NULL hematopoietic progenitors. Finally, the knock-down of the NuRD subunit Mi2 reveals that the occupancy of the NuRD complex at transcribed regions of genes favors the relief of POL II promoter-proximal pausing and thereby, promotes transcription elongation.IKAROS is a critical regulator of hematopoietic cell fate and its dynamic expression pattern is required for proper hematopoiesis. In collaboration with the Nucleosome Remodeling and Deacetylase (NuRD) complex, it promotes gene repression and activation. It remains to be clarified how IKAROS can support transcription activation while being associated with the HDAC-containing complex NuRD. IKAROS also binds to the Positive-Transcription Elongation Factor b (P-TEFb) at gene promoters. Here, we demonstrate that NuRD and P-TEFb are assembled in a complex that can be recruited to specific genes by IKAROS. The expression level of IKAROS influences the recruitment of the NuRD-P-TEFb complex to gene regulatory regions and facilitates transcription elongation by transferring the Protein Phosphatase 1α (PP1α), an IKAROS-binding protein and P-TEFb activator, to CDK9. We show that an IKAROS mutant that is unable to bind PP1α cannot sustain gene expression and impedes normal differentiation of Ik Perturbation of the expression level of IKAROS, a transcription factor critical during hematopoiesis, is associated with malignant transformation in mice and humans. The importance of IKAROS expression levels for the control of target-gene regulation was addressed in hematopoietic progenitor cells. The collaboration between IKAROS and the Nucleosome Remodeling and Deacetylase (NuRD) complex can promote gene activation or repression. IKAROS can also interact with the Positive-Transcription Elongation Factor b (P-TEFb) and the Protein Phosphatase 1 (PP1), an important P-TEFb regulator. Immunoaffinity purification of IKAROS interacting proteins and Fast Protein Liquid Chromatography analysis revealed a dynamic interaction between IKAROS, PP1 and the newly defined NuRD-P-TEFb complex. This complex can be targeted to specific genes in cells expressing high levels of IKAROS to promote productive transcription elongation. Based on our results we suggest that, in addition to P-TEFb, the NuRD complex and PP1 are required to facilitate transcription elongation of IKAROS-target genes and normal differentiation of hematopoietic progenitor cells. Ikaros null mutation (IkNULL) display severe defects in lymphocyte development and function, and develop leukemias and lymphomas with complete penetrance The tumor suppressor IKAROS is a transcription factor critical for hematopoietic multi-lineage priming, cell fate and lineage determination IKAROS controls chromatin organization mainly through association with the Nucleosome Remodeling and Deacetylase (NuRD) complex Here, we sought to define the importance of these protein associations for IKAROS and NuRD to function as transcriptional activators. We demonstrate that IKAROS is an adaptor molecule required for the recruitment of the newly identified NuRD-P-TEFb complex to IKAROS-target genes. IKAROS binding to the promoter region of specific genes is also associated with the local recruitment of the CDK9/P-TEFb activator, PP1α. Interestingly, the Mi2/NuRD occupancy at IKAROS-target genes is enhanced when transcription elongation is proficient, and the release of POL II promoter-proximal pausing is decreased in the absence of Mi2. Our data also reveal that the dynamic interaction of IKAROS with the NuRD-P-TEFb complex depends on IKAROS protein levels. Low-levels of IKAROS suffice for Mi2/NuRD recruitment to promoters, but higher levels of IKAROS are required for NuRD-P-TEFb and PP1α to chromatin, CDK9/P-TEFb activation, and productive transcription elongation. Finally, we demonstrate that IKAROS-PP1α protein interaction is required for normal hematopoietic differentiation of primary HPCs.Several studies have demonstrated physical interaction between IKAROS and NuRD To determine whether IKAROS interactions with Mi2/NuRD and CDK9/P-TEFb define a single complex or multiple complexes, Flag-HA-Ik immunoaffinity-purified complexes were analyzed by size exclusion chromatography. Flag-HA-Ik co-fractionated with NuRD and P-TEFb components as a major peak of ∼2 MDa . AdditioIkaros knock-down Jurkat cells  experiments were carried out in at cells . Co-IP rat cells . This suProtein interactions between NuRD and P-TEFb components were then assessed by protein co-IP of Jurkat nuclear extracts. The NuRD-associated proteins Mi2, RBBP4, MTA2, and MBD3 interacted with the P-TEFb components CDK9 and CYCT1 , S1F, whc-Kit and Flt3 (NULL HPCs −) HPCs obtained from Ikaros wild type (IkWT), Ikaros heterozygote null (IkHT) or Ikaros homozygote null (IkNULL) mice We examined whether IKAROS could be a licensing factor for NuRD-P-TEFb complex recruitment to chromatin and whether the variation of IKAROS thresholds, which naturally occurs during hematopoiesis and Flt3 , which ac-Kit and Flt3 expression correlated with the level of Ikaros expression in lin− HPCs  was not reduced in IkNULL or Ikaros knock-down G1E2 cells  assays. ChIP experiments indicated that IKAROS was recruited to critical regulatory regions of the c-Kit locus e.g., the −114 Kb enhancer, the c-Kit Transcriptional Start Site -TSS-, and +1 Kb, +5 Kb as well as +58 Kb regions of the Open Reading Frame (ORF) in IkWT lin− HPCs and G1E2 cells  c-Kit TSS together  were obtained in Cdk9 knock-down G1E2 cells (Cdk9 knock-down) does not affect loading of IKAROS, CDK9/P-TEFb and Mi2/NuRD at c-Kit TSS region, but impedes P-TEFb and, surprisingly, IKAROS and NuRD recruitment within the gene ORF.Since IKAROS, CDK9 and Mi2 were detected at kWT HPCs , we test reduced , S3B. Fld  promotes CDK9/P-TEFb activation in order to release promoter-proximal paused POL II.The role of the IKAROS-PP1α interaction for CDK9/P-TEFb activation was further delineated by exploring the influence of Calyculin A, a PP1/PP2A inhibitor ll death , but indnscripts , whereasnscripts , D, whiced cells . In genenot vary . Similart3 locus –H. Calyct3 locus . These bex vivo assays in methylcellulose (WT and IkNULL lin− HPCs were transduced with the control pMSCV empty vector (IkWT/GFP or IkNULL/GFP); IkNULL lin− HPCs were also transduced with pMSCV/Ik1  or pMSCV/Ik1ΔPP1 . Ik1ΔPP1 is an IKAROS mutant unable to bind PP1 and therefore, resistant to dephosphorylation NULL/Ik1ΔPP1 HPCs by single-cell immunofluorescence (IF) analysis. Since these transgenes possess a Flag-HA tag, IF analysis was carried out with anti-HA antibodies. IF studies indicated that Ik1ΔPP1 accumulates in the nucleus of Ik1ΔPP1-infected lin− HPCs , whereas expression of c-Kit and Flt3 genes was only increased to 33.3% (for c-Kit) and 11.5% (for Flt3) upon pMSCV/Ik1ΔPP1 infection  activity (1/166 IkNULL lin− HPCs vs. 1/33 IkWT lin− HPCs) as well as reduced colony size, which suggests reduction of cell proliferative potential. IkNULL lin− HPCs produced less mixed CFC (CFU-GEMM) and increased erythroid/megakaryocyte (BFU-E/Mk) colonies  as well as FLT3 ligand (FL). We observed that IkNULL lin− HPCs were refractory to SCF and FL treatments. This functional limitation was demonstrated by the absence of: (i) induced cell proliferation  c-nduction ; and . The gain in CFU-GEMM might be caused by an advantageous transduction ability of multipotent progenitor cells, whereas the gain in megakaryocyte vs. erythroid component, as indicated by increased BFU-Mk/E colonies, might be related to the Erythropoietin deprivation during the infection procedure (NULL lin− HPCs with Ikaros1 significantly augmented CFC activity of transduced HPCs (NULL lin−vs. IkNULL/Ik1 lin− HPCs). Interestingly, CFC activity of IkNULL/Ik1ΔPP1 lin- HPCs was lower than in IkNULL/Ik1 lin− HPCs (NULL/GFP lin− HPCs and IkNULL/Ik1ΔPP1 lin− HPCs was very similar (NULL/GFP lin−vs. IkNULL/Ik1ΔPP1 lin− HPCs). Thus these results suggest that the Ikaros1ΔPP1 mutant is defective in restoring hematopoietic functions and that IKAROS interaction with PP1, which is required for productive transcription elongation, is important for normal hematopoiesis.To define the biological contribution of IKAROS-dependent control over transcription elongation, we studied the hematopoietic potential of Ikrocedure . Infecticed HPCs , table, in− HPCs , table, P-TEFb can be recruited to gene promoters either by the general P-TEFb activator BRD4 Size fractionation analysis of nuclear extracts and IKAROS immunoaffinity-purified protein complexes revealed that a modest but significant percentage of nuclear CDK9 and CYCT1 (P-TEFb) is stably associated with IKAROS and NuRD components. This association resembles to the small and highly active portion of P-TEFb that is included in the Super Elongation Complexes (SECs), known to stimulate transcription elongation rate c-Kit elongation, FACT components were not identified by LC-MS/MS analysis of IKAROS-associated proteins. Instead, our data indicate that POL II release and the rate of transcription elongation of these genes are affected by Mi2 knockdown. In addition, Mi2/NuRD occupancy at c-Kit and Flt3 ORF decreased when transcription elongation was blocked by the CDK9 inhibitor Flavopiridol. Therefore, our results suggest that in HPCs, the NuRD complex can act as a chromatin complex that both destabilizes and restores nucleosomal structure in order to assist and facilitate the passage of POL II during transcription elongation. Accordingly, the NuRD complex can support nucleosome remodeling through the Mi2-associated helicase activity and histone deacetylation through the HDAC-associated activity Nucleosomes form barriers for the elongating POL II. The main remodeling complex known to facilitate POL II passage through nucleosomes is the histone chaperone complex FACT i.e., NuRD-dependent chromatin remodeling associated with gene priming and PIC assembly Initially regarded as a co-repressing complex because of its association with histone deacetylases and transcriptional repressors, the NuRD complex has also been associated with permissive chromatin and gene activation WT lin− HPCs and G1E2 cells, a fraction of IKAROS associates with P-TEFb and NuRD and this complex is recruited to c-Kit and Flt3 genes. In IkHT lin− HPCs and Ikaros knockdown G1E2 cells, IKAROS, Mi2/NuRD and CDK9/P-TEFb are recruited less efficiently to gene regulatory regions and transcription elongation is affected although transcription initiation occurs normally and POL II is in a promoter-proximal paused configuration. Finally, in IkNULL HPCs, transcription initiation occurs normally but P-TEFb and NuRD recruitment to c-Kit and Flt3 loci is impaired. Then, the enrichment of elongation-competent Ser2-phosphorylated POL II at gene ORF drops to almost background values. As a result, transcription elongation is profoundly reduced.During differentiation of B-cell progenitors, the dynamic