Volume 13, Issue 1, Pages 55-65 (January 2004) Coordination of Transcription, RNA Processing, and Surveillance by P-TEFb Kinase on Heat Shock Genes  Zhuoyu Ni, Brian E. Schwartz, Janis Werner, Jose-Ramon Suarez, John T. Lis  Molecular Cell  Volume 13, Issue 1, Pages 55-65 (January 2004) DOI: 10.1016/S1097-2765(03)00526-4

Figure 1 P-TEFb Reduction by RNAi Decreases Induced hsp70 RNA Accumulation (A) Western blot of whole-cell lysates from Kc cells after indicated RNAi treatments followed by incubation at 37°C (HS) or 23°C (NHS) for 30 min. The specific Cdk9 band is indicated by an arrow and those of CyclinT and HSF are as labeled. (B) RNA measurement of accumulated hsp70 and rp49 RNA from cells described in (A) using PERT (primer extension following reverse transcription), which is described in the Experimental Procedures. Molecular Cell 2004 13, 55-65DOI: (10.1016/S1097-2765(03)00526-4)

Figure 2 FP Inhibits the Kinase Activity of Drosophila P-TEFb In Vitro In vitro kinase assay was performed using 25 nM of P-TEFb in each reaction in the absence or presence of various amounts of FP. The phosphorylated MBP fusion proteins containing the C-terminal domains of the largest subunit of Pol II (MBP-dCTD) and Spt5 (MBP-dSpt5C) are as labeled. MBP protein alone is not phosphorylated (data not shown). Molecular Cell 2004 13, 55-65DOI: (10.1016/S1097-2765(03)00526-4)

Figure 3 FP Inhibits the Kinase Activity of Drosophila P-TEFb In Vivo Salivary glands dissected from fly third instar larvae (Z243 strain for [A] and [C] and cdk7ts line for [B]) were incubated in insect cell media without (−FP) or with 500 nM of FP (+FP) followed by 10 min heat shock (HS, [A] and [B]) or none (NHS, [C]). The epitopes detected by indirect immunofluorescence with specific antibodies are labeled on each panel. Exposure times for each −/+FP pair were the same except for “Ser2-P” of the “+FP” experiment in (A), which is a 10× longer exposure than the corresponding “−FP” image. The chromosomes shown are representative examples of a large set of nuclei examined. From 8 to 36 nuclei under each condition were photographed at multiple exposures, and the variation for at least 90% of nuclei was estimated visually to be less than a factor of two. (A) Ser2-P but not Ser5-P forms of Pol II decreases dramatically on heat-induced loci. Major heat shock loci are indicated with thin arrows; only hsp70 loci (87A and C), hsp26 locus (67B), and the hsp70 transgene-inserted locus (59D) are labeled. (B) Ser2-P and Ser5-P decrease dramatically on heat-induced hsp70 loci (87A and 87C as labeled) on polytene chromosomes from cdk7ts larvae at nonpermissive temperature. The lack of a detectable decrease in Ser5-P staining in the −FP larvae may be a consequence of P-TEFb partially compensating for cdk7 activity at the nonpermissive temperature (Schwartz et al., 2003a; Zhou et al., 2000). (C) Ser2-P form but not total Pol II decreases dramatically on non-heat-induced chromosomes. One of the ecdysone-induced puffs 75B is labeled in the +FP images. Molecular Cell 2004 13, 55-65DOI: (10.1016/S1097-2765(03)00526-4)

Figure 4 Effects of FP on Heat Shock Gene RNA Accumulation and the Distribution of Selected Transcription Factors and Pol II on hsp70 and hsp26 Genes (A) Experimental scheme: the concentration of P-TEFb in Drosophila Kc cell nuclei was estimated to be about 500 nM by Western blots (data not shown), and we also empirically titrated the FP concentration for Kc cell treatment and found 500 nM FP is sufficient for effective inhibition of hsp70 RNA production and for maintaining specificity (Figure 3). Drosophila Kc cell cultures were treated with 500 nM FP or mock treated at 23°C and then heat shocked at 37°C for 5 min. About 1 × 107 cells were used for RNA measurements (B), 1 × 108 cells for run-on analysis (D), and the rest for ChIP analysis (C). (B) RNA analyses of hsp70 and hsp26 RNA using rp49 RNA as an internal control. On the left is the PERT analysis (four experiments) of hsp70 RNA as described (Figure 1B). On the right are the S1 protection assays using probes complementary to 3′ coding region of hsp70 (four experiments) and hsp26 RNA (five experiments). Assays on decreasing amounts (Amt.) of untreated samples show that the detection is within the linear range. Protected probes corresponding to specific RNAs are indicated by black dots, and the undigested probes are labeled with arrowheads. Controls of yeast RNA (yRNA) with or without S1 digestion are shown on the right of each panel. (C) ChIP analysis showing the distribution of different phosphorylation states of Pol II and selected transcription factors on induced hsp70 (top panel) and hsp26 (bottom panel) genes in the absence (solid bar) or presence (hatched bar) of FP. The diagrams of hsp70 and hsp26 genes are shown on the right of each chart with the arrow indicating the location of the transcription unit relative to PCR fragments shown underneath each gene with the same grayscale as in the histogram. For the hsp70 gene, upstream promoter “Up” (−200/−108), 5′ (+4/+112), and 3′ (+1649/+1754) fragments are shown in gray, white, and black, respectively. For hsp26 gene, the 5′ promoter (−22/+63) and the 3′ region (+580/+669) are shown in white and black. Immunoprecipitation was done with antibodies as labeled on the x axes. The y axes represent the percentage of input material. The right hand y axes are for Spt5 and HSF, and the left hand axes are for the others. The two groups are separated by a vertical black line. Each ChIP result is shown as an average of four experiments with the standard error of mean (SEM). (D) Nuclear run-on analysis of the density of transcribing Pol II on hsp70 and hsp26 genes. The arrow on the schematic gene diagrams indicate the location of transcription units relative to DNA fragments used for hybridization: 5′ (−5/+412), “mid” (+1003/+1420), and 3′ (+1758/+2182) fragments for hsp70, and (+485/+910) fragment for hsp26. We used 1% of the run-on transcripts to probe an rDNA gene, thereby providing a normalization standard, and the rest was used to probe heat shock gene fragments. Background (BG) from an untranscribed region is subtracted from each signal. Reactions with or without 0.6% sarkosyl (Sark) and from the control or FP-treated cells (FP) are labeled. The results were plotted as a bar graph with the y axis representing the geometric mean of the ratio of polymerase density from FP-treated cells to that of mock-treated cells; a ratio of 1 indicates no change. The error bars are drawn as calculated from the geometric SEM of four (no sarkosyl) or three (0.6% sarkosyl) independent experiments. Molecular Cell 2004 13, 55-65DOI: (10.1016/S1097-2765(03)00526-4)

