuscript should be rewritten in light of this fact.CDKN1A) is located 2.25 kb upstream of NM_001220778 (short form CDKN1A) TSS. However, the chromatin looping and identified MAF1 binding site by ChIP is 2.65 kb upstream of TSS. Therefore, to address the possibility of enhancer activity at this region, we cloned the 2.25 ∼ 2.65 kb upstream region into the promoter of CDKN1A (CDKN1A-S) used in our original luciferase assay. The promoter activity was analyzed by luciferase assay and the results showed that the cloned 5′ region, both in sense (labeled as “LE(+)”) and antisense direction (labeled as “LE(-)”) does not exert enhancer activity on CDKN1A promoter after MAF1 knockdown as shown below . Both quantitative RT-PCR and western blot showed that the expression of CDKN1A is induced after MAF1 knockdown in p53-null HCT116 cell line (CDKN1A is still induced after MAF1 knockdown, indicating the induced transcription is independent of p53. Consistent with this result, we also showed that induction of CDKN1A expression in MDA-MB-231 cell line, which carries mutation in p53 (The other concern raised by the editors is that we failed to test p53 level by western blot to determine whether the induced


The basic helix-loop-helix transcription factor Twist1 has well-documented roles in progenitor populations of the developing embryo, including endocardial cushions (ECC) and limb buds, and also in cancer. Whether Twist1 regulates the same transcriptional targets in different tissue types is largely unknown.Chst11, Litaf, Ror2, and Spata5 also bound the potential Twist1 cofactor RREB1. Pathway analysis of the genes associated with Twist1 binding suggests that Twist1 may regulate genes associated with the Wnt signaling pathway in ECCs and limb buds.The tissue-specificity of Twist1 genomic occupancy was examined in mouse ECCs, limb buds, and peripheral nerve sheath tumor (PNST) cells using chromatin immunoprecipitation followed by sequencing (Chip-seq) analysis. Consistent with known Twist1 functions during development and in cancer cells, Twist1-DNA binding regions associated with genes related to cell migration and adhesion were detected in all three tissues. However, the vast majority of Twist1 binding regions were specific to individual tissue types. Thus, while Twist1 has similar functions in ECCs, limb buds, and PNST cells, the specific genomic sequences occupied by Twist1 were different depending on cellular context. Subgroups of shared genes, also predominantly related to cell adhesion and migration, were identified in pairwise comparisons of ECC, limb buds and PNST cells. Twist1 genomic occupancy was detected for six binding regions in all tissue types, and Twist1-binding sequences associated with Together, these data indicate that Twist1 interacts with genes that regulate adhesion and migration in different tissues, potentially through distinct sets of target genes. In addition, there is a small subset of genes occupied by Twist1 in all three tissues that may represent a core group of Twist1 target genes in multiple cell types.The online version of this article (doi:10.1186/1471-2164-15-821) contains supplementary material, which is available to authorized users. Twist1 is a highly conserved basic-helix-loop-helix (bHLH) transcription factor, which regulates cell migration, proliferation, and differentiation in progenitor populations, such as embryonic neural crest cells, cranial mesoderm, limb buds, and endocardial cushions (ECCs). In addiTwist1 expression is robust in the mesenchymal cells of the ECC and is subsequently down-regulated during remodeling and differentiation of the valve leaflets).Table of E12.5 ECC Twist1 ChIP-seq peak determination by MACS analysis. Twist1 ChIP-seq was performed in E12.5 ECCs and sequencing results were analyzed by MACS version 1.4.2 with default settings. The table contains the peak chromosomal location (chromosome (chr), start, and end), length of peak, summit of peak, number of tags at peak location, p value, fold enrichment, distance from nearest downstream gene and gene name, distance from nearest upstream gene and gene name. (XLS 2 MB)Additional file 1:Table of E10.5 limb bud Twist1 ChIP-seq peak determination by MACS analysis. Twist1 ChIP-seq was performed in E10.5 forelimb buds and sequencing results were analyzed by MACS version 1.4.2 with default settings. The table contains the peak chromosomal location (chromosome (chr), start, and end), length of peak, summit of peak, number of tags at peak location, p value, fold enrichment, distance from nearest downstream gene and gene name, distance from nearest upstream gene and gene name. (XLS 1 MB)Additional file 2:Table of peripheral nerve sheath tumor (PNST) cell Twist1 ChIP-seq peak determination by MACS analysis. Twist1 ChIP-seq was performed in PNST cells and sequencing results were analyzed by MACS version 1.4.2 with default settings. Table contains the peak chromosomal location (chromosome (chr), start, and end), length of peak, summit of peak, number of tags at binding peak location, p value, fold enrichment, distance from nearest downstream gene and gene name, distance from nearest upstream gene and gene name. (XLS 1 MB)Additional file 3:E-box containing motifs are significantly enriched in Twist1 ChIP-seq peaks for E12.5 ECCs, E10.5 limb buds, and PNST cells. PscanChIP analysis E12.5 ECCs, and and MEME-ChIP analysis was performed on peaks for E10.5 limb buds, and PNST cells following MACS analysis. The position weight matrix of the most enriched E-box containing motif (underlined) with associated p-values in E12.5 ECCs and PNST cells and the e-value associated with E10.5 limb buds are represented. (TIFF 1 MB)Additional file 4:Overlap of genes associated with Twist1 ChIP-seq peaks and genes expressed E12.5 ECCs, E10.5 limb buds, or PNST cells. Venn diagrams are shown for A. Gene probe sets corresponding to genes expressed in E12.5 ECCs versus genes associated with binding regions within 50 Kb of a TSS from Twist1 ChIP-seq in E12.5 ECCs. B. Gene probe sets corresponding to genes expressed in E10.5 limb buds versus genes associated with binding regions within 50 Kb of a TSS from Twist1 ChIP-seq in E10.5 limb buds. C. Gene probe sets corresponding to genes shared between mouse PNST cells and human MPNST cells versus genes associated with binding regions within 50 Kb of a TSS from