change of IKAROS expression level has been identified as a key mechanism for multiple target gene expression A large set of hematopoietic lineage-specific genes are characterized by permissive chromatin organization and PIC assembly at their promoters in HPCs c-Kit and Flt3 genes, and PP1α recruitment to these TSS is highly influenced by IKAROS concentration, it can be argued that IKAROS contributes to chromatin recruitment of PP1α at the TSS of target genes hence, facilitating CDK9/P-TEFb association through dephosphorylation of CDK9 Thr-186 and possibly, Ser-175 PP1α is involved in CDK9 dephosphorylation at Thr-186 and Ser-175 c-Kit and Flt3 promoters but did not affect TBP and POL II recruitment. Thus, PP1α activity is critical for IKAROS as well as NuRD and P-TEFb recruitment to these promoters and for transcription elongation, while being dispensable for PIC formation. Second, the rescue of IkNULL HPCs attempted with the Ik1ΔPP1 mutant, which interacts with Mi2, MTA2, CYCT1 and CDK9  as well as gene transcription elongation (P-TEFb) activities. IKAROS is important for CDK9 and Mi2 interaction and combined recruitment to actively transcribed genes in hematopoietic cells. We demonstrate that low IKAROS expression level does not preclude appropriate promoter organization but impairs productive elongation, whereas higher IKAROS levels are necessary to relieve promoter-proximal paused POL II and efficient transcription elongation of target genes . Our resThese assays were done essentially as previously reported Protein complexes were purified from Jurkat nuclear extract or IKAROS immunoaffinity-purified complexes on an AKTA Purifier FPLC System  using a Superose 6 10/300GL column .et al.Flag-HA sequential immunoaffinity (or Tap-Tag) purification was carried out as described in Nakatani NULL) and heterozygote (IkHT) Ikaros null mice were genotyped by PCR as described −) HPCs were purified using the easySep mouse hematopoietic progenitor enrichment kit (StemCell Technology).Homozygote . Retroviral infection of lin− HPCs was carried out as described − HPCs were cultured for 3 days in medium containing 50 ng/ml SCF, 10 ng/ml IL3, 10 ng/ml IL6 and 5×10−5 M β-mercaptoethanol, without Erythropoietin. Transduced lin−/GFP+ HPCs were isolated on a FACS Aria III sorter (BD Biosciences) based on green fluorescence. HPCs were seeded in triplicate on complete methylcellulose medium (MethoCult Stemcell Technologies). Colonies were scored at day 14.pMSCV/Ik1 and pMSCV/Ik1ΔPP1 vectors were generated by cloning the murine t-test was used to determine the level of statistical significance .Unpaired Student's Additional Materials and Methods information can be found in Figure S1A) Western blot assays of total cell lysates of stably transfected Jurkat clones; Ik1 and Ik2 indicate the endogenous full-length and functional IKAROS isoforms B) Protein co-immunoprecipitation on increasing amount of G1E-2 whole cell extracts (WCE). Immunoprecipitations were performed with HEXIM1 antibodies or isotype-matched IgG control (IgG) as indicated at the top of the panels; immunoblots were probed with IKAROS or HEXIM1 antibodies; Input samples represent 2% of protein extracts; filled dot: non-specific bands; C) Purification of Flag-HA-Ik associated proteins. Nuclear extracts from Jurkat cells carrying the pOZ-N-Flag-HA-IKAROS-IRES-ILR2 vector and expressing a double tagged IKAROS (Flag-HA-Ik) or Jurkat cells expressing the pOZ-N-Flag-HA-IRES-ILR2 empty vector (Mock) were used for sequential immunoaffinity purification using Flag- followed by HA-conjugated matrix. A fraction of the purified complexes were loaded on SDS-PAGE and silver stained; E1: first HA elution; E2: second HA elution; MW: molecular weights (in KDa); putative CDK955 and CDK942 isoforms are indicated by arrows; compared to the D) Protein expression analysis. Western blot assays of total cell lysates were performed with non-target (Nt-sh) or Ikaros-specific (Ik-sh) sh-RNA Jurkat clones; fold decrease of IKAROS protein levels in Ik-sh vs. Nt-sh samples was calculated as indicated at the bottom of the image, using ACTIN and PARP-1 as internal controls; densitometry analysis of Western blot gels was performed using Multi Gauge software (Fuji Film); E) Protein co-immunoprecipitation of whole cell extracts isolated from Ikaros knock-down (Ik-sh) or non-target control (Nt-sh) Jurkat cells. Immunoprecipitations were performed with the antibodies or isotype-matched IgG control (IgG) indicated at the top of the panels; immunoblots were probed with the antibodies indicated at the right-side of the panels; Input samples represent 2% of protein extracts; F, G) Protein co-immunoprecipitation of Jurkat cell nuclear extracts. Immunoprecipitations were performed with the antibodies or isotype-matched IgG control (IgG) indicated at the top of the panels; immunoblots were probed with the antibodies indicated at the right-side of the panels; Input samples represent 2% of protein extracts; asterisks: IgG heavy and light chains; H) Protein co-immunoprecipitation of Jurkat cell nuclear extracts performed with lysis buffer containing 1 µg/ml DNase1.IKAROS expression and protein co-immunoprecipitation in Jurkat clones. Protein expression analysis. (TIF)Click here for additional data file.Figure S2Flt3 locus in lineage negative hematopoietic progenitors and G1E2 cells. A) Schematic representation of the murine c-Kit and Flt3 loci. Amplicon positions are indicated with arrows c-Kit locus, they correspond to: −114 Kb enhancer region; −0.5 Kb region; the Transcriptional Start Site (TSS); +1 Kb, +5 Kb, +9.7 Kb, +16.9 Kb and +58 Kb Open Reading Frame (ORF) regions; for the Flt3 locus, the TSS and the ORF +0.5 Kb, +2 Kb and +8.5 Kb regions; filled circles: TGGGAA IKAROS consensus binding sites that IKAROS specifically and directly binds in crude nuclear extracts of hematopietic cells B) Protein expression analysis. Western blot assays of total cell lysates of Ikaros knock-down G1E2 clones; ACTIN was used as internal control; Nt-sh: non-target sh-RNA G1E2 clones; Ik-sh: Ikaros-specific sh-RNA (Ik-sh1 and Ik-sh2) G1E2 clones; C–E) Chromatin immunoprecipitation (ChIP). ChIP assays were carried out with the antibodies indicated on the top of each panel; POL II: is an antibody against the N-terminal region of the large subunit of POL II and binds POL II in a phosphorylation-independent manner; PCTD: is an antibody against the CTD repeats phosphorylated at Ser2; y-axis: fold enrichments of Flt3 or Alas2 regions relative to Thp promoter and input samples are plotted as the mean ± Standard Deviation (SD) of the measurements; a value of 1 (dotted lines) indicates no enrichment; n≥4; the amplicons tested recognized the c-Fos TSS, the Flt3 TSS and ORF +2 Kb regions as well as the Alas2 −1 Kb, TSS and +20 Kb ORF regions. Since the Alas2 gene is not expressed in G1E2 cells, it was used as negative control; the Alas2 values shown in panel C correspond to the Alas2 values displayed in −: bone marrow-derived lineage negative hematopoietic progenitor cells (HPCs); IkWT lin−: Ikaros wild type lin− HPCs; IkHT: Ikaros heterozygote null lin− HPCs; IkNULL lin−: Ikaros homozygote null lin− HPCs; Nt-sh: non-target sh-RNA G1E2 clones; Ik-sh: Ikaros-specific sh-RNA (Ik-sh1 and Ik-sh2) G1E2 clones. *: P≤0.05 by Student's t-test.Factor recruitment to the (TIF)Click here for additional data file.Figure S3Flt3 and c-Kit loci in Flavopiridol-treated and Cdk9 knock-down G1E2 cells. In panels A–C, G1E2 cells were treated for 2 h either with 0.01% DMSO (control) or 100 nM Flavopiridol. A) Protein co-immunoprecipitation of G1E2 total cell lysates. Immunoprecipitations were performed with an IKAROS-specific antibody and immunoblots were probed with the same antibody in order to exclude any significant variation of IKAROS phosphorylation levels upon Flavopiridol treatment; Input samples represent 2% of total cell lysates; filled dot: non-specific band; B) Gene expression profiles. RNA samples were retro-transcribed with random oligonucleotides to amplify nascent transcripts, which were used as template for qPCR with intron-specific Flt3  or Alas2  primer sets; Rnu2-1, a Flavopiridol-insensitive gene was used as internal control; y axis: relative nascent transcript enrichment levels; ratios are plotted as the mean ± Standard Deviation (SD) of the measurements; n≥4; C, F) Chromatin immunoprecipitation (ChIP). ChIP assays were carried out with the antibodies labeled on the top of each panel; POL II: is an antibody against the N-terminal region of the large subunit of POL II and binds POL II in a phosphorylation-independent manner; PCTD: is an antibody against the CTD repeats phosphorylated at Ser2; y-axis: fold enrichments of Flt3 TSS or +2 and +8.5 ORF regions, c-Kit and Alas2 TSS, relative to Thp promoter and input samples are plotted as the mean ± SD of the measurements; a value of 1 (dotted lines) indicates no enrichment; n≥4; D) Protein expression analysis. Western blot assays of total cell lysates of non-target (Nt-sh) or Cdk9-specific (Cdk9-sh1 and Cdk9-sh2) sh-RNA G1E2 clones; the antibodies used are indicated at the bottom of the panels; ACTIN was used as internal control; E) Gene expression profiles. mRNA samples were retro-transcribed with oligo-dT nucleotides; cDNA was used as template for qPCR with gene-specific primer sets; β-Actin was used as internal control; y axis: relative mRNA enrichment levels; ratios are plotted as the mean ± SD of the measurements; n≥4. *: P≤0.05 by Student's t-test.Factor recruitment to the (TIF)Click here for additional data file.Figure S4Flt3 locus in Calyculin A-treated G1E2 cells. A) Protein co-immunoprecipitation of COS-7 total cell lysates. COS-7 cells, which do not express any IKAROS protein, were transiently transfected with an expression vector encoding the murine Ikaros1 cDNA with a Flag and HA tag at its N-terminal region (FH-Ik1); immunoprecipitations were performed with PP1α antibodies or isotype-matched IgG control (IgG); immunoblots were probed with antibodies directed against HA  or PP1α (lower panel); Input samples represent 2% of nuclear extracts; B) CMX Ros staining of DMSO- (upper panels) or Calyculin A-  treated G1E2 cells. Flow cytometry; FCS: Forward scatter; SSC: Side scatter; CMX Ros: red fluorescence emission from CMX Ros; C, D, G) Gene expression profiles of G1E2 cells. RNA samples were retro-transcribed with random oligonucleotides to amplify nascent transcripts, which were used as templates for qPCR with intron-specific c-Kit , Flt3 (+2 Kb and +67 Kb regions) or Gapdh  primer sets; y axis: relative nascent transcript enrichment levels; ratios are plotted as the mean ± Standard Deviations (SD) of the measurements; n≥4; in panel C, the experiments were performed on G1E2 cells treated for 30 min with 1 µM Okadaic Acid or its diluent as control; in panel D, the experiments were performed on G1E2 cells treated for 