Transcriptional activation triggers deposition and removal of the histone variant H3.3 - PubMed (original) (raw)

Transcriptional activation triggers deposition and removal of the histone variant H3.3

Brian E Schwartz et al. Genes Dev. 2005.

Abstract

DNA in eukaryotic cells is packaged into nucleosomes, the structural unit of chromatin. Both DNA and bulk histones are extremely long-lived, because old DNA strands and histones are retained when chromatin duplicates. In contrast, we find that the Drosophila HSP70 genes rapidly lose histone H3 and acquire variant H3.3 histones as they are induced. Histone replacement does not occur at artificial HSP70 promoter arrays, demonstrating that transcription is required for H3.3 deposition. The H3.3 histone is enriched in all active chromatin and throughout large transcription units, implying that deposition occurs during transcription elongation. Strikingly, we observed that the stability of chromatin-bound H3.3 differs between loci: H3.3 turns over at continually active rDNA genes, but becomes stable at induced HSP70 genes that have shut down. We conclude that H3.3 deposition is coupled to transcription, and continues while a gene is active. Repeated histone replacement suggests a mechanism to both maintain the structure of chromatin and access to DNA at active genes.

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Figures

Figure 1.

Figure 1.

RI deposition of H3.3 in active chromatin. Salivary polytene chromosomes from larvae that contain either constitutively expressed H3-GFP (A,B), H3.3-GFP (C,D), or H3.3core-GFP (E,F) fusion genes. (A,C,E) Whole-spread merged images; arrowheads indicate the heterochromatic chromocenters. (B,D,F) Split and merged high-magnification views of chromosome tips with histone-GFP; arrows indicate a condensed DAPI-stained band, and arrowheads indicate an interband. (A,B) H3-GFP localizes to DAPI-stained bands as well as to the chromocenter. (C,D) H3.3-GFP is mostly localized to interbands, although occasional bands are also labeled. H3.3-GFP also stains the chromocenter. (E,F) H3.3core-GFP is specific for interbands and shows little detectable staining of the chromocenter. DAPI-stained DNA is in red, and histone-GFP in green.

Figure 2.

Figure 2.

Gene induction triggers histone replacement. Heat-shock induction triggers rapid puffing and transcription of the HSP70 genes at polytene bands 87A and 87C. Polytene chromosomes from larvae that contain a constitutively expressed H3-GFP construct (A_–_D,J,K), or a constitutively expressed H3.3core-GFP construct (E_–_I) were induced for the indicated times (in minutes) and stained with antibodies against phosphorylated RNA polymerase II (blue), which marks activated puffs. Arrows in A and E indicate the positions of the HSP70 loci at 87A and 87C before induction, while the puffs (B_–_D,F_–_H) are bracketed. Asterisks indicate chromosome bands that were used as internal standards for quantifying the summed intensities of histone-GFP signals in puffs. (B,C) Puffs contain some H3 in the first 5 min of induction, but have much less by the time puffs reach their maximal size (D). (F_–_H) H3.3core-GFP in expanding puffs rapidly increases. (I) After 20 min of heat-shock induction, many active HSP puffs (arrows) have large amounts of H3.3core-GFP, while staining throughout the arms is undiminished. (J,K) Histone H3 modifications that mark heat-shock puffs do not overlap with H3-GFP. DNA is in red, and histone-GFP in green.

Figure 3.

Figure 3.

Ecdysone-induced genes are broadly labeled with H3.3. (A,B) Chromosome divisions spanning the large ecdysone-responsive genes E74 and E75 from larvae constitutively expressing H3.3core-GFP before (A) and after (B) induction. (C,D) The same chromosomal region before and after induction from larvae constitutively expressing H3-GFP. Both regions show moderate amounts of H3.3core-GFP and H3-GFP before induction, but H3.3core is increased while H3 is depleted during transcription. Lines indicate the position of landmark DAPI bands that flank the E74 and E75 genes. DNA is in red, histone-GFP in green, and RNA polymerase II is in blue. (E,F) Fluorescence in situ hybridization of DNA probes (blue) to the 3′-end (E) and 5′-end (F) of the induced E75 gene. Brackets delineate the gene. H3.3core-GFP (green) is enriched in the intervening body of the gene.

Figure 4.

Figure 4.