Twist1 ChIP-seq in PNST cells. Note that multiple gene probe sets are present for many genes in the microarray analysis. (TIFF 2 MB)Additional file 5:The locations of Twist1 binding regions detected by ChIP-seq are typical for transcription factor binding. A. The genomic locations of Twist1 binding regions detected by ChIP-seq were analyzed relative to the nearest transcriptional start site using Genomatix RegionMiner. ChIP-seq peak location analysis was performed for cell-type specific peaks  and shared peaks . (TIFF 809 KB)Additional file 6:Table of gene lists for the four most predominant gene ontology (GO) categories associated with Twist1 binding regions as determined by ChIP-seq in E12.5 ECCs. The name and description of genes in the four gene ontology categories, cell-cell signaling, cell adhesion, cell morphogenesis, and neurogenesis, from the expressed genes associated with Twist1 binding regions within 50 Kb of a TSS in E12.5 ECCs. The presence of an E-box was not verified in this list. GO analysis was performed using ToppGene. (XLSX 59 KB)Additional file :Table of gene lists for the four most predominant gene ontology (GO) categories associated with Twist1 binding regions as determined by ChIP-seq in E10.5 limb buds. The name and description of genes in the four GO categories, kinase activity, cell migration, neurogenesis, and cytoskeletal proteins, from the expressed genes associated with binding regions within 50 Kb of a TSS in E10.5 limb buds. The presence of an E-box was not verified in this list. GO analysis was performed using ToppGene. (XLSX 55 KB)Additional file 8:Table of gene lists for the four most predominant gene ontology (GO) categories associated with Twist1 binding regions as determined by ChIP-seq in PNST cells. The name and description of genes in the four GO categories, cell adhesion, skeletal system development, cell recognition, and ventricular septum morphogenesis, from the expressed genes associated with binding regions within 50 Kb of a TSS in PNST cells. The presence of an E-box was not verified in this list. GO analysis was performed using ToppGene. (XLSX 42 KB)Additional file 9:Twist1 ChIP-seq binding regions have distinct candidate cofactor consensus binding sites in ECC, limb buds and PNST cells. Twist1 ChIP-seq binding regions associated with genes from the top four gene ontology (GO) categories for E12.5 ECCs, E10.5 limb buds, or PNST cells Additional file 11:


The transcription factor NFATc2 regulates dendritic cell (DC) responses to microbial stimulation through the C-type lectin receptor dectin-1. But the genetic targets of NFATc2 and their effects on DC function remain largely unknown. Therefore we used ChIP-seq to conduct genome-wide mapping of NFATc2 target sites in dectin-1-activated DCs. By combining binding-site data with a comprehensive gene expression profile, we found that NFATc2 occupancy regulates the expression of a subset of dectin-1-activated genes. Surprisingly, NFATc2 targeted an extensive range of DC-derived cytokines and chemokines, including regulatory cytokines such as IL2, IL23a and IL12b. Furthermore, we demonstrated that NFATc2 binding is required to induce the histone 3 lysine 4 trimethylation (H3K4me3) epigenetic mark, which is associated with enhanced gene expression. Together, these data show that the transcription factor NFATc2 mediates epigenetic modification of DC cytokine and chemokine genes leading to activation of their expression. Candida albicans and Aspergillus fumigatus  signaling pathway  are antigen presenting cells that express Toll like receptors (TLRs) and C-type lectin receptors (CLRs) that mediate the detection of microbial pathogens and stimulate DCs to induce appropriate immune responses against bacteria and fungi. Dectin-1 belongs to the CLR family of receptors and mediates DCs recognition of β-glucan carbohydrates, which are produced by various species of fungi including  pathway . Alterna pathway –8. Whilein vivo -stimulated DCs, which is an essential mechanism for limiting inflammatory responses to microbial pathogens   with theBy integrating ChIP-seq data with gene expression profiles, we demonstrate that NFATc2 occupancy is responsible for curdlan-mediated gene activation, and we present the first exhaustive list of NFATc2-regulated genes in DCs. The NFATc2 target genes incorporated an extensive range of DC-derived cytokines and chemokines, including regulatory cytokines such as IL2, IL23a and IL12b. In addition, our data reveal that NFATc2 recruitment is essential for the induction of the H3K4me3 epigenetic mark which is associated with enhanced gene expression in curdlan-stimulated DCs, thus indicating that this epigenetic modification is associated with NFATc2 binding.The murine DC line (D1) was cultured as previously described ,24 In brBone marrow cells from 8–12-week-old C57BL/6 mice were cultured in IMDM  (Life Technologies), penicillin 100 U/ml, streptomycin 100 μg/ml and 10% B16-GMCSF growth supernatant for 7 days. On day 8, mature bone marrow-derived DCs (BMDCs) were stimulated with 10-μg/ml curdlan in the absence or presence of 200-ng/ml FK506 for experiments.D1 cells were seeded into 8 well μ-slides (Ibidi) and rested for 2 h before stimulation with curdlan (10 μg/ml) for various times prior to fixation in 2% paraformaldehyde (PFA). The cells were then re-suspended in permeabilization buffer , immunolabeled with antibodies against NFATc2 (Thermo Scientific) or V5 tag (Invitrogen) and finally counter-stained with 4', 6-diamidino-2-phenylindole (DAPI) and anti-Alexa488 antibody (Life technologies).ChIP assays were conducted using D1 cells that were stably expressing NFATc2 with a C-terminal V5 tag ,30. BrieChIP-seq libraries were prepared from a total of 10 or 20-ng starting DNA using the Illumina TruSeq DNA version 2 kit according to the manufacturer's protocol, with the following modifications; 30X adaptor dilution and 18 PCR cycles of amplification. Batches of five to eight samples were sequenced as a multiplex in individual lanes of an Illumina HiSeq 2000 apparatus using a single-end run (1×51) together with the SBS kit v3. Reads were mapped to the mouse genome (version mm9) with bowtie 1  using deThe promoter regions including the Zfp206-binding sites for IL2, IL12b and IL23a were cloned into pGL3-basic vector (Promega). A dual luciferase system  was