2 h with 1 µM Cyclosporin A or its diluent as control; in panel G, the experiments were performed on G1E2 cells treated for 30 min with 100 nM Calyculin A or its diluent as control; E, F, H) Chromatin Immunoprecipitation (ChIP). ChIP assays were carried out with the antibodies labeled on the top of each panel; POL II: is an antibody against the N-terminal region of the large subunit of POL II and binds POL II in a phosphorylation-independent manner; PCTD: is an antibody against the CTD repeats phosphorylated at Ser2; y-axis: fold enrichments of Flt3 TSS or +2 Kb and +8.5 Kb ORF regions, Alas2 TSS or Gapdh TSS regions (used as negative controls) relative to Thp promoter and input samples are plotted as the mean ± SD of the measurements; a value of 1 (dotted lines) indicates no enrichment; n≥4; lin−: bone marrow-derived lineage negative hematopoietic progenitor cells (HPCs); IkWT lin−: Ikaros wild type lin− HPCs; IkHT lin−: Ikaros heterozygote null lin− HPCs; IkNULL lin−: Ikaros null lin− HPCs; Nt-sh: non-target sh-RNA G1E2 clones; Ik-sh: Ikaros-specific sh-RNA (Ik-sh1 and Ik-sh2) G1E2 clones. *: P≤0.05 by Student's t-test.IKAROS-PP1α interaction and factor recruitment to the (TIF)Click here for additional data file.Figure S5A) Single-cell immunofluorescence analysis. Ikaros homozygote null bone marrow-derived lineage negative hematopoietic progenitor cells (IkNULL lin− HPCs) were transduced with pMSCV/Ik1 (IkNULL/Ik1), pMSCV/Ik1ΔPP1 (IkNULL/Ik1ΔPP1) or the empty pMSCV vector (IkNULL/GFP) and then cytospun and fixed on slides; infected cells were recognized as GFP+ cells; IKAROS1 or IKAROS1ΔPP1 proteins were detected with mouse anti-HA and Texas Red (TR)-conjugated anti-mouse antibody; representative lin− HPCs transduced cells are shown where infected cells are detected as green signals and IKAROS1- or IKAROS1ΔPP1-expressing cells are detected as red signals; B) Protein co-immunoprecipitation of 293T total cell lysates. 293T cells, which do not express any IKAROS protein, were transiently transfected with an expression vector encoding the murine Ikaros1 cDNA (FH-Ik1), the mutant Ikaros1ΔPP1 (FH-Ik1ΔPP1) cDNA or the empty vector (Mock), all with a Flag and HA tag at their N-terminal regions; immunoprecipitations were performed with FLAG antibodies; immunoblots were probed with IKAROS, Mi2, CDK9, CYCLIN T1 or MTA2 antibody; Input samples represent 2% of total cell lysates; C) In vitro hematopoietic differentiation of lineage negative (lin−) hematopoietic progenitor cells (HPCs). Clonogenic assays in methylcellulose of IkWT or IkNULL lin− HPCs grown for 3 days in cytokine-supplemented liquid cultures; lin− HPCs were seeded on methylcellulose and colonies were scored at day 14; CFU-GEMM: colony forming unit granulocyte, erythrocyte, macrophage, megakaryocyte; CFU-G/M/GM: collectively identifies granulo-macrophage colonies; BFU-E: burst-forming unit erythrocyte; BFU-E/Mk: immature erythroid colonies with elevated megakaryocytic content; BFU-Mk/E: almost pure megakaryocyte colonies that contain only few clusters of immature erythroid cells; CFU-DC: colony forming unit dendritic cell; the data shown are representative of three independent experiments.IKAROS1-ΔPP1 detection in hematopoietic progenitors and IKAROS1-ΔPP1 protein interactions. (TIF)Click here for additional data file.Table S1Flt3 POL II traveling ratio. Flt3 traveling ratio values  were obtained by chromatin immunoprecipitation with POL II antibody, which recognizes the N-terminal region of the large subunit of POL II and binds POL II in a phosphorylation-independent manner and indicate the enrichment levels of Flt3 +2/TSS regions relative to the control and the input samples ; Fvp: Flavopiridol-treated G1E2 cells (100 nM for 2 h).see also  legends;(DOCX)Click here for additional data file.Table S2Protein phosphatase 1 catalytic subunits identified by immunoaffinity purification and LC-MS/MS analysis of Flag-HA-IKAROS complexes. The percentage values indicate the sequence coverage of the identified proteins; False Discovery Rate (FDR): 0%.(DOCX)Click here for additional data file.Table S3Super Elongation Complex (SEC) and microprocessor components identified by immunoaffinity purification and LC-MS/MS analysis of Flag-HA-IKAROS complexes. False Discovery Rate (FDR): 0%.(DOCX)Click here for additional data file.Text S1Supplemental methods.(DOCX)Click here for additional data file.


Host cell differentiation-dependent regulation of human papillomavirus (HPV) gene expression is required for productive infection. The host cell CCCTC-binding factor (CTCF) functions in genome-wide chromatin organization and gene regulation. We have identified a conserved CTCF binding site in the E2 open reading frame of high-risk HPV types. Using organotypic raft cultures of primary human keratinocytes containing high-risk HPV18 genomes, we show that CTCF recruitment to this conserved site regulates viral gene expression in differentiating epithelia. Mutation of the CTCF binding site increases the expression of the viral oncoproteins E6 and E7 and promotes host cell proliferation. Loss of CTCF binding results in a reduction of a specific alternatively spliced transcript expressed from the early gene region concomitant with an increase in the abundance of unspliced early transcripts. We conclude that high-risk HPV types have evolved to recruit CTCF to the early gene region to control the balance and complexity of splicing events that regulate viral oncoprotein expression.IMPORTANCE The establishment and maintenance of HPV infection in undifferentiated basal cells of the squamous epithelia requires the activation of a subset of viral genes, termed early genes. The differentiation of infected cells initiates the expression of the late viral transcripts, allowing completion of the virus life cycle. This tightly controlled balance of differentiation-dependent viral gene expression allows the virus to stimulate cellular proliferation to support viral genome replication with minimal activation of the host immune response, promoting virus productivity. Alternative splicing of viral mRNAs further increases the complexity of viral gene expression. In this study, we show that the essential host cell protein CTCF, which functions in genome-wide chromatin organization and gene regulation, is recruited to the HPV genome and plays an essential role in the regulation of early viral gene expression and transcript processing. These data highlight a novel virus-host interaction important for HPV pathogenicity. Papillomaviruses are a highly diverse family of small DNA tumor viruses that specifically infect the mucosal and cutaneous epithelium. Human papillomavirus (HPV) types that infect the mucosal epithelium are subdivided into low-risk and high-risk groups, depending on their association with cancer development , 2.97 in HPV16 and P105 in HPV18 and HPV31) within the viral upstream regulatory region (URR). This drives expression of the E6 and E7 oncoproteins in the basal cells and stimulates continued cellular proliferation. The E7 gene products target members of the retinoblastoma family of proteins, pRb/p105  . RNA polpRb/p105  and p107pRb/p105 , stimulapRb/p105 . This alpRb/p105 , and thepRb/p105 . This inAll HPV transcripts are polycistronic. Alternative splicing and polyadenylation of transcripts further regulate HPV early gene expression and increase the repertoire of expressed proteins –17. Exac–CCCTC-binding factor (CTCF) is a ubiquitous host architectural protein that binds 10,000 to 50,000 sites within the human genome . Dynamic21–http://insulatordb.uthsc.edu/ and http://bsproteomics.essex.ac.uk:8080/bioinformatics/ctcfbind.htm) or using Storm analysis software. The position weight matrices (PWM) utilized by these analysis tools have been published previously  within the E2 coding region. The plasmid pDrive-SP6-His-CTCF was a gift from D. Farrar  and encodes human CTCF protein with a 10× histidine tag at the N terminus.pUC19-HPV6b, pBR322-HPV11, and pBR322-HPV16 were a gift from E.-M. de Villiers, DKFZ, Germany. pBR322-HPV31 was a gift from L. Laimins, Northwestern University, USA. pGEMII-HPV18 was a gift from F. Stubenrauch, University of Tübingen, Germany, and was used as a template for site-directed mutagenesis  to create pGEMII-HPV18-ΔCTCF that contains three conservative nucleotide substitutions (CCTCF antibody was purchased from Active Motif (Belgium). FLAG M2 and anti-cytokeratin 1/10 8.60 antibodies were purchased from Sigma-Aldrich (United Kingdom). Anti-cytokeratin 5 D5/16 B4 was purchased from Boehringer Mannheim Biochemica (Switzerland), and loricrin AF62 was from Covance Research Products (United Kingdom). Bromodeoxyuridine (BrdU) and p130 were purchased from Becton Dickinson (United Kingdom). Cyclin B1 H-433, HPV18 E6 (G-7), p53 DO1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz Biotechnology (USA). Phospho-histone H3  was purchased from Cell Signaling (USA), and HPV18 E7 (8E2) was from Abcam (United Kingdom). All fluorescent secondary antibodies were purchased from Invitrogen (United Kingdom). Rabbit polyclonal anti-HPV16 E2 antibody was obtained from F. Thierry (Singapore) . Monocloin vitro transcription translation reaction using the TNT SP6 high-yield wheat germ protein expression system .For electrophoretic mobility shift assay (EMSA), DNA fragments were amplified with a forward primer containing an M13-overhang (sequences are available upon request) using master mix S . The products of the first PCR then were amplified in a second PCR using a 6-carboxyfluorescein (FAM)-labeled M13 forward primer. CTCF protein was produced in an Two μl of FAM-labeled DNA was incubated with 1 μl CTCF protein in a 10-μl reaction mixture containing 0.5% NP-40, 50 mM KCl, 10 mM Tris-HCl, pH 7.5, 0.1 μg/μl poly(dI-dC), 5% Ficoll 400, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.1 mM dithiothreitol (DTT). Samples were incubated at room temperature for 1 h before separation on a 4.5% native polyacrylamide gel. FAM fluorescence was imaged at 520 nm using a Typhoon FLA7000 .TC) values were calculated at a constant threshold for each experiment, and the percentage of input DNA was calculated using the standard curve.Chromatin immunoprecipitation (ChIP) assays were carried out using the ChIP-IT express enzymatic ChIP kit (Active Motif) by following the manufacturer's instructions. Cells were fixed in 1% formaldehyde for 3 min at room temperature, and nuclei were released by 40 strokes in a tight dounce homogenizer. DNA was purified using a GenElute PCR cleanup kit (Sigma-Aldrich). ChIP efficiency was assessed by quantitative PCR (qPCR) using SensiMix SYBR master mix  using an MXPro 3000 (Agilent Technologies). Primer sequences used are available upon request. Cycle threshold . Five hundred μl growth medium and 50 μl CCK-8 reagent  were added to each well, and the plate was incubated at 37°C for 2 to 4 h. Absorbance