RNA polymerase II-mediated transcription at active loci does not require histone H3S10 phosphorylation in Drosophila

doi:10.1242/dev.024927

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First published online 30 July 2008
doi: 10.1242/dev.024927

Development 135, 2917-2925 (2008)
Published by The Company of Biologists 2008

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RNA polymerase II-mediated transcription at active loci does not require histone H3S10 phosphorylation in Drosophila

Weili Cai, Xiaomin Bao, Huai Deng, Ye Jin, Jack Girton, Jørgen Johansen and Kristen M. Johansen^*

Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa 50011, USA.

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Fig. 1. JIL-1 and histone H3S10ph antibody labeling of Drosophila salivary gland nuclei smush preparations. JIL-1 (green) and histone H3S10 phosphorylation (H3S10ph, red) co-localize on chromosomes from both female (top row) and male (middle row) wild-type (wt) nuclei. The middle row illustrates the characteristic upregulation of JIL-1 and H3S10ph labeling on the male X chromosome (X). Labeling of both JIL-1 and H3S10ph is absent in JIL-1^z2/JIL-1^z2 (z2) null mutant nuclei (lower row). The H3S10ph antibody used was from Cell Signaling.

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Fig. 2. Immunocytochemistry and immunoblot characterization of three different H3S10ph antibodies. (A-C) Acid-free polytene chromosome squash preparations from male and female third instar Drosophila larvae double labeled with antibodies to JIL-1 (green) and H3S10ph (red). H3S10ph labeling with antibody from (A) Cell Signaling (cs), (B) Epitomics (epi) and (C) Upstate (up). Composite images (comp) of the labelings are shown to the left. The labeling of all three H3S10ph antibodies shows co-localization with JIL-1 and upregulation on the male X chromosome (X). The Epitomics H3S10ph antibody, in contrast to the other two antibodies, showed strong labeling of the chromocenter (B, asterisks). The images in A are projection images from confocal sections. (D-F) Immunoblots of protein extracts from salivary glands from wild-type (wt), JIL-1^z2/JIL-1^z2 (z2), and JIL-1^z2/JIL-1^z2 Su(var)3-9⁰⁶ (z2, 3-9) larvae labeled with H3S10ph antibody from Cell Signaling (D), Epitomics (E) or Upstate (F). H3S10ph antibody labeling by all three antibodies is greatly reduced in JIL-1 null mutant backgrounds. Labeling with histone H3 (H3) antibody was used as a loading control.

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Fig. 3. JIL-1 is not upregulated at actively transcribed regions during the heat shock response. (A) Polytene chromosome squash preparations from wild-type Drosophila larvae (wt) triple labeled with Pol IIo^ser2 antibody (green), JIL-1 mAb 5C9 (red), and Hoechst (DNA, gray/blue), with (right column) and without (left column) heat shock treatment. At many sites that showed especially high levels of Pol IIo^ser2 staining, such as at developmental puffs, there were relatively low levels of JIL-1 (arrow). After heat shock treatment, Pol IIo^ser2 labeling was reduced at most sites while being dramatically upregulated at heat shock-induced puffs (boxed area), whereas there was no discernable redistribution of JIL-1. (B) Higher magnification of the boxed area from A, showing that there was no upregulation of JIL-1 at the 87A/C and 93D heat shock puffs (boxed).

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Fig. 4. Polytene chromosome distribution of H3S10ph in response to heat shock treatment. (A-F) Acid-free polytene chromosome squash preparations from female third instar Drosophila larvae triple labeled with antibodies to Pol IIo^ser2 (green), H3S10ph (red), and with Hoechst (DNA, blue/gray). H3S10ph labeling with antibodies from Epitomics (A), Cell Signaling (C) or Upstate (E) with (+HS) and without (-HS) heat shock treatment. (B,D,F) Higher magnification images of the heat shock-induced puffs 87A/C (boxed regions) labeled by H3S10ph antibodies from Epitomics (B), Cell Signaling (D) or Upstate (F). (G) Immunoblots of protein extracts from salivary glands from wild-type larvae without heat shock treatment (wt) and with heat shock treatment [wt (HS)] labeled with H3S10ph antibody from Cell Signaling, Epitomics or Upstate. Labeling with histone H3 (H3) antibody was used as a loading control. (H) Immunoblots of protein extracts from salivary glands from wild-type larvae without heat shock treatment (wt) and with heat shock treatment [wt (HS)] labeled with Pol IIo^ser2 antibody. Labeling with lamin antibody was used as a loading control.