used. For the luciferase assay in 293T cells, 4 × 105 cells were seeded into one well of a 24-well plate. After 18 h, 275 ng of luciferase reporter plasmid, 1 ng of plasmid pRL-SV40 and either 1 μg of NFATc2 overexpression vector or empty vector were co-transfected into the cells using Lipofectamine 2000 (Invitrogen). The pRL-SV40 plasmid served as an internal control for normalizing the transfection efficiency. After 40 h of transfection, the 293T cells were lysed, and the luciferase activity was determined with the dual luciferase system (Promega) using a Centro LB960 96-well luminometer (Berthold Technologies).Affymetrix Mouse Gene 1.0ST Arrays were used to profile gene expression in curdlan-stimulated D1 cells (2- or 4-h exposure) that had been treated or not with 200-ng/ml FK506 for the duration of culture. Total RNA was isolated by acid guanidinium thiocyanate-phenol-chloroform extraction  followed by Qiagen RNeasy clean-up procedure. Total RNA integrity was assessed by Agilent Bioanalyzer and RNA Integrity Number (RIN) was >9.1 for all samples. Target ssDNAs were prepared using WT Expression Kits (Ambion/Affymetrix) according to the Affymetrix protocol, then fragmented and hybridized to standard arrays for 17 h at 45°C. The arrays were washed and stained using the fluidics station prior to analysis using a GeneChip Scanner 3000.P-value <0.05 and a cut-off fold-change value of ≥1.5 were applied to identify genes that displayed statistically significant changes in expression.Data were normalized using the Robust Multi-Array Average approach  and diffP-value <0.05) with mode set to ‘One Occurrence per Sequence’  under accession numbers GSE59896 and GSE59998.The data from this study have been submitted to the NCBI Gene Expression Omnibus (GEO)  to identify physical binding of this transcription factor to specific regions of DNA ,38. WhilNFATc2-bound DNA fragments were obtained by ChIP using an anti-V5 antibody and were then subjected to ultra-high-throughput sequencing on the Illumina HiSeq 2000 platform. For NFATc2 ChIP-seq, 1.4 million sequence reads were uniquely mapped to the mouse genome, and a total of 10 187 binding peaks were identified in the proximity of 6887 target genes using the MACS algorithm. The distribution of NFATc2 binding sites was analyzed using a similar annotation to that described previously , and inhibition of NFAT translocation using FK506 reduced the recovery of these regions by ChIP , thus confirming the specificity of transcription factor binding to these sites. We therefore proceeded to determine the NFATc2 consensus binding sequence using the MEME algorithm, which generated three distinct binding motifs from three sets of 600 randomly selected peaks (each being located within ±100 bp of a binding peak max). Consistent with reports from other investigators, we identified the NFATc2 binding motif as 5-′GGAAAA-3′ Figure  42,43).,43.42,43DCs produce a plethora of cytokines and chemokines that play critical roles in immune regulation. Several key modulatory cytokines have been shown to depend on NFAT family members for their expression, so we hypothesized that at least a subset of DC-derived cytokines and chemokines were likely to depend on NFATc2 for their transcription. We therefore extracted a list of cytokines that were identified by NFATc2 ChIP-seq (Supplementary Table S1), and, consistent with previous findings, we observed that NFATc2 binds to several known target genes including IL-2, TNF, IL-10, GM-CSF, Egr2 and Egr3 Figure  44). We. We44). curdlan and NFAT inhibitors, and untreated control DCs. Using a cut-off fold-change value of 1.5 (P < 0.01), we identified a number of genes that were significantly differently expressed following curdlan treatment and/or by NFAT inhibition (Supplementary Table S2). We also discovered that the majority of NFAT-regulated genes required dectin-1 activation to modulate their expression . Using this approach, we identified a total of 21 cytokine and chemokine genes that were directly regulated by NFATc2 in DCs (Supplementary Table S3). In order to confirm NFATc2 regulation of the cytokine and chemokine genes identified by microarray, we next used qPCR to verify mRNA expression levels and successfully validated expression level changes in 16 of the 21 candidate genes . We also measured the expression levels of these cytokine genes in BMDCs to confirm that the results from D1 cells were representative of primary DCs. We found that IL2, IL12b and IL23a gene expression was similarly induced by curdlan stimulation and inhibited by Fk506 treatment , and calculated the proportion of activated genes in each fraction, we observed that the percentage of activated genes for NFATc2 peaks of medium to very high height (15–18%) was at least 2-fold greater when compared to ‘low’ peaks (6%) Figure . These dIn the current report we provide evidence that the transcription factor NFATc2 mediates epigenetic modification of DC cytokine responses to dectin-1 stimulation. Our data indicate that NFATc2 signaling is an essential mediator of myeloid cell responses to microbial antigens, consistent with previous reports that dectin-1 signaling in DCs is essential for effective host protection against fungal infections , and sugWe conducted genome-wide mapping of NFATc2 binding sites in dectin-1-stimulated DCs and integrated our findings with gene expression data to identify the directly regulated targets of this transcription factor. In addition to targets of NFATc2 such as the IL2 and Egr2/3 genes –51. SimiAmong the thousands of NFATc2 binding targets identified here, only a small proportion conferred increased gene expression upon recruitment of NFATc2, which appears to be a common feature of transcription factor binding ,38. InstAn additional influence on gene expression levels is chromatin structure, which is remodeled by epigenetic modifications that control the access of transcriptional machinery to the template DNA. Chromatin remodeling can define different cell lineages, developmental stages and cellular responses to environmental signals , but untDue to the established role of NFAT in regulating T-cell immunity, the calcineurin/NFAT inhibitors CsA and FK506 have been widely used to treat autoimmune diseases and suppress allograft rejection in human patients –60. Our Supplementary Data are available at NAR Online.