was read at 450 nm using an iMark microplate reader (Bio-Rad). Wells that contained J2 3T3 fibroblasts but not HFK were used as a blank for each plate.A total of 1 × 10Four-μm sections of organotypic cultures were placed on polylysine-coated slides and incubated at 50°C for 30 min. One section from each raft was stained with hematoxylin and eosin for morphological analysis. Antigens were retrieved using an agitated low-temperature method, as previously described , followiNuclei positive for HPV DNA amplification in raft sections were detected with a biotin-conjugated high-risk HPV DNA-specific probe using Leica Bond-Max technology, as described by the manufacturer .TC method . Five μg of RNA was treated with 1 U of RQ1 DNase (Promega) for 30 min at 37°C, which subsequently was inactivated for 10 min. Reverse transcription was performed using a Tetro cDNA synthesis kit (Bioline). Two μl of cDNA was used for the amplification of HPV transcripts using the primers listed in t test was used to determine statistical significance.A two-tailed, unpaired student's CTCF binding sites in the genomes of low-risk HPV types 6b and 11 and high-risk HPV types 16, 18, and 31 were predicted using open access databases and Storm analysis software . These m27–in silico analysis, CTCF binding was assessed in vitro by EMSA. Approximately 200-bp DNA fragments containing the predicted binding motifs were incubated with CTCF protein . No consistent differences were observed in the number of cells with amplified HPV genomes between WT and ΔCTCF HPV18 rafts, demonstrating that CTCF recruitment has a minimal role in viral genome amplification , early differentiation (keratin 1), and late differentiation (loricrin) alongside E1^E4, a marker of the productive phase of the HPV life cycle . ExpressBrdU incorporation was used to assess cell cycle entry and cellular DNA replication. BrdU-positive cells were confined to the basal layer in rafts derived from untransfected donor keratinocytes . Increas2 and mitotic entry, respectively  as markers of Gectively  and E. Iectively  and B, aectively  and D. Iectively  and F. TThe increased cell cycle entry and hyperproliferation observed in the organotypic raft cultures derived from HFK lines maintaining ΔCTCF HPV18 genomes could be due to an increase in the expression of E6 and E7 viral oncoproteins. Detection of these proteins by immunostaining currently is not possible; therefore, raft sections were stained with surrogate markers, p53 as a marker for E6 expression and pRb family member p130 for E7 expression . Cells sCTΔΔ method. Donor 1 was shown to have a 21.19-fold increase (±10.48-fold standard errors [SE]) and donor 2 had a 44.08-fold increase (±26.95-fold SE) in E6E7 transcript in the HPV18 ΔCTCF rafts compared to wild-type levels. In addition, Western blot analysis of protein extracts from raft cultures harvested at day 14 demonstrated a clear increase in E6 and E7 protein levels . As expected, the relative abundance of unspliced E6E7 transcripts in ΔCTCF HPV18 raft cultures was significantly increased compared to that of the WT  and B. En levels , which wThe increase in E6E7 unspliced transcript could be due to an increase in the activity of the early promoter. HPV-encoded E2 protein is known to repress the activity of this promoter, and changes in E2 expression could affect early promoter activity –45. To d–A diverse range of early transcripts is expressed from the HPV genome as a result of numerous alternative splicing events , 15. AltIn silico predictions were used to identify potential CTCF binding sites, a high frequency of which bound CTCF in vitro. The relative position of many of the CTCF binding sites is conserved. A cluster of CTCF binding sites was identified in the late gene region of all of the HPV types tested, and binding within the E2 ORF appears to be conserved in the high-risk types, indicating that recruitment of CTCF to this region is related to the ability of the virus to induce cellular transformation. This suggests that the recruitment of CTCF to these regions was an early evolutionary event and that CTCF is important for the virus life cycle. Furthermore, the frequency of CTCF binding sites within the genomes of the HPV types analyzed in this study show an enrichment of sites compared to the frequency of binding sites within the human genome . However, our data show that CTCF does not bind within the late gene region in HPV16 and HPV18 in cells grown in monolayer culture. It is possible that CTCF recruitment to this region is promoted by cellular differentiation, and this is important for capsid protein expression or viral genome amplification. Differentiation-induced loss of CpG methylation in the late region of episomal HPV16 genomes has been reported . CpG metSeveral host cell proteins are recruited to the HPV genome to regulate transcriptional control. The binding of host cell transcription factors to sequences within the URR to control early gene transcription has been well characterized. Transcriptional regulators, such as AP1 , SP1 4949, TFIID56–2 than progress through mitosis. Importantly, there were no discernible effects on the overall expression of E2, although expansion of the midlayers of the epithelium resulted in an apparent delay in E2 expression. In addition, viral genome replication and amplification were unaffected, suggesting that E1 protein levels were unaffected. This suggests that HPV18 and perhaps other oncogenic HPV types have evolved to bind CTCF in this region to regulate balanced and controlled E6 and E7 expression in the context of a productive infection. Interestingly, CTCF does not appear to bind to the site within the E2 ORF in integrated sequences in HeLa cells  and Kaposi's sarcoma-associated herpesvirus (KSHV). Mutation of sites to prevent CTCF binding has demonstrated that CTCF determines latency in these viruses by blocking epigenetic silencing of latency-associated promoter elements and mediating long-range interactions within the viral genome –68. This–CTCF binding within the host genome controls cotranscriptional alternative splicing events by creating a roadblock to processing RNA polymerase II and promoting inclusion of weak upstream exons . TherefoOur data suggest that CTCF recruitment to the E2 ORF binding site is a control mechanism for the expression of unspliced and alternatively spliced early transcripts in the HPV life cycle. It is interesting that the current model of CTCF-mediated splicing regulation predicts that DNA-bound CTCF pauses RNA polymerase II progression and promotes the inclusion of weak upstream exons by allowing the splicing machinery more time to process the nascent RNA strand . Our dat


Here we provide an updated map of the Drosophila melanogaster regulatory genome based on the location of 84 TRFs at various stages of development. This regulatory map reveals a variety of genomic targeting patterns, including factors with strong preferences toward proximal promoter binding, factors that target intergenic and intronic DNA, and factors with distinct chromatin state preferences. The data also highlight the stringency of the Polycomb regulatory network, and show association of the Trithorax-like (Trl) protein with hotspots of DNA binding throughout development. Furthermore, the data identify more than 5800 instances in which TRFs target DNA regions with demonstrated enhancer activity. Regions of high TRF co-occupancy are more likely to be associated with open enhancers used across cell types, while lower TRF occupancy regions are associated with complex enhancers that are also regulated at the epigenetic level. Together these data serve as a resource for the research community in the continued effort to dissect transcriptional regulatory mechanisms directing Drosophila development.Annotation of regulatory elements and identification of the transcription-related factors (TRFs) targeting these elements are key steps in understanding how cells interpret their genetic blueprint and their environment during development, and how that process goes awry in the case of disease. One goal of the modENCODE ( Drosophila melanogaster has been at the forefront of the biology of transcriptional regulation for decades, with polytene chromosome studies providing some of our earliest glimpses into genome-wide gene regulatory and protein–DNA interactions  Project, however, is to survey a diverse sampling of transcriptional regulators, both DNA-binding and non-DNA-binding factors, to get a broad view of the patterns by which transcriptional regulators target the genome. Here we provide an updated map of the Drosophila melanogaster regulatory genome based on the location of localization of 84 transcriptional regulators at various stages of development.The importance of focused studies of gene regulatory networks—studies exploring panels of TRFs working within the same network, or studies exploring the impact of cellular context on TRF–DNA interactions—cannot be overstated. The goal of the modENCODE , using both previously published data sets and new data generated by the modENCODE Project (http://intermine.modencode.org/) or Gene Expression Omnibus , represent 171 separate genome-wide ChIP experiments performed in duplicate or greater, 413,743 TRF-binding sites, and 50,336 unique TRF-binding regions.We describe in vivo genome-wide binding patterns of 84 cis-regulatory modules (CRMs)  . To get s (CRMs)  all bindDrosophila genome based on chromatin state. Despite using different cell types, experimental techniques, and chromatin factors these independent studies generated functionally consistent chromatin state maps  . HoweverThere are three ‘inactive’ chromatin states according to the five-state model: BLACK, BLUE, and GREEN. GREEN chromatin is repressive heterochromatin, primarily pericentric, and is targeted by HP1 and little else . HoweverDrosophila , and vestigial (vg), all previously characterized Pan targets  regions. Subtracting HOT regions from the calculation of TRF–TRF overlap significance leads to a more defined interaction matrix, often further highlighting the pairwise interactions described in the previous paragraph ; in keeping with the previous literature, we refer to these hotspots of TRF localization as HOT   . Indeed,Methods) . Indeed,Methods) . Thus, aAside from high TRF co-occupancy, do HOT regions have any other distinguishing features? To address this question we separated binding regions into three categories: HOT, WARM, and COLD; HOT regions are targeted by 14 or more TRFs, WARM regions are targeted by 4–13 TRFs, and COLD regions are targeted by 1–3 TRFs (see Methods). Relative to COLD regions, HOT regions occupy a much smaller fraction of the intergenic genome . The revWe next asked whether there are differences in DNA conservation across the range of TRF occupancies. Our measure of DNA conservation in this case is its phastCons