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Fig. 5. Polytene chromosome labeling by three different H3S10ph antibodies in response to heat shock treatment in JIL-1 null mutants. Acid-free polytene chromosome squash preparations from female JIL-1^z2/JIL-1^z2 third instar Drosophila larvae triple labeled with antibodies to Pol IIo^ser2 (green), H3S10ph (red), and with Hoechst (DNA, blue/gray) after heat shock treatment. H3S10ph labeling by antibodies from Cell Signaling, Epitomics and Upstate. Arrows indicate the likely position of the 87A/C heat shock puff regions.

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Fig. 6. Immunoblot analysis of H3S10ph in mutants of the 55 kd regulatory subunit of Protein phosphatase 2A. (A) H3S10ph antibody labeling of protein extracts from wild-type (wt) and tws⁰²⁴¹⁴/tws⁰²⁴¹⁴ (tws⁰²⁴¹⁴) whole third instar Drosophila larvae. (B) H3S10ph antibody labeling of protein extracts from wild-type and tws⁰²⁴¹⁴/tws⁰²⁴¹⁴ salivary glands. (C) H3S10ph antibody labeling of protein extracts from tws^P/tws^P (tws^P) and JIL-1^z2/JIL-1^z2 (z2) salivary glands with (HS) and without heat shock treatment. The H3S10ph antibody was from Epitomics (epi); labeling with histone H3 (H3) antibody was used as a loading control.

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Fig. 7. Immunocytochemical and immunoblot labeling of Pol IIo^ser2 in JIL-1 null mutant backgrounds. (A) Robust Pol IIo^ser2 antibody labeling (green) of chromosomes including the male X chromosome (X) in polytene chromosome squash preparations from JIL-1^z2/JIL-1^z2 (z2) and JIL-1^z2/JIL-1^z2 Su(var)3-9⁰⁶ (z2, 3-9) third instar Drosophila larvae. The DNA (blue/gray) was labeled by Hoechst. (B) Immunoblot of protein extracts from wild-type (wt), JIL-1^z2/JIL-1^z2 (z2), and JIL-1^z2/JIL-1^z2 Su(var)3-9⁰⁶ (z2, 3-9) larvae labeled with Pol IIo^ser2 antibody. Labeling with lamin antibody was used as a loading control.

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Fig. 8. Analysis of Pol IIo^ser2 distribution and transcription at active loci during the heat shock response in JIL-1 mutant backgrounds. (A) Polytene chromosome squash preparations from JIL-1^z2/JIL-1^z2 (z2) and JIL-1^z2/JIL-1^z2 Su(var)3-9⁰⁶ (z2, 3-9) Drosophila larvae triple labeled with Pol IIo^ser2 antibody (green), Hsf antibody (red) and Hoechst (DNA, gray/blue) after heat shock treatment. Arrows point to heat shock puff regions. (B) Immunoblot of protein extracts from salivary glands from JIL-1^z2/JIL-1^z2 (z2) and JIL-1^z2/JIL-1^z2 Su(var)3-9⁰⁶ (z2, 3-9) larvae with (+HS) and without (-HS) heat shock treatment labeled with Pol IIo^ser2 antibody. Labeling with lamin antibody was used as a loading control. (C) Transcript levels of Hsp70 mRNA in JIL-1 null mutant backgrounds in response to heat shock treatment. Hsp70 transcript levels were determined by qRT-PCR and normalized to the mRNA levels of the control non-heat shock protein Rp49 (Ribosomal protein 49) both without and after heat shock treatment. The data shown are the average from two independent experiments in which total RNA was isolated from wild-type (wt), JIL-1^z2/JIL-1^z2 (z2) and JIL-1^z2/JIL-1^z2 Su(var)3-9⁰⁶ (z2, 3-9) larvae and each determination of transcript levels was performed in duplicate. Error bars indicate the s.d.m.

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