Drosophila embryos to human cells. Release of paused Pol II is primarily mediated by the activated form of positive transcription elongation factor b (P-TEFb) initially sequestered in the inhibitory 7SK small nuclear ribonucleoprotein (7SK snRNP) complex. Many proteins and RNAs have been discovered and studied in detail to explain the process of the pausing and release of Pol II in relation to P-TEFb. At the functional level, promoter-proximal pausing regulates genes involved in stimulus-response and development in Drosophila. In mammalian stem cell biology, pausing is important for proliferation and signaling in embryonic stem cells and the formation of induced pluripotent stem cells. Other than this, however, little is known about the biological significance of pausing in mammalian cell differentiation. Further study on pausing mechanisms as well as its functions will contribute to the development of stem cell biology and its clinical applications.RNA polymerase II (Pol II) temporarily stops transcription after synthesizing 30–50 bases, and resumes elongation only after stimulations by various signaling molecules and developmental cues. This phenomenon, called promoter-proximal pausing, is observed in 10–50% of the entire genes from  PPP is highly prevalent; between 10% and 50% of entire genes simultaneously undergo PPP in many cell types, with only around a half of them proceeding to transcription elongation [Drosophila development and pluripotent stem cell differentiation, as they are the most widely studied models.After RNA polymerase II (Pol II) and general transcription factors are assembled to form the pre-initiation complex (PIC) at a promoter, it synthesizes a short mRNA of 30–50 bases and then pauses  –6 Figur. PPP is ongation –10. As eBy definition, the study of PPP requires the detection of the distribution pattern of Pol II within a given gene, which is commonly performed with chromatin immunoprecipitation (ChIP) followed by a microarray analysis (ChIP-chip) or next generation sequencing (ChIP-seq) . These aDefinitive evidence for paused transcription requires detection of transcript length. Global Run-On Sequencing (GRO-seq) is a genome-wide nuclear run-on assay, in which nascent transcripts are labeled with 5-bromouridine 5′-triphosphate (BrUTP) . The labCapping of mRNA with 7-methylguanosine takes place at the 5′ end during the initial stage of transcription . The capAmong these genome-wide approaches, ChIP-based approaches are more readily available to general cell biology laboratories than mRNA-based approaches, which require extensive optimization. A recent report showed that GRO-seq data are generally consistent with ChIP-seq data obtained using anti-Pol II antibody ; currentAbout half of P-TEFb in the nucleus is sequestered in 7SK snRNP . The relth position (Thr186) in the T-loop of Cdk9. This, in turn, leads to the release of P-TEFb from 7SK snRNP. The dephosphorylation of Thr186 is induced in a physiological context as well. For example, nuclear factor-κB (NF-κB) and the human immunodeficiency virus (HIV)-1 protein Tat recruit PPM1G  to promoters, which also dephosphorylates Thr186 [in vitro [in vitro process is accompanied by a conformational change of 7SK RNA and the release of Hexim1 from 7SK RNP.Exposure of cells to ultraviolet light or hexamethylene bisacetamide (HMBA) disrupts 7SK snRNP and releases P-TEFb . Both ags Thr186 . HoweverThe human I-mfa domain containing protein (HIC) interacts with CycT1 to inhibit P-TEFb-dependent transcription . In contRelease of P-TEFb is also directly linked with splicing of nascent mRNA. Serine/arginine-rich splicing factor 1 and 2 (SRSF1 and SRSF2), major components of the splicing machinery, bind 7SK snRNP at active gene promoters . When anAll of the previously described release mechanisms are reversible; however, an irreversible mechanism also exists. During megakaryocyte diferentiation, a calcium influx activates the protease calpain 2, causing the cleavage of the Mepce subunit of 7SK snRNP, promoting the release of P-TEFb and upregulating cytoskeleton remodeling genes . GATA1 iP-TEFb released from 7SK RNP must to be recruited to the promoters of target genes. This recruitment process is also diverse , frequenBrd4 is a member of the bromodomain and extraterminal domain (BET) family proteins ,23. Brd4c-Myc and hsp70 [Egr1 in mouse embryonic stem cells (ESCs) through a direct interaction with Cdk9 [P-TEFb is also recruited to promoters by Mediator through multiple mechanisms. Mediator is a protein complex composed of 26 core subunits serving as a physical and functional bridge between Pol II and DNA-bound transcription factors during transcription initiation and elongation . The MEDnd hsp70 . The recnd hsp70 . Mediatoith Cdk9 .The oncoprotein c-Myc directly binds Cdk9 and CycT1 at different domains and recruits them to target genes ,32. In mMyoD is a master regulator of muscle differentiation . It intehsp70 in a Drosophila cell line [Drosophila genes – was discovered in 2007 by applying the ChIP-chip technique [hsp70, which is rapidly activated upon a heat stimulus.PPP was discovered in 1986 at the heat shock gene ell line . Howeverechnique ,40. Whilechnique . These rDrosophila. The bone morphogenetic protein (BMP) pathway regulates dorsal-ventral patterning and 25 genes involved in this pathway are paused [Snail gene (sna), which is a key regulator for epithelial-mesenchymal transition and mesoderm formation [sna is expressed in around 1,000 presumptive mesoderm cells and synchronized activation of the gene is essential for coordinated invagination of the cells. When the tightly paused sna promoter was swapped with the moderately paused sog gene promoter, it resulted in a mixed embryonic population with normal and partial invagination. Replacement with the non-paused ths promoter completely abolished invagination in the majority of embryos, and normal invagination was rarely detectable.Paused developmental genes include those involved in embryonic axis formation and gastrulation in e paused . The segormation ,43. sna Drosophila also provided an unexpected insight into a novel mechanism of PPP. Knockdown of NELF was supposed to primarily upregulate gene expression by releasing Pol II; however, it upregulated 71 genes and downregulated 170 genes out of 18,500 genes in total [Drosophila embryos [Studies with in total . The dowin total ,45. This embryos . These fPluripotent stem cells and hematopoietic cells have been the main cell types used for the study of the developmental role of PPP in mammalian cells. The focus of this section will primarily be upon pluripotent stem cells due to the more in depth mechanistic and genome-wide studies compared to hematopoietic cells ,46–48. IDrosophila of a direct link between PPP and the regulation of development, there appears to be no definitive link in mammalian cells. The number of RefSeq genes paused