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. 2004 Feb 10;101(6):1525-30.
doi: 10.1073/pnas.0308092100. Epub 2004 Jan 19.

Histone H3.3 is enriched in covalent modifications associated with active chromatin

Erin McKittrick  1 , Philip R Gafken, Kami Ahmad, Steven Henikoff
Affiliations

Histone H3.3 is enriched in covalent modifications associated with active chromatin

Erin McKittrick et al. Proc Natl Acad Sci U S A. .

Abstract

Chromatin states can be distinguished by differential covalent modifications of histones or by utilization of histone variants. Chromatin associated with transcriptionally active loci becomes enriched for histones with particular lysine modifications and accumulates the H3.3 histone variant, the substrate for replication-independent nucleosome assembly. However, studies of modifications at particular loci have not distinguished between histone variants, so the relationship among modifications, histone variants, and nucleosome assembly pathways is unclear. To address this uncertainty, we have quantified the relative abundance of H3 and H3.3 and their lysine modifications. Using a Drosophila cell line system in which H3.3 has been shown to specifically package active loci, we found that H3.3 accounts for approximately 25% of total histone 3 in bulk chromatin, enough to package essentially all actively transcribed genes. MS and antibody characterization of separated histone 3 fractions revealed that H3.3 is relatively enriched in modifications associated with transcriptional activity and deficient in dimethyl lysine-9, which is abundant in heterochromatin. To explain enrichment on alternative variants, we propose that histone modifications are tied to the alternative nucleosome assembly pathways that use primarily H3 at replication forks and H3.3 at actively transcribed genes in a replication-independent manner.

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Figures

Fig. 1.
Fig. 1.
Histone variants are resolved by HPLC separation. Acid-extracted histones from Kc cells were injected onto a reverse-phase HPLC column in 30% acetonitrile/0.1% trifluoroacetic acid. All histone 3 eluted in the two peaks shown here, and the peaks were determined to be composed of pure histone 3 by SDS/PAGE and Coomassie staining. The relative abundance of the two peaks was determined by integrating the area under each curve (gray and hatched areas) and averaging over multiple measurements.
Fig. 2.
Fig. 2.
Identification of HPLC peaks by MALDI-TOF spectrometry of peptide 27-40. Pooled fractions from early and late histone 3 HPLC peaks were digested to completion with ArgC protease and subjected to MALDI-TOF MS. Peptides were identified based on their masses, which are indicated above the peaks. Peptide 27-40 contains an alanine at position 31 in H3, and serine in H3.3, for a 16-Da difference. The early HPLC peak (Upper) is composed of H3, and the late peak (Lower) is composed of H3.3. Five modified forms, with 2, 3, 4, 5, and 6 methyl groups, were identified on both H3 and H3.3.
Fig. 3.
Fig. 3.
H3.3 levels increase during Kc cell differentiation after ecdysone addition. After the indicated time at 25°C, histones were extracted and analyzed by HPLC. Curves were fit to HPLC traces (see Fig. 1), and peak areas were measured and averaged for two experiments.
Fig. 4.
Fig. 4.
Identification of modified forms by LC-ESI MS. Pooled fractions from the H3 or H3.3 HPLC peaks were digested to completion with the ArgC protease and subjected to LC-ESI MS. Peptides and their modified forms were identified by their masses, as indicated above the peaks. For peptide 73-83, doubly charged ions of unmethylated, monomethylated, and dimethylated forms were found in both the H3 and H3.3 spectra, corresponding to methylation at lysine 79. H3.3 is enriched for lysine 79 methylation.
Fig. 5.
Fig. 5.
LC-ESI MS determinations of relative abundances of lysine modifications on H3 and H3.3. Histograms show the averages for two to four experiments. Percentages of H3 (black bars) or H3.3 (gray bars) are shown for each modification. (A) Methyls on K79 (peptide 73-83), integrating areas under the first three isotopic peaks (e.g., Fig. 4). (B) Acetyl groups on K18 and/or K23 (peptide 18-26). (C) Methyl groups on K27, K36 and/or K37 (peptide 27-40). (D) Methyl groups and/or acetyl groups on K9 and/or K14 (peptide 9-17).
Fig. 6.
Fig. 6.
Differential enrichment of modifications on H3 and H3.3 detected by using antibodies. (A) Examples of “active” modifications detected by using antibodies to probe slot blot filters after applying equal protein amounts of H3 and H3.3 from pooled fractions, ascertained by Coomassie blue staining of gel samples (not shown). Two-fold dilutions from top to bottom are shown for acetyl-K9 and dimethyl-K79, and 4-fold dilutions are shown for acetyl-K14 modifications. (B) HPLC trace showing fractions (designated 1-4) used for the Western blot analysis in C, where acid-extracted histone from the column input was loaded in the first lane, and HPLC fractions in equal volumes (protein amounts differ) were loaded in remaining lanes. The blot was successively probed with antibodies to the indicated modifications.
Fig. 7.
Fig. 7.
Relative enrichment of modifications on H3.3 vs. H3 determined by ELISA. Histogram bars show enrichment on H3.3 (above zero) or on H3 (below zero) of each indicated modification.
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