Figure 5 FP Inhibition of P-TEFb Kinase Activity Does Not Affect Pol II Entry or Progression on the hsp70 Gene Drosophila Kc cells mock treated (−FP, gray bar) or treated with 500 nM FP (+FP, hatched gray bar) were subject to 2 min heat shock at 37°C before ChIP analysis. A schematic diagram of hsp70 gene is shown on the upper left with the midpoint of each real-time PCR fragment shown underneath (approximately drawn to scale). The antibodies used for immunoprecipitation are labeled on each panel. The x axes show the midpoint of each PCR fragment along hsp70 gene and the y axes represent the percentage of input (SEM of three experiments is shown). Molecular Cell 2004 13, 55-65DOI: (10.1016/S1097-2765(03)00526-4)

Figure 6 Effects of FP on the Accumulation of Total and Processed Heat Shock RNA RNAs from mock-treated (−FP) and 500 nM FP-treated (+FP) cells after a 5 min heat shock at 37°C were used for S1 nuclease protection assay. (A) Poly (A)− and poly (A)+ RNA from −FP (5 μg total RNA) and +FP (25 μg total RNA) cells were analyzed using S1 probes (see Supplemental Data available on Molecular Cell's website) corresponding to both 5′ and 3′ sequences of hsp70 and hsp26 RNAs. The probes and the distance between the 5′ and 3′ probes relative to each gene coding region are shown on the top. The y axes show the ratio of PolyA−/A+ RNA with the left hand one for the hsp70-5′ and the right hand one for the others as separated by a thick black line. The geometric mean of each PolyA−/A+ ratio is shown with the SEM of four (hsp70) or five (hsp26) independent experiments. (B) Relative amounts of 5′ and 3′ RNA sequences of hsp70 and hsp26 in total RNA (10 μg) from −FP and +FP cells are measured with S1 probes as in (A). RNA amount from untreated samples are set at 100 and the amount from FP-treated samples relative to the control are shown as the geometric mean with the SEM of four (hsp70) or five (hsp26) independent experiments. (C) Total RNA from −FP (5 μg) and +FP (50 μg for hsp70 and 25 μg for hsp26) cells were probed with the 3′-UTR probes corresponding to hsp70 and hsp26 genes. The bands corresponding to uncleaved and cleaved heat shock RNA products are labeled as “U” and “C.” The signals labeled with “*” correspond to digested probes caused by breathing within the RNA-DNA probe hybrid at the 14 nucleotide-long AU region within the 3′-UTR and near the cleavage site of hsp70 RNA. The ratio of cleaved to uncleaved RNA is calculated as “C/U.” The geometric mean of the C/U fold effect, (−FP)/(+FP), caused by FP treatment is shown with the SEM from six (hsp70) or four (hsp26) independent experiments. The range of each effect can be obtained with >95% confidence by dividing and multiplying the geometric mean with the SEM. Molecular Cell 2004 13, 55-65DOI: (10.1016/S1097-2765(03)00526-4)

Figure 7 Model for Transcription-Coupled 3′ End Processing and RNA Surveillance at the Heat-Induced hsp70 Gene The model summarizes hsp70 RNA expression in the absence (−FP) or presence (+FP) of FP. Only 5′ and 3′ regions of the gene are shown and are indicated by the thick black lines. The transcription start site is represented as a right turn arrow and the polyadenylation signal “AATAAA” as an arrowhead. Only factors related to this study are shown in the figure as labeled. The black and gray “P” symbols represent phosphorylation of Pol II CTD on Ser2 and Ser5, respectively. The 3′ symbol represents the 3′ processing machinery. The number of P labels on the Pol II CTD, the height of the polymerase symbol, and the number of capped RNA transcripts represent the relative amounts of these species in control and FP-treated cells. “X” indicates the loss of P-TEFb kinase activity and the 3′ processing machinery activity on hsp70 gene. Molecular Cell 2004 13, 55-65DOI: (10.1016/S1097-2765(03)00526-4)