in mouse ESCs and MEFs, 39% and 34% respectively, is quite similar [In contrast to the initial findings in  similar . In both similar . The catMouse ESCs have traditionally been cultured in the presence of serum; however, a serum-free condition called the 2i system, which uses two kinase inhibitors, has seen widespread use as a more chemically-defined culture system recently . After bIn a more functional sense, the manipulation of PPP during reprogramming of MEFs to iPSCs can provide a new approach to improve the quality and efficiency of cell reprogramming. During reprogramming there are two waves of gene activation, the first being genes related to proliferation, metabolism, and cytoskeleton whereas the second is primarily the pluripotency genes and their regulators . During Drosophila and pluripotent stem cells, very little is known about the roles of PPP in the differentiation of many other mammalian lineages. HIF1A-mediated pause release, mentioned above, is evidence that PPP is also involved in cancer and potentially other pathological conditions. In cardiomyocytes, PPP is present at metabolism-related genes, controlling energy homeostasis [Following its discovery, the study PPP has resulted in as many new questions as it has answers. As technology continues to improve, our genome-wide picture of PPP becomes more complex and diverse. The major protein and RNA players have been uncovered; however, our understanding of the mechanisms behind the release and recruitment of those molecules is still incomplete. While the developmental roles for pausing have become more apparent in eostasis . These s


PRDM (PRDI-BF1 and RIZ homology domain containing) protein family members are characterized by the presence of a PR domain and a variable number of Zn-finger repeats. Experimental evidence has shown that the PRDM proteins play an important role in gene expression regulation, modifying the chromatin structure either directly, through the intrinsic methyltransferase activity, or indirectly through the recruitment of chromatin remodeling complexes. PRDM proteins have a dual action: they mediate the effect induced by different cell signals like steroid hormones and control the expression of growth factors. PRDM proteins therefore have a pivotal role in the transduction of signals that control cell proliferation and differentiation and consequently neoplastic transformation. In this review, we describe pathways in which PRDM proteins are involved and the molecular mechanism of their transcriptional regulation. N-terminal PR (PRDI-BF1 and RIZ1 homology) domain. The PR domain shares high homology with the catalytic SET  domain that defines a group of histone methyltransferases [PRDM family genes is the expression of different molecular forms by alternative splicing or by the action of different promoters. Furthermore, some genes of this family are expressed as two alternative forms, one lacking the PR domain (PR-minus) but otherwise identical to the other PR-containing product (PR-plus)  [PRDM6, PRDM9) [The PRDM (PRDI-BF1 and RIZ homology domain containing) protein family is characterized by the presence of an sferases . PRDM pr PRDM16) ,5,6,7. O, PRDM9) ,9. Recen, PRDM9) .PRDM1 and PRDM2, initially identified as Blimp-1 (B lymphocyte-induced maturation protein-1) and RIZ (Retinoblastoma interacting zinc finger protein) respectively, have two promoters that encode for a PR-plus and a PR-minus isoform. PRDM1 promoters are localized upstream of exon 1 and exon 4 respectively. These transcriptional start sites at two promoters guide: PRDI-BF1 (Positive regulatory domain I-binding factor 1) α (PR-plus) e PRDI-BF1β (PR-minus) that differ only by the PR domain presence [PRDM2 is located upstream of the open reading frame in a region including exon 1a and a second promoter is located within intron 5 and exon 6 [PRDM1, PRDM2 expresses two proteins, PRDM2a/RIZ1 (PR-plus) and PRDM2b/RIZ2 (PR-minus), by differential transcription initiated by the two promoters.presence . One proPRDM16 encodes a Zn-finger protein (MEL1) that shares 63% sequence similarity to PRDM3/MECOM . Like PRDM3, two mRNAs coding for PR-plus and PR-minus protein are transcribed from this locus: PRDM16/MEL1 (MDS1/EVI1-like gene 1), the PR-plus form, with the PR domain coded from codon ATC91 (exon 2) to codon CCC223 (exon 5) and PRDM16/MEL1S, the PR-minus form, initiated from an internal codon ATG599 (exon 9) [(exon 9) ,13.PRDM1 encodes also for an alternatively spliced transcript lacking exon 7; this variant (Blimp-1Δexon7) lacks DNA binding activity and fails to bind G9a or HDAC1/2, but retains the ability to interact with Prmt5 (protein methyltransferase 5) [PRDM1 alternative splicing variants is regulated during development by chromatin structure modification and fine-tunes PRDM1’s functional capabilities [erase 5) . This evbilities .PRDM3/MECOM is a complex locus containing EV1 and MDS1 genes, located on chromosome 3q26. This complex locus encodes for different gene products generated by alternative splicing or by intragenic splicing [splicing .N-terminal seven-zinc finger domain, a central transcription repression domain, a second zinc finger domain with three finger motifs and a C-terminal acidic region. One EVI1 mRNA splice variant, identified as PRDM3/EVI1/Δ324, is a protein that lacks zinc fingers 6 and 7 as well as part of the transcription repression domain. The PRDM3/EVI1-Rp9 variant is abundant both in humans and mice and lacks 9 amino acids in the repression domain. The variant, PRDM3/EVI1/Δ105, is a protein truncated of 105 aminoacids at its C-terminus and is detected only in murine but not in human cells.The major and most studied protein, EVI1 (Ecotropic virus integration site 1 protein homolog), also named MECOM (E) is a 1051 aminoacid protein , that coEVI1 may form a fusion transcript with the MDS1 gene located upstream. The use of alternative transcriptional start sites generates mRNA combining sequences derived from the MDS1 (Myelodysplasia syndrome-associated protein 1) gene, which is located upstream of EVI1, and the EVI1 sequences starting from exon 2. The derived protein, called MDS1/EVI1 or MECOM (ME), from this mRNA contains a 188 amino acid extension encoding a PR domain at its N-terminus, but is otherwise identical to the EVI1 protein [ protein ,15,17.Prdm6 encodes for four isoforms referred to as Prdm6/4#, 3#, 33# and 36#, produced by alternative splicing. Prdm6/4# has a PR/SET domain in the central region and four Zn-finger domains at its C-terminal region. Prdm6/3# and Prdm6/33# have an additional sequence of 31 residues produced by retention of the first intron, absent in the Prdm6/4# transcript. Similarly, Prdm6/36# has a single amino acid insertion if compared to Prdm6/4#, derived from the recognition of an alternative splicing site 3 bp upstream of the intron 1/exon 2 boundary. Prdm6/33# is a PR-minus isoform, obtained