score, which measures the probability that an individual base is part of a stretch of base pairs  that is conserved. One implication from such analysis is that DNA with a higher phastCons score is under purifying selection and more likely to be functional. Mean phastCons values were calculated for the center ±1 kb for all HOT, WARM, or COLD regions . IntriguP < 10−15). Additionally, non-housekeeping categories including ‘developmental process’ and ‘transcription regulator activity’ are also enriched (both P ≤ 10−25) among HOT target genes. However, comparing the gene sets targeted by HOT, WARM, and COLD regions becomes complicated because many loci are associated with more than one type of binding event. Thus, we instead focused on genes targeted by various combinations of HOT and COLD binding to explore potential differences in regulatory logic across unique gene sets. We broke genes down into loci associated only with a HOT binding region, loci associated with both HOT and COLD binding, and loci associated only with COLD binding. GO analysis of these gene categories revealed an interesting pattern for genes associated with HOT regions  analysis of the genes associated with HOT regions: Categories such as ‘metabolic process’ and ‘cellular component biogenesis’ are highly significant  was used to identify two primary types of enhancers (Multiple results described thus far are consistent with a model in which lower occupancy binding regions (COLD and WARM) represent the traditional developmentally regulated enhancer—these regions are conserved, often distal to the promoter, and tend to be associated with genes involved in developmental patterning. However, HOT regions can be associated with these patterning genes but are also associated with ubiquitous genes and tend to be promoter-proximal. It is important to point out that HOT regions are capable of driving patterned expression, as was recently demonstrated by nhancers . The firnhancers . Overallnhancers . Warm renhancers . Thus, aIn this study we provide analysis of new and previously published ChIP data, in combination with published chromatin state and genome-wide enhancer characterization data, to provide a window into the principles governing genomic gene regulatory networks and TRF–DNA interactions.From a gene regulatory network perspective, this work highlights numerous interactions that may warrant further exploration. Thousands of TRF–DNA interactions are observed within these data, with 5823 binding events overlapping DNA regions that act as transcriptional enhancers , suggestA broad range of binding strategies and preferences are clear from the TRFs analyzed in these data. Although a majority of the factors tend to bind proximal promoter regions, many important developmental regulators are significantly bound to intronic and promoter-distal intergenic regions, likely representing targeting of enhancers controlling genes subject to complex developmentally regulated transcriptional controls. TRFs also differ in their association with various types of chromatin. Most TRFs preferentially bind one of the two ‘active’ chromatin states , consistent with the overall accessibility of these chromatin types and their association with expressed genes.os , upd2, and upd3—fall within a large domain of BLUE chromatin and are confirmed Pc targets . Consistent with the promoter bias of HOT regions, some evidence suggests that SNP density increases in the proximal promoter in D. melanogaster and humans and, additionally, SNPs in the proximal promoter show a biased signature of transversions in humans , whereas lower-occupancy regions are more likely to target less accessible enhancers that tend to be further regulated at the epigenetic level. In combination with the results described above, this provides another piece of evidence that lower-occupancy regions represent traditional enhancers, which tend to be subject to more complex spatial and developmental regulation, while HOT regions represent DNA regions with context-independent, and possibly less complex, regulatory functions.http://intermine.modencode.org). A number of antibodies were generous contributions from members of the Drosophila research community: Ken Cadigan (Pan/TCF), Andy Dingwall (Cmi/Lpt), Eric Lai (Insv), Erika Bach (Stat92E), Jim Kadonaga (TBP), Ken Irvine (Yki), Claude Desplan (Prd), Stephen Crews (Sc), Sean Carroll (Dll), Richard Mann (Hth), and Scott Hawley (Trem). Immunoprecipitated DNA was prepared for Illumina sequencing either as described in Chromatin collection and chromatin immunoprecipitation were performed as described previously . TransgeP-value threshold of 1 × 10−3 to obtain a maximum of 30-k peaks, and peaks were ranked according to their P-value scores. Replicates were then pooled and MACS2 was again used to call peaks at a P-value threshold of 1 × 10−3. Peaks from the pooled set that overlapped at least one peak in both individual replicate sets were retained. From this set of replicate-reproducible peaks, we obtained two independent rankings based on the P-values from each replicate; this pair of ranked lists was used as input for the IDR framework. Cross-replicate and pseudoreplicate rank thresholds at an IDR of 5% were generated, and the better of the two was used to generate the final set of rank consistent and reproducible peaks. Details of the IDR framework are available at https://sites.google.com/site/anshulkundaje/projects/idr. All data sets are available through the modENCODE Data Coordinating Center .ChIP-chip peak calls are as previously described . For ChIChIP peaks were annotated as overlapping genomic features according to the FlyBase r5.34 gene structure annotation and the following categories: promoter , coding (coding sequence), downstream , intron, intergenic regions. Peaks were assigned to target genes based on the nearest transcription start site . Peaks were assigned to chromatin states based on their overlap with the five states defined in Z-score was assigned to each pair to indicate whether the co-occurrence was significantly higher or lower than expectation (see http://www.encodestatistics.org/). Regions of significant cobinding—HOT regions—were defined following the algorithm described in D. melanogaster genome, we took centers of all peaks of all regulators to represent their genomic coordinates and calculated the density genome widely using 300-bp bandwidth Kernel Density Estimation. We then scanned the density scores peak wide and denoted each peak a HOT region candidate. The complexity (occupancy) of each HOT region candidate was calculated by summing the Gaussian kernalized distance from the peak to peaks of each other regulator that contributed at least 0.1 to this strength. Finally, we named these candidate regions as HOT if the complexity was ≥15, as COLD if the complexity was ≤3, and as WARM for all the rest. HOT, WARM, and COLD regions were assigned to genomic features, target genes, and chromatin states as described above for individual TRFs. Only level 5 Gene Ontology (GO) categories in which the enrichment P-value (Bonferroni corrected) was ≤1 × 10−5 for at least one of the three categories  were used for GO category clustering , modMine (http://intermine.modencode.org), or both under GEO accession nos. GSE49768–GSE49780, GSE49899, or the modMINE DCC IDs 2627, 2629, 2630, 2633, 2634, 2636, 3234, 3240, 3390–3396, 3399–3401, 3403. See Supplemental Table S1 for all data set submission information.Data from this study have been submitted to the NCBI Gene Expression Omnibus (GEO;


We have used PAR-CLIP to map the position of Pol II genome-wide in living yeast cells after depletion of components of either the pA or non-pA termination complexes. We show here that Ysh1, responsible for cleavage at the pA site, is required for efficient removal of Pol II from the template. Depletion of Ysh1 from the nucleus does not, however, lead to readthrough transcription. In contrast, depletion of the termination factor Nrd1 leads to widespread runaway elongation of non-pA transcripts. Depletion of Sen1 also leads to readthrough at non-pA terminators, but in contrast to Nrd1, this readthrough is less processive, or more susceptible to pausing. The data presented here provide delineation of  Transcription termination is an important regulatory event for both non-coding and coding transcripts. Using high-throughput sequencing, we have mapped RNA Polymerase II's position in the genome after depletion of termination factors from the nucleus. We found that depletion of Ysh1 and Sen1 cause build up of polymerase directly downstream of coding and non-coding genes, respectively. Depletion of Nrd1 causes an increase in polymerase that is distributed up to 1,000 bases downstream of non-coding genes. The depletion of Nrd1 helped us to identify more than 250 unique termination regions for non-coding RNAs. Within this set of newly identified non-coding termination regions, we are further able to classify them based on sequence motif similarities, suggesting a functional role for different terminator motifs. The role of these factors in transcriptional termination of coding and/or non-coding transcripts can be inferred from the effect of polymerase's position downstream of given termination sites. This method of depletion and sequencing can be used to further elucidate other factors whose importance to transcription has yet to be determined. Termination of RNA Polymerase II (Pol II) transcription plays an essential role in the transcription cycle and has been the subject of several recent reviews In addition to mRNAs, Pol II transcribes a diverse set of non-coding RNAs including snoRNA, some snRNAs and several classes of ncRNA with unknown functions. In yeast, this pervasive transcription Pol II terminates coding and non-coding transcripts by different mechanisms Yeast Pol II terminators contain short RNA sequences that bind proteins within large complexes associated with the elongating Pol II. Loosely conserved pA signal sequences downstream of protein-coding genes bind to components of the CF1 complex leading to assembly of the cleavage and polyadenylation machinery Several possible mechanisms for Pol II termination have been proposed. The “torpedo” model postulates that cleavage at the pA site exposes an uncapped 5′ end on the nascent transcript that acts as a substrate for the 5′→3′ RNA exonuclease Rat1 in yeast or Xrn2 in metazoans Part of the uncertainty in delineating termination mechanisms is identifying which factors operate at which terminators in vivo. In this study we have used PAR-CLIP to map Pol II on the yeast genome in living cells RPL13A leading to rap-dependent depletion of FRB-tagged protein from the nucleus. Previous work has shown that a Pol II subunit RPB1-FRB strain shows greater than 90% depletion of Pol II at the PMA1 gene within 40 min NRD1-FRBGFP or YSH1-FRBGFP strains that also