Histone H3 variants specify modes of chromatin assembly

Abstract

Histone variants have been known for 30 years, but their functions and the mechanism of their deposition are still largely unknown. Drosophila has three versions of histone H3. H3 packages the bulk genome, H3.3 marks active chromatin and may be essential for gene regulation, and Cid is the characteristic structural component of centromeric chromatin. We have characterized the properties of these histones by using a Drosophila cell-line system that allows precise analysis of both DNA replication and histone deposition. The deposition of H3 is restricted to replicating DNA. In striking contrast, H3.3 and Cid deposit throughout the cell cycle. Deposition of H3.3 occurs without any corresponding DNA replication. To confirm that the deposition of Cid is also replication-independent (RI), we examined centromere replication in cultured cells and neuroblasts. We found that centromeres replicate out of phase with heterochromatin and display replication patterns that may limit H3 deposition. This confirms that both variants undergo RI deposition, but at different locations in the nucleus. How variant histones accomplish RI deposition is unknown, and raises basic questions about the stability of nucleosomes, the machinery that accomplishes nucleosome assembly, and the functional organization of the nucleus. The different in vivo properties of H3, H3.3, and Cid set the stage for identifying the mechanisms by which they are differentially targeted. Here we suggest that local effects of "open" chromatin and broader effects of nuclear organization help to guide the two different H3 variants to their target sites.

Figure 1

Drosophila produces three versions of H3 histones. H3 and H3.3 are nearly identical throughout their N-terminal tail and histone fold domains (HFD), with only four amino acid residue differences. Cid is much more diverged, and can only be aligned to H3 in the HFD (49). Black boxes indicate identities to H3.

Figure 2

The three versions of histone H3 determine the mode of nucleosome assembly. The deposition of H3 is strictly replication-coupled (RC), and H3 is recruited to replication forks for chromatin doubling. Deposition of Cid (blue) is exclusively replication-independent (RI), and normally occurs only at centromeres. H3.3 (green) undergoes RC deposition, and RI deposition at active loci. Open chromatin at centromeres and at active genes may promote histone replacement. Transcriptional activity, chromatin remodeling factors, and RNA polymerases (orange) will unfold the chromatin fiber and disrupt nucleosome (gray) structure (open chromatin). Transcriptionally inactive regions are not subjected to these forces and remain in a closed configuration. Flanking heterochromatin and H3-containing blocks within the centromeric domain presents the H3K9me epitope, thereby binding HP1 (red) and resulting in a compacted, closed chromatin structure. Cid-containing nucleosomes cannot be methylated in this way, and thus remain comparatively open. The specialized N-terminal tail of Cid may alter the linker DNA between nucleosomes, also contributing to the open chromatin configuration. RI deposition of H3.3 is limited to the open chromatin at active genes and RI deposition of Cid is limited to the open chromatin in centromeric domains.

Figure 3

Drosophila Kc cells. (A) The cell cycle is ≈20 h long, and S phase has two distinct periods: early S phase, when all euchromatin (gray) replicates, and late S phase, when all heterochromatin (black) replicates. Eighty percent of cells show ≈15 chromosomes, and this karyotype has been stable for >2 years. (B) The morphology of a Drosophila interphase nucleus. All heterochromatin typically associates into a chromocenter (black). Centromeres (red) are enclosed within the chromocenter, with the nucleolus (light gray) next to it. The active rDNA genes (green) are located within the nucleolus.

Figure 4

Centromeres replicate with euchromatin in tetraploid Kc cells and in larval diploid neuroblasts. (A) Mitotic X chromosomes from cells pulsed with dig-dUTP nucleotide analog (green) and then chased for 4 h show heavy labeling in the heterochromatin surrounding centromeres (Cid, red), as expected for incorporation during late S phase. (B) Mitotic X chromosomes from cells pulsed with dig-dUTP and then chased for 10 h were in early S phase at the time of the pulse, because they show heavy labeling in the euchromatic arms. There are also foci of incorporation corresponding to both sister centromeres. (C) Pulse-labeling and imaging of interphase Kc cells shows that centromeres replicate in the early S-phase period when euchromatin is also replicating. We tracked cell survival and S-phase progression over a 5-h period, and mitotic index over a 25-h period in all labeling experiments. These parameters were indistinguishable from control, untreated cultures. In labeled cultures after 7 h we observed ≈98% labeling of mitotic figures, indicating that virtually all cells in S phase at the time of the pulse received the nucleotide analog. (D) Neuroblast centromeres are contained within one to three heterochromatic chromocenters (H3K9me, blue). Pulse-labeling with dig-dUTP reveals foci of DNA replication in two centromeric spots and in euchromatin. Cultured cells and dissected larval brains were labeled and prepared as described (8).