by an alternative splicing event that results in the deletion of exons 3–5 of transcript encoding for Prdm6/4#. Prdm6/36#, missing the fourth Zn-finger domain, derives from an alternative splicing in intron 7 of Prdm6/4# transcript that includes an in-frame stop codon [In mice, op codon .Prdm9 murine gene encodes for three isoforms generated by alternative splicing: one isoform has a PR domain in its N-terminal region and a Zn-finger motif in its C-terminal portion. The other two isoforms generated by alternative splicing lack the Zn-finger domain responsible of the nuclear localization [The lization .C-terminal region of Prdm10/tristanin, identified two molecular forms of this protein of 50 kDa and 25 kDa respectively. This finding corroborates the hypothesis that there are also different molecular variants encoded by the gene Prdm10 [In 2002 Siegel, analyzing the protein extracted from mouse brain by Western blot with an antibody to the e Prdm10 .The functional relevance of the different variants has not yet been elucidated. PRDM protein are involved in the transduction of many signals that are responsible for proliferation and differentiation control. PRDM proteins, through the formation of chromatin remodeling complexes, regulate gene expression acting generally as transcription repressors ,22,23,24cis-acting hormone-regulatory elements [Nuclear receptors act as ligand-dependent transcription factors, modulating gene expression by direct interaction with well conserved consensus sequences of target genes: elements .PRDM2 gene product PRDM2a/RIZ1 is a downstream effector of estrogen action and is related to estrogen-regulated cell proliferation in classical estrogen target tissues. PRDM2 proteins interact with estrogen receptor (ER) through a LXXLL motif and their interaction is dependent on 17β-estradiol treatment [in vitro histone H3K9 methyltransferase activity and is a weak activator or a repressor of transcription [et al. in fact demonstrated that PRDM2a is able to bestow estrogen inducibility to a promoter containing an incomplete ERE and a G/C TTGGC motif [Several findings suggest that the reatment ,30,31. Pcription ,32,33. Icription . Medici GC motif .et al. have shown the fundamental role of histone methyltransferase (HMT), including PRDM2a, in maintaining in off-state the promoters regulated by nuclear receptors, such as ERα or androgen receptor (AR). However, the H3-K9 methylation-mediated down-regulation allows the action of lysine-specific demethylase 1 (LSD1) molecules recruited by steroid nuclear receptors ligand, complexed to the same genes [minus) form is unable to produce enhancer effects in presence of estradiol, as observed with ERE-Luc reporter assay experiments in vitro [Garcia Bassets me genes ,36. Baseme genes  and of Ome genes . This woin vitro , despitein vitro . It mighin vitro ,30.PRDM2 gene products are endowed with DNA-binding as well as transcription factor-binding activities. In fact PRDM2 was independently isolated as a retinoblastoma-binding protein (RIZ) [Moreover, in (RIZ) , a DNA-bin (RIZ) . MTB-Zf in (RIZ) . G3B (PRin (RIZ) .PRDM genes. In breast cancer cells (MCF-7 cell line), 17β-estradiol stimulation specifically modulates expression of PRDM2 gene products (PRDM2a and PRDM2b), inducing a shift in the balance of their intracellular concentrations; in particular 17β-estradiol induced a selective decrease in PRDM2a transcript and an increase in total PRDM2 mRNA, accounted by an increase in the PRDM2b form [PRDM2 gene contains an estrogen responsive element (ERE) endowed with enhancer activity that is recognized by ERα [PRDM2 promoter (promoter 2) and the polyadenylation site. Formation of loops has been implicated not only in bringing together far upstream or downstream regions with regulatory or transcribed gene regions, but also in establishing contacts between the 5' and 3' ends of genes, [PRDM2 gene locus to create a loop for the mRNA transcription with poliA-exon 9a, leading to the production of oncogenic variants [Nuclear receptor ligands modulate expression of several M2b form . In factd by ERα . Moreovef genes, ,45, in af genes, . In the variants . In non-variants ,49.PRDM2 is an androgen responsive gene, because there is an androgen responsive element (ARE) at -361 bp, in the upstream regulatory region of the promoter 1 of PRDM2 gene [PRDM2 proteins might also be mediators of androgen effects. In EPN cells, 5α-dihydrotestosterone (DHT) induced a slight increase in cell growth, related to a sharp increase of PRDM2a mRNA and protein concentration. Further investigation could confirm whether DM2 gene ,50.PRDM2 proteins might also be active for retinoid action. In fact, in a human promyelocytic leukemia cell line (HL60) treatment with retinoic acid induced a selective expression of PRDM2a and a redistribution of the protein within the nucleus, correlated to the granulocytic differentiation. In HL60 cells, PRDM2a expression was also induced by activation of a retinoid receptor-independent maturation pathway based on retinoid X receptor agonist and protein kinase A synergism .Similarly to PRDM2 acting as co-activator of ERα, PRDM16 stimulates adipogenesis by binding and co-activating, in a ligand-dependent manner, the peroxisome-proliferator-activated receptor γ (PPAR-γ) . PRDM16 p450c17c coding for a component of cytochrome P450 family, and of Luteinizing Hormone Receptor gene when steroidogenesis was induced in mouse Leydig cells (TM3 cell line) by LH treatment [LH stimulates testosterone synthesis in Leydig cells inducing the expression of cytochrome P450 enzymes, 3β-hydroxysteroid dehydrogenase and LH receptor. Prdm8 is a transcriptional repressor that specifically methylates lysine 9 of histone H3. The overexpression of Prdm8 wild-type protein or its mutant deletion, lacking the PR domain, induced a reduction in the expression levels of the steroidogenic enzyme gene reatment . This evIGF-1 expression through a direct binding to its promoter, increasing histone H3K9 methylation. PRDM2a also positively controls insulin-like growth factor-binding protein 2 (IGFBP-2) and SPARC expression [PRDM2a expression in chronic myelogenous leukemia-blast crisis (CML-BC) cell lines decreases activation of IGF-1 receptor and of the downstream signaling components ERK 1/2 and AKT.PRDM2a acts as a repressor of a subgroup of genes involved in IGF-1 signaling. A chromatin immunoprecipitation (ChIP) assay showed that PRDM2a down-regulates pression . MoreovePrdm4 gene product binds to the p75 neurotrophin receptor and provides a downstream transducer for the effects of nerve growth factor (NGF) through this receptor. In fact, NGF treatment of the monkey kidney fibroblast-like cell line (COS) induces a translocation of Prdm4/SC-1 from the cytoplasm to the nucleus that is related to a reduction in bromodeoxyuridine (BrdU) incorporation. The translocation of Prdm4/SC-1 to the nucleus