contain, RPB3-TAGRFP, Nrd1 and Ysh1 are depleted from the nucleus with similar kinetics  system kinetics . We werekinetics . Given ty slowly .600 to 0.7 OD600 as measured by BioTek scanner) indicating that 4tU has a less drastic effect on yeast growth than it does in mammalian cells treated for a longer time Growing yeast cultures were cross-linked as previously described RPB2-HTB NRD1-FRB strain grown in the presence or absence of rap. We also created libraries of Rpb2-bound RNA from SEN1-FRB and the parental RPB2-HTB strains grown in the presence of rap and a single library from YSH1-FRB. Replicate libraries were multiplexed and sequenced by Illumina Hi-seq. Each library yielded between 15–39 million unique reads  as previously described ue reads . Biologiue reads . The WT ue reads . Thus, PGAL7 and CYC1 pA sites To map pA-dependent termination we carried out PAR-CLIP on Rpb2 after depletion of Ysh1, the factor that has been shown to be required for cleavage of the nascent mRNA at the RNA14 and DBP2 genes show an increase of Pol II toward the 3′ end of the coding region indicating the presence of a Ysh1-dependent terminator upstream of the main pA site (RNA14 is located in the same region as the 3′ end of the shorter (1 kb) mRNA  mRNA .DBP2 gene is unique in yeast in having a long (1 kb) intron localized toward the 3′ end of the coding region and previous work has shown that the gene is autoregulated through sequences in the intron Dbp2p is a DEAD-box RNA helicase that plays a role in assembly of mRNP complexes and in RNA quality control Previous work has shown that Nrd1p is required for proper termination of ncRNAs like snoRNAs and CUTs as well as premature termination of coding transcripts of genes regulated by attenuation To map the position of termination we used an approach similar to that used to calculate a travelling ratio Once these regions were identified, we created plots of the difference between the reads in the control and reads with rap . Nrd1 desnR34 and the antisense CUT to the LEO1 gene demonstrating how the termination sites were identified. We have carried out this analysis for 49 snoRNAs and for 144 CUTs showing the highest level of readthrough transcription  which in general are not AT-rich. We propose that these U-rich regions constitute part of the non-poly(A) terminator.In NA genes . Taken tSNR13 readthrough transcripts are present at less than one copy per cell in wild-type cells and this is consistent with the low level of readthrough transcripts seen in the absence of rap in the Northern blot shown in SNR13  tag as described previously NRD1, YSH1, and SEN1 were C-terminally tagged with FRB and FRBGFP as described in Haruki et al. GAL1 promotor using a cassette from pFA6a-kanMX6-PGAL1-HBH Yeast strains for anchor away were constructed using the parental strain HHY168 600 of 0.1 from a 5 ml or 50 ml overnight culture. The cells were then incubated at 30°C to an OD600 of 1. 1 µg/ml of rap (LC laboratories) was then added to the cultures and the cells were allowed to grow for the indicated times in the experiment. For growth curves, a BioTek Infinity 2 (BioTek) was used to incubate a 24 well plate containing CSM media with and without 1 µg/ml rap (added after 6 hrs of growth) with orbital shaking . Cells were diluted to an OD600 of 0.1 per well and an OD600 was measured every 10 min for 24 hours.Cells were grown in Complete Synthetic Media (CSM) supplemented with 2% glucose or galactose and 40 mg/l adenine. For RNA analysis, cells were seeded to an ODTotal RNA was extracted from yeast with hot acid phenol as previously described NRD1-FRBGFP, YSH1-FRBGFP, or GALpSEN1-FRBGFP, RPB3-TAGRFP were grown to an OD600 of 1 at 30°C. Live cell imaging was done on a Deltavision microscope (Applied Precision) as described previously The strains containing 600 of 0.1 and incubated at 30°C until an OD600 of 1. Crosslinking was carried out by the protocol described in 2) from an LC-L5 LED UV Lamp (Hamamatsu) for 15 min with gentle stirring. The top ∼2 mm of the culture appears luminescent indicating that the UV light does not penetrate further into the culture. Assuming an even mixture of the culture we calculate that the average cell is cross-linked for only ∼10 seconds within the 15 min crosslinking period. The cultures were then filtered through 0.45 micron nitrocellulose filters (Millipore) and cells were scraped into 5 ml Buffer 1  and frozen in liquid nitrogen.Cells were grown overnight 30°C in 50 ml of YPD (Yeast Extract Peptone with 2% glucose) media. Four 500 ml flasks of sterile cross-linking media . The protein was eluted off of the Ni-NTA agarose with 5 ml Buffer 2+250 mM immidazole and into 5 ml Buffer 2+120 µl Protease inhibitor Cocktail VII+100 µl of hydrophilic streptavidin magnetic beads (NEB). The slurry was allowed to incubate for 4 hours at room temperature. The strepavadin magnetic beads were then resuspended in 500 ul of Buffer 3 , transferred to a 1.5 ml siliconized tube and washed with 3×1 ml of Buffer 3 followed by 3×1 ml of T1 Buffer . Beads were resuspended in 200 µl of T1 buffer. 0.15 U/µl of Rnase T1 (Fermentas) was added to the bead slurry and allowed to incubate at 25°C for 15 min. The beads were then washed 3×1 ml T1 wash buffer  followed by 3×1 ml washes in PNK Buffer . The beads were resuspended in 200 µl of TSAP reaction solution  and incubated at 37°C for 30 min. The beads were washed 1×1 ml Buffer 3, 2×1 ml T1 Wash Buffer, and 3×1 ml PNK Buffer.HTB tagged Rpb2 purification was adapted from our previously published protocol 32P γ-ATP, 5 mM DTT in PNK Buffer) and incubated at 37°C for 30 min. 1 mM of cold ATP was added to the reaction and incubated at 37°C for 10 min. The beads were washed 1×1 ml Buffer 3, 1×1 ml T1 wash, 3×1 ml T4 RNA Ligase Buffer . The beads were resuspended in 44 µl of T4 RNA Ligase 2 reaction solution  and incubated at 25°C for 4 hours. The beads were then washed with 1×1 ml Buffer 3 and 5×1 ml T4 RNA Ligase Buffer. The beads were resuspended in 50 ul of T4 RNA Ligase reaction solution  and incubated at 16°C overnight. The beads were then washed 5×1 ml Proteinase K Buffer . The beads were resuspended in 200 µl of Proteinase K Buffer +2% SDS and 12 µl of Proteinase K (NEB). The suspension was incubated for 30 min at 37°C at which point the supernatant was removed and saved and the beads were resuspended in 200 µl of Proteinase K Buffer +2% SDS and 12 µl of Proteinase K. The beads were then incubated 30 min at 37°C and the supernatants were pooled. The RNA was recovered by acid phenol/chloroform extraction followed by two serial ethanol precipitations. The resulting pellet was allowed to dry and resuspended in 15 µl of Nuclease-Free Water [Invitrogen]. The RNA was split into 5 µL aliquots and frozen at −80°C for storage.Streptavadin beads were resuspended in 200 ul of PNK reaction solution . For PCR analysis, biological repeats were multiplexed with the following primers. The forward primer was AATGATACGGCGACCACCGAGATCTACACGTTGAGAGTTCTACAGTCCG*A (where a * denotes and phosphorothioate bond). The first reverse primer was CAAGCAGAAGACGGCATACGAGATATTGGCGTGACTGGAGTTCAGACGTGTGCTCTTCGGATC*T for the first biological repeat and the second reverse primer was CAAGCAGAAGACGGCATACGAGATTACAAGGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T for the second biological repeat (IDT).The Reverse Transcription, PCR, and gel extraction of the cDNA Library were carried out as described previously 7 reads. PAR-CLIP datasets have been submitted to GEO with the accession number GSE56435.Sequencing and demultiplexing was done at UC Riverside on an Illumina HiSeq. Trimming of the resulting sequences as described previously To calculate the best polyA sites per gene we used the sites provided by Moqtaderi et al. Global readthrough percentage was calculated for each point in the genome as the percentage readthrough (reads downstream 500 bp/reads downstream 500 bp + reads upstream 500 bp) in the treated sample minus control sample. All points with a percentage readthrough of greater than 10%, at least a total of 1000 reads in either sample, and greater reads in the treatment vs the control were extracted. The list was further refined by taking the difference of every point 2 kb around non-overlapping termination sites , graphing the region, and then fitting a spline function over a 25 bp window to find the best point of inflection. These points of inflection were then used as a focal point for the meta gene analysis.Figure S1A. Growth of indicated FRB strains in CSM media with and without rap. OD600 was taken every 10 min for 20 hours. The grey bar represents the timeline of the experiment. B. Timeline of the PAR-CLIP protocol.(EPS)Click here for additional data file.Figure S2A–D. Replicate pair-wise comparison of number of reads corresponding to each gene in our PAR-CLIP biological replicates. Spearman coefficient is represented by rho.(EPS)Click here for additional data file.Figure S3A–C. Pair wise comparison of number of Rpb2-HTP cross-linked reads in each annotated gene vs. GRO-seq reads (EPS)Click here for additional data file.Figure S4A–D. Histograms representing normalized reads with Ysh1 (grey) and without Ysh1 (black) at the given genomic locations. Genes and pA sites are represented below each graph and the length of the genome depicted is given in the lower right hand corner. Below are qPCR at different times after addition of rap using the amplicon highlighted in red.(EPS)Click here for additional data file.Figure S5Relative position on Schulz et al. termination points compared to our PAR-CLIP determined inflection points.(EPS)Click here for additional data file.Figure S6A comparison of NET-Seq data from Churchman and Weissman (EPS)Click here for additional data file.Figure S7Comparison of PAR-CLIP data to Schulz et. al. 4tU-seq data (EPS)Click here for additional data file.Figure S8Histograms representing normalized reads with Sen1 (grey) and without Sen1 (black) at the given genomic locations.(EPS)Click here for additional data file.Figure S9Histograms representing normalized reads with Nrd1 (grey) and without Nrd1 (black) for NRD1 and TYE7. The same region is also represented in as a histogram below each graph with Sen1 (grey) and without Sen1 (black).(EPS)Click here for additional data file.Table S1Read counts at various stages of bioinformatic manipulation.(EPS)Click here for additional data file.Table S2Top 266 points of inflection after spline function refinement. CUT annotations are defined in Neil et. al. (PDF)Click here for additional data file.Table S3Top 49 points of inflection 3′ of snoRNAs after spline function refinement. snoRNA annotations come from the saccharomyces genome database.(EPS)Click here for additional data file.Table S4Yeast strains used in this study.(EPS)Click here for additional data file.Table S5Primers used in this study.(EPS)Click here for additional data file.