Figure 5

Replication within centromeric domains. (A) Cid and H3 appear to be interspersed as alternating blocks in the centromeric domain. Replication within the domain occurs before replication of the flanking heterochromatin. We consider three possible arrangements of replication origins within this domain. 1: Multiple origins coincide precisely with each block of Cid-containing chromatin. Firing of these origins would replicate every Cid block first, after which replication forks proceed into H3-containing chromatin. 2: Multiple origins are distributed throughout the domain, without regard to the kind of chromatin. At any one time, some Cid-containing and some H3-containing chromatin would be replicating. 3: A single origin lies in the centromeric domain, and bidirectional replication duplicates the entire region. Labeling from short nucleotide analog pulses can distinguish these arrangements. (B) A pulse-labeled centromeric domain fiber. Kc cells were labeled with dig-dUTP (8) and fibers prepared according to (30), except that a high salt buffer described in (60) was used. The stretched fiber shows an array of Cid spots (red). In this case, the nucleotide analog (green) has incorporated in the intervening gaps between Cid chromatin. These replication tracts are scattered throughout the centromeric domain, and this pattern demonstrates that multiple origins are scattered throughout the domain. The statistically significant association of replication tracts with non-Cid chromatin across the domain suggests that origins have a fixed relationship to the chromatin blocks, and that H3-containing blocks replicate out of phase from Cid-containing blocks.

Figure 6

Overexpression of Cid mis-localizes to euchromatin by RI deposition. Kc cells were transfected with a HS-CidGFP construct (3) with a modified translational start sequence (61). Cells were induced to produce high levels of Cid-GFP (red), and then immediately pulse-labeled with nucleotide analog (green) to identify cell cycle stages. The heterochromatic compartment is labeled with an anti-HP1 antibody (blue; ref. 61). (A and B) Overexpressed Cid incorporates at centromeres but also mis-incorporates throughout euchromatin. Mis-incorporation in euchromatin occurs by a replication-independent process, because it occurs both in early S phase (A) and in late S phase (B) cells. (C) After a chase of 6 h, mitotic chromosomes that were induced during S phase show labeling at both sister centromeric foci (arrowhead), and throughout the euchromatic arms.

Figure 7

RI deposition allows switching of heritable chromatin states. Nucleosomes in silent heterochromatin are distinctively modified by methylation, and thereby recruit the HMT Suvar3-9. The silencing epitope can be perpetuated by Suvar3-9 through the cell cycle by the methylation of H3 after replication-coupled deposition (vertical arrow). A gene can be activated (rightward arrow) at any time in the cell cycle, and the unraveling of methylated nucleosomes and RI deposition of H3.3 will remove the silencing epitope. This abolishes Suvar3-9 recruitment and allows stable activation. RI deposition of H3.3 will continue as long as the gene is transcribed. Switching from an active to a silent state (leftward arrow) can occur by repressing transcription and methylating the N-terminal tail of H3.3 at Lys-9, once again recruiting the Suvar3-9 complex.

Figure 8

The relationship of the canonical H3 histones in animals and fungi. Three residues in the HFD differ between H3 and the replacement H3.3 histone in metazoans. Basidiomycete fungi (Cryptococcus) also have two canonical H3 histones that can be classed as an H3 and a replacement histone. Ascomycetes (Saccharomyces, Neurospora, and Schizosaccharomyces) have only one type of canonical H3, resembling the replacement histone in basidiomycetes. This phylogeny identifies that ascomycetes have lost their H3 histone, and retain only a replacement variant.