was specific for p75, as NGF binding to the TrkA receptor prevented the nuclear localization of Prdm4/SC-1  cells . In muriPRDM14 is essential for the maintenance of the pluripotent state of human and, potentially, murine ESC, but not for the murine epiSCs (derived from post-implantation epiblast cells), and enhances epigenetic reprogramming of human and murine somatic cells to induced pluripotent stem cells (iPSC) .Co-expression of PRDM1 and PRDM14 is obligatory for the establishment of germ cell lineage . In mammOther epigenetic changes, associated with PRDM1 expression, allow PGC to escape the somatic pathway: PGCs show low levels of DNA methylation and H3K9me2 histone marks while acquiring high levels of H3K27me3 modifications . The expIn vitro, the ES cells are capable of differentiating in germ cells [rm cells  and thesrm cells . In embrrm cells . In thesrm cells . PRDM1 irm cells .CCND1), a Wnt/β-catenin downstream target gene whose product binds CDK4, was markedly decreased when PRDM5 was overexpressed [CDK4 and TWIST1 and PRDM5 expression resulted in significantly decreased levels of active transcription marks H3K4me3 and acetyl-histone H4 in CDK4 and TWIST1 promoters.Aberrant activation of Wnt/β-catenin signaling is also frequently involved in cancers, accompanied with elevated levels of active β-catenin. In addition to genetic defects, epigenetic silencing of Wnt/β-catenin antagonists also leads to aberrant Wnt/β-catenin signaling in tumors . PRDM5 axpressed . By ChIPThe nervous system of mammals contains a large number of neurons in a diverse array of neuron classes. Transcription factors play central roles in generating this complexity by controlling neural progenitor cell proliferation, patterning, and defining neuron fate ,86. One Hes factors that then function as DNA-binding repressors and antagonize the expression of proneural bHLH genes [Drosophila homolog of mammalian Prdm3/Evi1 and Prdm16, controls olfactory receptor neuron (ORN) development fate by modifying the cellular response to the Notch signals. Ham up-regulating H3K27me3 and down-regulating H3K4me3 directs chromatin-modification events at specific Notch targets, altering the accessibility for Su(H) binding at the enhancer. In nascent ORNs, Ham activity erased the Notch state that was inherited from the parental pNa intermediate precursor cell. This permitted a new and modified response of Notch targets in the subsequent round of Notch signaling [in situ hybridization analysis showed that in the developing murine telencephalon, Prdm family genes are expressed at high level in a spatially and temporally restricted manner. The Notch-Hes pathway controls their expression: in particular Hes positively or negatively regulated expression of Prdm16 and Prdm8, respectively. In fact, in Hes-null telencephalon neural differentiation is enhanced, Prdm8 expression is up-regulated, and Prdm16 expression is down-regulated. Conversely, electroporation of Hes1 into the developing telencephalon in utero up-regulates Prdm16 expression .The Transforming growth factor β (TGF-β) signaling regulates central cell processes, such as proliferation and extracellular matrix production during development of the orofacial region ,99,100. Prdm16 is expressed in the murine embryonic secondary palate [Prdm16 knockout murine embryos display a completely penetrant cleft palate [Prdm16−/− fetuses, chromatin immunoprecipitation-promoter microarray analysis (ChIP-Chip) has revealed a gene expression change of markers for bone (Opn) and muscle (Myf-4) development. The expression of Opn, [Prdm16 to myo-, chondro- and/ or osteogenesis in the developing orofacial region, in addition to regulating other processes of normal development. Prdm16 knockout could cause an abnormal muscle and/or bone development leading to altered morphogenesis of the nascent palatal processes with the failure of reorientation and subsequent separation of the oral and nasal cavities [y palate  where itt palate . In Prdm of Opn, , linked cavities .PRDM1 is a transcription repressor that plays a critical role in terminal differentiation of B cells into antibody-secreting plasma cells . PRDM1/BInterleukin 21-producing T helper lymphocytes are central to humoral immune response because this cytokine is required for the antibody production induced by IL-6. In B cells, IL-21-treatment induces the expression of signal transducer and activator of transcription 3 (STAT3), required for optimal immunoglobulin production and an up-regulation of PRDM1, the master plasma cell factor .PRDM1 is induced by IL-2 signaling and inhibits IL-2 production in a negative feedback loop [H2 differentiation and inhibits the TH1 differentiation, which induces the down-regulation of IL-2. PRDM1 is an IL-4 responsive gene that potentiates the IL-2 inhibition. In fact, IL-4-mediated IL-2 suppression was less pronounced in activated, PRDM1- deficient T helper cells [PRDM1 plays also a crucial role in controlling T cell homeostasis ,111. In ack loop . In naiver cells .PRDM1 expression level and, consequently, the secretion of pro-inflammatory cytokines was regulated not only at transcriptional level by activation of T helper cells but also at post-transcriptional level, by enhanced miR-9 expression. The miR-9 is particularly abundant in activated human T helper cells and controls expression of PRDM1 and B cell lymphoma-6 protein (Bcl-6). In fact, suppression of miR-9 led to increased expression of PRDM1 and Bcl-6, which subsequently resulted in diminished secretion of IL-2 and IFN-γ [Recent studies revealed that the nd IFN-γ .FH cells), by Bcl-6 to maintain the expression of several TFH genes implicated in lineage commitment [H1-specific T box transcription factor, to maintain the TH1 gene-expression profile [PRDM1 is directly targeted by Bcl-6 and is responsible for the repression of a subset of TFH signature gene in TH1 cells [H1 and TFH-like gene-expression regulated by T-bet-Bcl-6 complex, through the activation or repression of PRDM1. In TH1 cells, the variations of the ratio between Bcl-6 and T-bet are regulated by the low or high concentration of IL-2. In this way, low concentration of IL-2 enables the Foxo transcription factor to activate Bcl-6 transcription; Bcl-6 in turn represses the PRDM1 expression, promoting the expression of TFH signature genes [Another microRNA gene cluster is repressed, in T follicular helper cell  [In addition to controlling the fate of effector T helper cells, PRDM1 cooperates with transcription factor IRF4 for the differentiation of natural Tat Lys27 . In thes of IRF4 ,121. Theuence 9) ,123.PRDM2 is involved in the regulation of inflammatory response in host defense and might play an important role in inflammatory diseases. In the murine leukemic monocyte macrophage cell line (RAW 267.4) PRDM2 is a lipopolysaccharide (LPS)-responsive gene that increases the production of TNF-α (Tumor necrosis