Phosphorylation of histone H3 correlates with transcriptionally active loci

Abstract

Posttranslational modifications of the N-terminal tails of the core histones within the nucleosome particle are thought to act as signals from the chromatin to the cell for various processes. The experiments presented here show that the acetylation of histones H3 and H4 in polytene chromosomes does not change during heat shock. In contrast, the global level of phosphorylated H3 decreased dramatically during a heat shock, with an observed increase in H3 phosphorylation at the heat shock loci. Additional experiments confirm that this change in phosphorylated H3 distribution is dependent on functional heat shock transcription factor activity. These experiments suggest that H3 phosphorylation has an important role in the induction of transcription during the heat shock response.

Figure 1

Distribution of Lys 14 acetylated histone H3 does not change during heat shock. (A,B) FITC image of anti-Lys 14 acetylated H3 immunostaining. (C,D) 4′,6-diamidino-2-phenylindole (DAPI) counterstaining of chromosomes shown in A and B. (E,F) Composite merge of FITC and DAPI channels, antibody staining shown in yellow, DAPI shown in blue. (A,C,E) Antibodies specific for Lys 14 acetylated H3 stain multiple discrete sites in polytene chromosomes prepared from third instar larvae grown at 22°C. (C,E) Ecdysone-induced developmental puff 62A and nonpuffed chromosomal subdivision 89B both display Lys 14 acetylated H3 staining (insets). Heat shock locus 93D is acetylated but not puffed. (B,D,F) Anti-Lys 14 acetylated H3 staining after heat shock is observed at the 87A and 87C puffs, which contain the hsp70 gene cluster. (B,F) Chromosomal subdivisions 89B and 62A, which do not contain heat shock genes, are not puffed after heat shock but contain histones that remain acetylated (insets).

Figure 2

Distribution of diacetylated histone H3 and Lys 8 acetylated H4 does not change during heat shock. In all panels, antibody staining is shown in yellow, chromosomes are counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). (A) Antibodies specific for H3 acetylated at lysines 9 and 14 stain region 89B, the developmental puff at 34A, and loci 87A, 87C, and 93D, which contain heat shock genes before heat shock. (B) Fragment of chromosome 3R isolated from heat shocked third instar larva showing the 87A, 87C, and 93D heat shock puffs, stained with anti-Lys 9,14 acetylated H3. Subdivision 89B, which does not contain any heat shock genes, is acetylated but not puffed after heat shock. (C) Anti-Lys 8 acetylated H4 antibodies stain discrete regions in polytene chromosomes before heat shock, including the developmental puff at 62A, and nonpuffed region 89B. Heat shock loci 87A and 93D are acetylated before heat shock. (D) Lys 8 acetylated H4 is detected at the 87A and 87C heat shock puffs following heat shock. Chromosomal subdivision 89B, which does not contain heat shock genes, remains H4 acetylated during heat shock.

Figure 3

Distribution of phosphorylated H3 changes during heat shock. (A–C) FITC image of anti-Ser 10 phosphorylated H3 immunostaining. (D–F) 4′,6-diamidino-2-phenylindole (DAPI) counterstaining of chromosomes shown in (A–C). (G–I) Composite merge of FITC and DAPI channels, antibody staining shown in yellow, DAPI shown in blue. (A,D,G) Antibodies specific for Ser 10 phosphorylated H3 stain multiple discrete regions of polytene chromosomes isolated from third instar larvae at 22°C, including several of the indicated ecdysone-induced developmental puffs. (B,E,H) After heat shock, phosphorylated H3 staining is restricted to subdivisions 87AC, 63BC, and 67B, which contain the hsp70 gene cluster, hsp83 gene, and the small hsp gene cluster (hsp22, hsp23, hsp26, and hsp27), respectively. (C,F,I) Image of a section of chromosome 3R showing the change in staining of three of the heat shock loci before and after heat shock. Heat shocked chromosome is on the right.

Figure 4

Distribution of antiphosphorylated histone H3 staining changes dynamically during and after recovery from heat shock. Visible major heat shock loci are indicated. In all panels, antibody staining is shown in yellow, chromosomes are counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Polytene spreads were prepared from Oregon R larvae incubated for (A) 0 min, (B) 1 min, (C) 5 min, (D) 10 min, and (E) 20 min at 37°C. Larvae were removed from 37°C and allowed to recover for (F) 5 min, (G) 10 min, (H) 20 min, and (I) 30 min at room temperature.

Figure 5

Analysis of H3 and H4 acetylation and H3 phosphorylation in hsf⁴ mutants during heat shock. In all panels, antibody staining is shown in yellow, chromosomes are counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). (A) H3 phosphorylation before heat shock appears identical to wild-type distribution. (B) After heat shock, no H3 phosphorylation can be detected. (C) The distribution of Lys 14 acetylated histone H3 does not appear to change before or (D) after heat shock. (E) The distribution of diacetylated H3 in hsf⁴ mutant chromosomes before and (F) after heat shock does not appear to change during heat shock. (G) In addition, histone H4 acetylated at lysine 8 does not change in hsf⁴ mutants before and (H) after heat shock.

Figure 6

Model of H3 phosphorylation and transcription initiation during heat shock. (A) The paused RNA polymerase complex present at a representative heat shock locus. (B) During heat shock, the heat shock transcription factor (HSF) binds to acetylated H3 and H4 residues at the heat shock promoter elements. (C) Bound HSF recruits a histone kinase, which (D) phosphorylates histone H3 at the Ser 10 position.