Fig. 1

GAF modulates different phosphoisoforms of RNA-Pol at the Hsp70 locus. a Molecular map of the Gaf locus. Two isoforms of GAF, GAF-519 and GAF-581, share the same BTB/POZ (yellow box) and Zn-finger domains (green box), but differ in the Gln-rich domain (blue box). The GAF antibody is raised against the shared region (pink box). The P-element insertion lines, Gaf ^13C and EP(3)3184, are indicated by triangles. The deletion in Gaf ^DH34, shown by the dotted box, extends to 218 bp upstream of 3′end of the gene. b Quantification of GAF. Extracts from salivary glands of third-instar larvae were immunoblotted with GAF and α-tubulin antibodies. The relative abundance of GAF from duplicate experiments is shown. c Gene map of Hsp70. A simplified map is shown with approximate locations of the GAF and HSF binding sites, TATA box, and four sets of primers used for qPCR. The relative position to the TSS for the midpoint of each PCR fragment is indicated. d–f. Cytogenetic studies of RNA-Pol at Hsp70 loci. Polytene chromosomes from WT and mutants before (d–f, upper panels) or after a 30-min heat shock (d–f, lower panels) were simultaneously stained with antibodies against RNA-Pol (Hypo-p, Ser-5p or Ser-2p in red), GAF (in blue) and BEAF-32 (in green). The color is removed for panels showing individual staining for clarity. The region from 87A to 87C is marked by five sections (1–5) based on BEAF-32 staining. Two Hsp70 clusters are located in sections 2–3 and 4–5 and are indicated by *. d’–f’. Quantitative measurement of RNA-Pol on Hsp70. Chromatin samples prepared from imaginal tissues of WT or Gaf mutant larvae before (non-HS) or after treatment at 37 °C for 5 min (HS) were subjected to immunoprecipitation with RNA-Pol antibodies against Hypo-p (d’), Ser-5p (e’), or Ser-2p (f’), followed by qPCR using four primer sets shown in c. The amounts of qPCR products from each set of measurements are expressed as a percentage of input. The average of three ChIP experiments is shown. Statistically significant differences are indicated (t test, *p < 0.05; **p < 0.01; ***p < 0.001)