Histone replacement uses newly produced H3.3. Late-third-instar larvae containing a heat-shock-inducible histone-GFP construct were heat-shocked for 1 h to induce the construct and endogenous HSP genes, and then allowed to recover for 4 h. (A_–_C) DNA (red) and histone-GFP (green) images are shown as merges on the left, and the histone-GFP signal alone are shown on the right. (A) Full-length H3.3-GFP has deposited specifically at the heat-shock loci (arrowheads indicate the 87A, 87C, 95D, 63E, and 67F loci). (B) H3-GFP is not incorporated into chromatin. H3-GFP fills the interchromatin space within unfixed salivary gland nuclei after induction, but remains diffuse (inset). (C) Spreads with H3.3-GFP and two HSP70 promoter transgene arrays show intense H3-GFP signals at HSP loci but little signal at the arrays. Arrows in A and C indicate the 43E region without an array (A) and with an array (C). (Insets) Magnified images of 43E are shown. (D) Chromosomes from larvae carrying P[H3.3-GFP]B6 and the HSP70 promoter transgene arrays at positions 30A and 43E during a 20-min heat-shock induction. The endogenous HSP70 loci (arrowheads) bind moderate amounts of HSF activator (green), while arrays (arrows) bind large amounts. Conversely, HSP70 loci have large amounts of engaged RNA polymerase II (blue), while arrays have little.

Figure 5.

Figure 5.

Chromatin-bound H3.3 is unstable in post-mitotic cells. (A_–_C) Adult flies carrying a heat-shock-inducible H2B-GFP (A), H3-GFP (B), or H3.3-GFP (C) construct were frozen without heat shock or at the indicated times (in hours) after induction. Samples were extracted with 400 mM NaCl and centrifuged to separate soluble from pelleted chromatin-bound histones. Ten percent of the supernatant and 20% of the pellet fractions were separated by SDS-PAGE, and GFP-tagged histones were detected with anti-GFP antibody. Anti-H2A antibody was used as an internal control for the total nucleosome content at each time point (hours after induction). The chromatin-bound (black bars) and soluble forms (empty bars) of each histone-GFP protein are graphed as the ratio of GFP signal to H2A signal (arbitrary units). (A) A pulse of H2B-GFP rapidly enters chromatin, and almost all soluble H2B-GFP is used up within 8 h of production. The bulk of H2B-GFP is stable in chromatin. (B) Soluble H3-GFP is produced, but there is no detectable incorporation into chromatin. (C) A pulse of H3.3-GFP incorporates into chromatin, and reaches its maximum 8 h after production. Soluble H3.3-GFP has disappeared by 16 h, and the bulk of chromatin-bound tagged protein is gone by 48 h.

Figure 6.

Figure 6.

H3.3 is rapidly displaced from highly active chromatin. (A_–_D) Kc cells transfected with the HS-H3.3-GFP construct were heat-shocked to induce the construct. Samples were collected 2 h (A), 8 h (B), 14 h (C), and 20 h (D) later and lightly extracted with Triton X-100 to remove soluble proteins before fixation. The location of the nucleolus is marked by antibody detection of the nucleolar protein fibrillarin (red). Arrowheads indicate the position of H3.3-GFP nucleolar foci. Line profiles show the pixel intensities of the GFP signal (arbitrary units) through the nucleus. The ratio of H3.3-GFP spot intensities within the nucleolus to that of the brightest site in euchromatin was used as a single-cell measure of the amount of histone deposited at the active rDNA genes. (A) Two hours after induction many cells have intensely labeled rDNA foci. (B_–_D) H3.3-GFP signals in nucleoli become dimmer over time, and by 20 h nucleolar foci are rare. Euchromatic signals reach their maximum 8 h after production, and then diminish slowly. (E) Quantitation of H3.3-GFP nuclear patterns and intensities over time. Twenty-five GFP-positive cells were counted for each sample. Both the frequency of nuclei with nucleolar foci and the intensity of those foci drop over time. The decrease in focal intensity is statistically significant (P < 0.001, comparison of ratio of means). (F) Constitutive expression of H3.3-RFP (red) in Kc cells gives similar intensities in nucleolar foci and in euchromatin. In the same cells, a pulse of H3.3-GFP (green) 2 h earlier intensely labels the same nucleolar foci. (G) Twenty hours later, nucleolar H3.3-GFP signal is dimmer than the constitutive H3.3-RFP signal.

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