A prominent feature of demyelinating diseases such as multiple sclerosis (MS) is the degeneration and loss of previously established functional myelin sheaths, which results in impaired signal propagation and axonal damage. However, at least in early disease stages, partial replacement of lost oligodendrocytes and thus remyelination occur as a result of resident oligodendroglial precursor cell (OPC) activation. These cells represent a widespread cell population within the adult central nervous system (CNS) that can differentiate into functional myelinating glial cells to restore axonal functions. Nevertheless, the spontaneous remyelination capacity in the adult CNS is inefficient because OPCs often fail to generate new oligodendrocytes due to the lack of stimulatory cues and the presence of inhibitory factors. Recent studies have provided evidence that regulated intracellular protein shuttling is functionally involved in oligodendroglial differentiation and remyelination activities. In this review we shed light on the role of the subcellular localization of differentiation-associated factors within oligodendroglial cells and show that regulation of intracellular localization of regulatory factors represents a crucial process to modulate oligodendroglial maturation and myelin repair in the CNS. The central nervous system (CNS) is composed of two major classes of cells—neurons and glial cells—the latter of which can be subdivided into astrocytes, microglia and oligodendrocytes. While neurons provide the basis for signal transduction and information processing, glial cells account for a wide range of specific functions. Oligodendrocytes generate myelin sheaths surrounding axons, and they are therefore referred to as myelinating glial cells. Myelin sheaths are imperative for stabilization, protection, and electrical insulation of axons, which enables accelerated electrical signal propagation ,2,3. ThiOlig1/2 and Nkx2.2 [However, demyelination can be counteracted to a certain extent via recruitment and activation of resident adult oligodendroglial precursor cells (OPCs) . These cd Nkx2.2 ,15,26. Fd Nkx2.2 ,27.in vivo models, such as those based on cuprizone-mediated demyelination or upon direct injection of toxins such as lysolecithin or ethidium bromide [Remyelination can be very effective in experimental  bromide . Neverth bromide ,29,30,31Intracellular protein shuttling is essential for protein function and for functional and spatial diversity  because i.e., chromosome region maintenance 1, CRM1; also referred to as exportin1 or Xpo1) and Ran-GTP complex, and this heterotrimeric complex can then diffuse through the NPC into the cytoplasm. On the cytoplasmic side, the cargo dissociates from the complex by means of GAP-mediated hydrolyzation of GTP to GDP. CRM1 and Ran-GDP then diffuse back to the nucleus where GEFs exchange GDP to GTP  and subsequent cargo release to the dedicated subcellular compartment . The actP to GTP .Because nuclear localization of proteins such as transcription factors is key to their function, and an adequate localization of signaling proteins close to their downstream targets can be imperative for many signal transduction pathways, unbalanced nucleocytoplasmic shuttling can result in their inactivation. Undesired gain-of-functions can thus cause a variety of cellular malfunctions related to signaling, metabolic or structural properties . NucleocEven though the restricted capacity of the CNS to regenerate myelin sheaths is linked to different regulatory factors/pathways, a broad range of regulators share common features. Not only do their expression and presence appear to limit glial maturation, but their subcellular localization within the oligodendroglial cells is assumed to constrain the regeneration process as well. Hence, precise and timely nucleocytoplasmic transport of these regulators across the nuclear membrane represents an essential mechanism for controlling their activity. For this reason, nuclear transport could be considered as a putative therapeutic target with potential implications for neurodegenerative diseases. This review therefore focuses on aberrant/dysregulated nucleocytoplasmic protein shuttling within oligodendroglial cells, its implication/contribution to the restricted capacity of the CNS to repair damaged tissue, and the description of possible molecular targets that could be used to develop therapeutic strategies.In order to generate functional oligodendrocytes, cell cycle progression and differentiation must be tightly controlled. The shift between proliferation and differentiation depends on regulatory complexes involving cyclins, cyclin-dependent kinases (CDK), and cyclin-dependent kinase inhibitors (CDKIs) as well as phosphorylation-dephosphorylation events, all responding to intracellular and extracellular signals and influencing their expression levels and intracellular localization . ProlifeFurthermore, nuclear activity of CDKs, especially of CDK2 maintaining the G1/S-phase checkpoint together with cyclin E, was shown to be necessary to retain cell cycle progression in mammals . Loss ofCDK5 absence led to nuclear translocation of the transcription factor FOXO1 and subsequently facilitated neuronal cell death ; it alsoin vitro and in vivo is accompanied by nuclear export of E2F1 [E2F1, a member of the E2F family of transcription factors, plays a critical role in coordinating early cell cycle progression; in its unbound state, it directs the transcription of genes required for entry into the S phase . Unbound of E2F1 . E2F1 waCDKIs negatively regulate cell cycle progression by binding and inactivating cyclin/CDK complexes. Based on their CDK binding specificity, CDKI proteins fall into two subfamilies-namely, the inhibitor of kinase 4 (INK4) family and the CDK interacting protein/kinase inhibitory protein (Cip/Kip) family . The INKin situ analysis of active MS lesions revealed increased expression of p53 in apoptotic oligodendrocytes suggesting it to be a critical pro-apoptotic effector [In proliferating OPCs, the expression of the tumor suppressor p53 is low, and the protein is likely not functional based on its cytoplasmic localization . Transloeffector . Accumulin vitro  proteins and important positive regulators of OPC differentiation  Table 2Table 2. The achaete-scute complex homolog 1 (Ascl1/Mash1) protein encodes another transcriptional regulator important for oligodendroglial specification, differentiation, and myelination ,107,108 The zinc-finger myelin transcription factor 1 (Myt1) is a DNA-binding protein that regulates proteolipid (PLP) transcription. . InteresInhibitor of differentiation (Id) proteins are non-DNA-binding transcriptional regulators, located downstream of the BMP signaling, that exert strong inhibitory effects on oligodendroglial differentiation. Nuclear accumulation of Id2 and Id4 results in complex formation with Olig1, Olig2, or Ascl1/Mash1 proteins for subsequent sequestration ,113. The35–55 induced experimental autoimmune encephalomyelitis (EAE), GPR17 appears to be up-regulated [In 2009 the G-protein coupled receptor 17 (GPR17) was found to be transiently expressed during oligodendrogenesis and to interfere with oligodendroglial differentiation via Id2 ,115 Tab. In pathegulated , and itsegulated .in vivo [The nuclear presence of members of the Wnt/β-catenin signaling cascade in MS lesions, as well as recent observations on inhibited myelination and differentiation of oligodendrocytes following constitutive expression of β-catenin, suggests an active role regarding remyelination failure ,117,118.in vivo . Stabiliin vivo ,123. In in vivo . In the in vivo ,126.2+ concentrations were found to reduce SMAD2 nuclear translocation [SMADs are downstream effectors of bone morphogenic proteins (BMPs), secreted proteins that form the largest subclass of the transforming growth factor-β (TGFβ) superfamily and participate in multiple steps during nervous system development  Table 2Table 2. location ,140.The genes encoding bHLH transcription factors hairy and enhancer of split-1 and -5 (Hes1 and Hes5) have been implicated as critical targets of the Notch signaling pathway, that was shown to be involved in oligodendroglial differentiation and also in T helper (Th) cell activation and differentiation ,142 Tab. Notch r35–55 induced EAE was able to recapitulate defective remyelination in vivo [Olig2 expression and thus oligodendroglial differentiation. It was therefore assumed that HDAC1 and HDAC2 compete with β-catenin for Tcf4/Lef interaction to regulate downstream genes involved in oligodendroglial differentiation [Acetylation and deacetylation of histones essentially contribute to the regulation of gene expression and are catalyzed by histone acetyltransferases or histone deacetylases (HDACs), respectively. Acetylation is associated with active open chromatin and an initiated gene expression, whereas deacetylation blocks the expression of, for example, inhibitory transcription factors such as Hes5, Id2 and Id4, Tcf7l2 and Tcf4 and can thus promote myelination ,149,150. in vivo ,152. Thintiation . Studiesntiation .As yet another structural protein modification process, citrullination of arginine residues is catalyzed by peptidylarginine deiminases (PADs). In normal-appearing white matter of MS patients and in a ND4 transgenic mouse model for chronic progressive primary demyelination, TNFα-dependent citrullination of histones by PAD4 was observed . Enhancein vitro knockdown approaches for every member of TET family enzymes resulted in up-regulation of differentiation inhibitors such as Hes1 or Id2 and down-regulation of MBP and PLP gene expression [TET2 in peripheral blood mononuclear cells was found to be significantly down-regulated and aberrant DNA hydroxymethylation resulted in decreased 5hmC levels, suggesting a relevant role in MS pathophysiology [The ten-eleven translocation (TET) family of methylcytosine dioxygenases catalyzes oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and promotes DNA demethylation. TET proteins have been demonstrated to regulate promoter activities in embryonic stem cells . Three Tpression . In MS pysiology .As describe above, expression of inhibitory transcription factors Hes1/5 is controlled by Notch signaling. Of note and in contrast to many membrane receptors that transmit signals to the nucleus via complex cascades, Notch receptors can also act on gene expression by translocating themselves to the nucleus. Activation of the non-canonical Notch signaling pathway by the axonal F3/contactin, for example, triggers nuclear translocation of NICD and has been demonstrated to promote both generation as well as differentiation of oligodendroglial cells ,158. In The axon-derived ligand neuregulin 1 type III (NRG1) interacts with ErbB receptor tyrosine kinases expressed on oligodendroglial cells and affects their survival, maturation, and myelination ,164,165 2 vitamin D3 (VDR), the peroxisome proliferator-activated receptors (PPARs), and many orphan receptors.Nuclear receptors belong to a superfamily that consists of a large number of ligand-activated transcription factors . The supMBP, PLP, MAG, and 2′,3′-cyclic nucleotide 3′-phosphodiesterase [Upon ligand binding TRs as well as RARs exhibit nuclear localization and interact with the transcriptional machinery in the nucleus ,171. Furesterase —hence presterase .RXRs are a family of nuclear receptors previously shown to be important regulators of differentiation  Table 3Table 3. The PPARs function as transcription factors to regulate target genes that are involved in lipid metabolism, and they have been shown to play a significant role in oligodendroglial differentiation ,178,179 Apart from a number of proteins for which the subcellular localizations is arranged during oligodendrogenesis, strong evidence also exists for the subcellular transport and distribution of selected RNA molecules. Notably, mRNAs for myelin proteins such as MBP are transported from the nucleus to terminal processes of oligodendroglial cells, where they are locally translated at the site of developing myelin sheaths . In olig-acting factors heterogeneous nuclear ribonucleoproteins, including hnRNPA2, -A3 and quaking (QKI) [RNA granule formation, cargo selection, and cytoplasmic transport are tightly controlled by RNA-binding proteins referred to as the transng (QKI) ,191. Mutng (QKI) ,193. Strng (QKI) ,195. Thung (QKI) .However, MBP protein variants, resulting from different mRNA splice variants, have been shown to be differently distributed within oligodendroglial cells, depending on which isoforms are encoded by exons I-VII . It was Finally, lamins-the structural components of the filamentous protein meshwork essential to inner nuclear membrane structure-are also functionally engaged in maturation processes. Overexpression of lamin B1 leads to a disturbance of inner nuclear membrane proteins and of chromatin organization, mislocalization of NPCs, and impaired nuclear pore transport ,202. As Since deregulated nucleocytoplasmic transport and the subsequent aberrant intracellular distribution of proteins appears to be critically involved in oligodendroglial differentiation failure and in myelin repair deficits , the ide