factor α) and IL-6 by nuclear factor-κB (NF-κB) activation. LPS, in fact, augments the PRDM2a expression via the activation of PI3K/Akt/NF-κB pathway. In turn, PRDM2a increases TNF-α expression level and IL-6 cytokine enhancing NF-κB activation. PRDM2a knock-down by RNA interference led, in fact, to the inactivation of NF-κB in response to LPS. TNF-α induced PRDM2 expression by the activation of NF-κB and AKT signaling. PRDM2a negatively regulates the proliferative activity of TNF-α-treated human monocytic leukemia cells via activation of p53. In fact, PRDM2a forced expression produces an increase of p53 protein expression and silencing of RIZ1 prevented it. On the other hand, a p53 inhibitor enhanced the TNF-α-induced PRDM2a expression [pression ,125.PRDM5 is probably involved with the regulation of hematopoiesis. PRDM5 is in fact able to interact with Growth factor independent 1 (Gfi1) transcription factor, essential for hematopoiesis [opoiesis , whose iopoiesis ,128,129.opoiesis  to repreNOTCH2, IL6R, MYB and c-MYC, whose transcriptional regulation is also controlled by Gfi1, suggesting that Gfi1-PRDM5 interaction activates rather than represses transcription. Neutropenia-associated PRDM5 sequence variants interfere with its transcriptional activity.PRDM5 can also activate some target genes, such as Prdm9-null mice showed arrest of gametes in meiotic prophase I and impaired double-strand break repair [PRDM9, also referred as Meisetz (Meiosis-induced factor containing PR/SET domain and Zn-finger motif), is a histone methyltransferase acting as a transcription activator of meiosis-specific genes in murine germ cell lineage. PRDM9 has catalytic activity only for trimethylation of lysine 4 of histone H3 and its transactivation activity depends on the methylation activity. The methylation of lysine 4 of histone H3 is a well-characterized feature of transcriptionally active genes . PRDM9 ik repair .Histone posttranslational modifications (PTMs) and sequence variants regulate genome function. H3K9 methylation occurs prior to histone incorporation into chromatin. Notably, initial modifications on non-nucleosomal H3 variants can potentiate the action of enzymes as exemplified with SUV39H1 HMT to produce H3K9me3 found in pericentric heterochromatin .Prdm3 and Prdm16 prevents the nuclear lysine methylation of histone 3 by the SUV39H1 enzyme that reinforce heterochromatin, resulting in disintegration of heterochromatic foci and disruption of the nuclear lamina [It has recently been demonstrated that in mouse embryonic fibroblasts, Prdm3 and Prdm16 are redundant H3K9me1-specific lysine methyltransferase enzymes (KMT) that direct cytoplasmic H3K9me1 methylation. Combined impairment of r lamina .Prdm4/SC1 and Prtm5 are located both in the nucleus and cytoplasm of neuroepithelial cells, suggesting that they might act similarly to Prdm3 and Prdm16, inducing methylation of a cytoplasmic pool of newly synthesized histones.Prdm5 is specifically expressed in the osteoblastic compartment of developing bones and exerts its function along the osteogenic lineage by promoting osteogenic differentiation in culture. Prdm5 targets extracellular matrix (ECM) gene families such as those encoding for collagens and small leucine-rich proteoglycans. Prdm5-bound genes were trimethylated on lysine 9 or 4 of histone 3. The methylation level was higher on lysine 4 than on lysine 9. By association with RNA polymerase II, probably affecting its ability to bind DNA during transcription, Prdm5 sustains the transcription of collagen I genes while the regulation of Decorin expression is mediated by binding to a distal enhancer-like element [Bone is composed of a highly specialized, mineralized collagenous matrix that provides tensile strength to the skeletal system . Prdm5 i element .PR domain in smooth muscle), regulates SMC phenotypic plasticity by suppressing differentiation and maintaining the proliferative potential of vascular SMCs. Prdm6 acts as a transcriptional repressor by interacting with a class I histone deacetylase, heterochromatin protein-1 (HP1-B), a H3K9 specific transcriptional repressor, and the G9a, a ubiquitous H3K9 and K27 methyltransferase, repressing Prdm1-mediated transcription. Prdm6 interacts with transcriptional activators in addition to repressors such as p300, a powerful transcriptional co-activator with intrinsic histone acetyltransferase activity [Prdm6 protein, also named PRISM  [plus form or overexpression of the PR-minus one. Nevertheless, expression of forms missing the PR domain is not only limited to neoplastic transformation and tumor progression. Actually, the significance of the balance between the different forms and the mechanism controlling the ratio is unknown.ariants) ,136. An PRDM genes are grouped in five subfamilies and the genes lying in sister branches of the tree maintain similar gene organization, splicing patterns, and functions. For example, PRDM2 and PRDM5, belonging to the same subfamily , have histone methyltransferase activity and are involved in cell cycle progression regulation [PRDM family expanded in vertebrates in parallel with the increased complexity of the genome in higher organisms. gulation ,30.PRDM genes with their expression in human tissues, it is evident that the newer genes have a lower expression than the older genes and acquire tissue specificity, suggesting a progressive specialization and/or a tighter regulation of their functions. Could the concomitant expression of old and new genes in a tissue suggest a cooperation in the establishment of the phenotype? This behavior is shown by PRDM1 and PRDM14, cooperating during germ cell development, and by PRDM3 and PRDM16, participating to maintain mammalian heterochromatin integrity. We hypothesize that the cooperation is a common characteristic of the PRDM gene family. Moreover, we observed (data non published) that PRDM2 gene siRNA silencing did not produce major phenotypic changes but increased the expression level of other PRDM-family proteins, suggesting that these could have a vicarious role.By comparing the evolutionary features of PRDM proteins are localized in the nucleus where they participate in the transcriptional regulation of gene expression. However, the function of the PRDM protein in the cytosolic compartment is not completely clarified. Recently it has been demonstrated that PRDM3 and PRDM16 methylate H3K9me1 in the cytosol. Moreover, PRDM2a and Prdm4/SC-1 translocate from the cytosol to the nucleus after retinoic acid and NGF treatment respectively. We hypothesize that other than the role in histone code PRDM proteins targets other cytosolic proteins and control their function. PRDM protein as PRDM2 and PRDM16 are co-activators of the nuclear receptor superfamily and participate in the steroid genomic pathway. No clues are available about the involvement of PRDM proteins in the steroid non genomic pathway.