Schizosaccharomyces pombe displays a large transcriptional response common to several stress conditions, regulated primarily by the transcription factor Atf1. Atf1-dependent promoters contain especially broad nucleosome depleted regions (NDRs) prior to stress imposition. We show here that basal binding of Atf1 to these promoters competes with histones to create wider NDRs at stress genes. Moreover, deletion of atf1 results in nucleosome disorganization specifically at stress coding regions and derepresses antisense transcription. Our data indicate that the transcription factor binding to promoters acts as an effective barrier to fix the +1 nucleosome and phase downstream nucleosome arrays to prevent cryptic transcription. Schizosaccharomyces pombe, the MAP kinase Sty1 and the basic leucine zipper (bZIP) transcription factor (TF) Atf1 regulate up to 400 genes in response to several stress conditions  tracts ,12. How,sty1 promoter. Briefly, the full length or truncated atf1 coding sequences were polymerase chain reaction (PCR)-amplified from an S. pombe cDNA library using specific primers. Each PCR product was digested with BglII/SmaI and cloned into the plasmid p386 (sty1 promoter (0.8 kb from ATG) fused to the HA coding sequence, digested with BamHI/SmaI. The p413 integrative plasmid encodes the DNA-binding domain of budding yeast Gal4  and a FLAG epitope (DYKDDDDK) (sty1 promoter. The integrity of each of these constructs was confirmed by DNA sequencing.The p428 and p428.bZIP integrative plasmids encode the HA-tagged Atf1 full-length protein (566 amino acids) or only the C-terminal region of Atf1 containing the bZIP domain respectively (from 396 to 566 amino acids); the chimeric genes are under the control of the constitutive mid p386  that conYKDDDDK) . The GalYKDDDDK) ,16, digeS. pombe strain 972 (−h) and 975 (+h) and mutants thereof. The origins and genotypes of strains in this study are indicated in Supplementary Table S1. Strains EP203 and EP203.bZIP were constructed integrating the plasmid p428 and p428.bZIP respectively in the EP193 strain at +leu1 locus. To construct strains EP184 and EP255 with CRE-to-G4BS substitutions, we first deleted 160 bp of the ctt1 or gpd1 promoters in the strain CH1364 (ura5-294 lys7-2 leu1-32) with a cassette containing both the +ura5 and the +lys7 genes, as recently described by the group of Hoffman ; we double checked lysine auxotrophy in the selected clones. The resulting strains were crossed out to wild-type strains to eliminate auxotrophies, yielding strains EP184 and EP255. In EP184, the CRE site (ATGACGT) at ctt1 promoter was replaced by a Gal4 binding site by substitution of −435 to −419 from the translational start site by the following G4BS: CGGAAGACTCTCCTCCG. In the EP255 strain, the CRE site (TTACGTCA) at gpd1 promoter was replaced by substitution of −329 to −312 from the translational start site by the G4BS. Strains EP213 and EP286 were constructed by integrating the plasmid p413 at the +leu1 loci of strains EP184 and EP255, respectively. Strains EP212.ΔbZIP and EP287.ΔbZIP were constructed by integrating the plasmid p411.ΔbZIP at the +leu1 loci of strains EP184 and EP255, respectively. Cells were grown in liquid- or solid-rich medium (YE5S) or in synthetic minimal medium (MM) as described previously . Cultures were then centrifuged, cell pellets washed from TSA, resuspended in rich media, and growth proceeded performing one culture dilution with rich media, to reach in ∼20 h an OD600 of 0.5 after eight doublings. For survival on solid plates, cells before, during or after TSA withdrawal were serial diluted and spotted on YE5S plates containing or not 1 mg/ml of 5-FOA, or on minimal media plates lacking uracil.We used the wild-type (WT)  Hoffman . Then, w600 of 0.5 and then cross-linked with formaldehyde ) for 20 min at 25°C. Cells were then digested with Zymolyase 20T (Amsbio), and spheroplasts were treated with increasing concentrations of micrococcal nuclease . Purified DNA was separated electrophoretically, and samples displaying 80–90% mononucleosomal DNA without subnucleosomal fragments (faster migration than mononucleosomes in the electrophoresis) were further analysed by qPCR with a set of overlapping primer pairs (see Supplementary Table S2). For each primer pair, numbers in Y-axis correspond to the relative value to the input, which was obtained using as a template DNA from cells not treated with MNase, and received a value of 1.Mononucleosomes were obtained as described before ,11,19, aΔatf1 cells expressing HA-bZIP, while two full biological replicates (including duplicate sequencing reactions) were performed for Δatf1 nucleosomes. Short reads produced in this work (26–50 millions of which 98% were uniquely mapped) were aligned against the genome of S. pombe 972 using the Bowtie2 software (http://genome.ucsc.edu/goldenPath/help/wiggle.html) and then we developed an in-house R [R Core Team (2013)] script to produce a data matrix in which each row is the genomic coordinate of the +1 nucleosome for the selected genes and each column is the nucleR's processed signal value for a certain genomic coordinate located in the region spanning ±500 nucleotides around the +1 nucleosome.Fixed cells were subjected to chromatin isolation and MNase digestion as described above for nucleosome-scanning analysis ,19. Agaisoftware  with defsoftware  and thensoftware  was used32P]dCTP-labelled gpd1, hsp9, ctt1, sty1 or act1 probes.Total RNA from cultures was obtained, processed and transferred to membranes as described previously . MembranTo analyse the amount of Atf1, trichloroacetic acid extracts were prepared as previously described . ImmunobThe indicated strains were grown in minimal media, and chromatin isolation and immunoprecipitation was performed as described previously .RNA was purified, treated with DNAse and subjected to reverse transcription  using primers complementary to either forward or reverse transcripts. Reverse-transcribed cDNAs were amplified by real-time PCR using gene-specific primers (Supplementary Table S3).gpd1 and ctt1) by nucleosome scanning. Briefly, we treated chromatin from wild-type and Δatf1 strains   Figure . As showt Figure . The nucs Figure . CRE sits Figure .ade6 gene  and promote hotspot meiotic recombination  assay showed specific binding of both proteins (G4 and G4-Atf1) to G4BS-containing gpd1 or ctt1 promoters, but not to other stress promoters . Since the new nucleosome around the G4BS at gpd1.G4BS promoter seems to be strongly positioned  cells fully re-established silencing demonstrating de novo nucleosome deposition . As already reported for the nmt1 and fbp1 genes . However, after eight generations of TSA withdrawal, mRNA expression levels returned to normal . Consistently, during TSA treatment our nucleosome scanning analysis failed to detect proper nucleosome positioning around the gpd1 gene . As expected, the map of nucleosome positions at the gpd1 gene was completely re-established after TSA withdrawal . This binding of G4 and G4-Atf1 precluded histones from binding to the gpd1.G4BS promoter: cells expressing G4 or G4-Atf1 proteins showed a dramatic decrease in nucleosome occupancy . After mild cross-linking and MNase digestion, nucleosomal DNA fragments were isolated, purified from agarose gels and subjected to single-end sequencing using an Illumina platform . We also applied to the S. pombe genome the program designed by Segal et al., which is supposed to infer the position of nucleosomes based exclusively on DNA sequence  were aligned at their first nucleosome (+1) and the average of their log2 nucleosome occupancy data was plotted , even though the average NDR at stress genes was less pronounced in this wild-type dataset. Interestingly, Δatf1 cells showed a wider +1 nucleosome position with a shoulder getting into the NDR, indicating a defect defining the position of the first nucleosome . Howeve the TSS ,27,30,31d Figure . Wild-tys Figure . Furthers Figure , and anaS. pombe, the ATP-dependent chromatin remodellers, Hrp1 and Hrp3, have been described to mediate the formation of a nucleosome array downstream of the TSS, with this correct nucleosome array formation being required for (i) preventing cryptic promoter activity, (ii) silencing antisense transcription and (iii) promoting sense transcription .In cription ,30,31. Ts Figure . Strikins Figure , suggestn nucleosomes in a given gene . HoweveS. cerevisiae, these sequences occur with less frequency in NDRs than elsewhere in the S. pombe and human genomes  is a strong nucleosome exclusion signal in  genomes ,12. Furte genome , this pr Figures , and gens Figure .Using both our genome-wide data and nucleosome scanning of specific Atf1 target genes, we can conclude that binding of the TF to its specific DNA site competes with histones and generates NDRs. In cells lacking Atf1, narrower NDRs are still present at these promoters which may be explained by the fact that stress genes are regulated by multiple TFs . Moreoveper se.Interestingly, the loss of Atf1 at promoters of stress genes causes an ‘unlocked’ +1 nucleosome, an irregular nucleosome array in coding regions and an increase in antisense transcripts Figure . Severalatf1 cells .RNA polymerase II has also been proposed to influence nucleosome positioning within coding regions in budding yeast . Howeverin vivo. Further studies will be required to demonstrate that the role of Atf1 as a barrier can be extended to other TFs which, by high affinity interaction with their DNA sites, should establish the boundaries towards which nucleosomes are ‘pushed’ by chromatin remodellers.Our results prompt us to speculate that TFs may function as effective nucleosome barriers to promote proper deposition of the +1 nucleosome. We propose here that Atf1 competes with histones to bind DNA, and functions as an effective barrier that blocks the +1 nucleosome positioning and in turn allows the proper organization of the downstream nucleosome array that is essential for preventing cryptic transcription events. It has been speculated previously that DNA bound factors, such as TF, may function as nucleosome barriers , but toSupplementary Data are available at NAR Online.