BIOLOGICAL OVERVIEW

Chromatin assembly is a cell process of central importance, quite literally: it takes place at the interface of DNA replication, regulation of gene expression, and progression through the cell cycle. The fundamental repeat structure of chromatin is the nucleosome core particle, comprising 146 base pairs of DNA wrapped around an octamer of histone proteins consisting of two molecules each of the four core histones: H2A, H2B, H3 and H4. Packaging of DNA into nucleosomal structures is generally inhibitory to processes requiring access to DNA, including transcription. Conversely, activation of transcription is correlated with increased accessability to DNA and an alteration of chromatin structure. How is chromatin assembled, and how does this assembly process assure a spacing of nucleosomes that will, in turn, assure proper gene function?

Two processes must be distinguished: chromatin assembly and nucleosome restructuring. The former takes place during DNA replication, and involves Chromatin assembly factor 1 (CAF-1) and Nucleosome assembly protein 1 (NAP1). The latter involves other multiprotein complexes related to yeast SWI/SNF proteins (See ISWI and Brahma). Although there may be some overlap between the processes, these points of hypothetical overlap have yet to be discovered. For example, access of transcription factors to DNA may be regulated by histone acetylation, and this process might be controlled during both assembly and remodeling. But for the time being, the two processes will be thought of as separate.

In Drosophila two factors (CAF-1 and NAP1) mediate assembly of regularly spaced nucleosomal arrays when combined with DNA, purified core histones, and ATP (Ito, 1996a). In humans, CAF-1 is a three-subunit protein complex that assembles histone octamers into replicating DNA. Both human complex and Drosophila CAF-1 protein mediate chromatin assembly with newly replicated DNA as substrate, in preference to unreplicated DNA as substrate (Kamakaka, 1996). Human CAF-1 colocalizes with DNA replication foci during S phase. The single-strand-binding protein Replication Protein A (RPA) also localizes to these sites. During DNA replication, CAF-1 assembles new nucleosomes in a two-step reaction. Coupled to DNA replication, histones H3 and H4 are deposited first in a reaction that is strictly dependent upon CAF-1. In a later step, histones H2A and H2B are added to this immature nuclesome precursor. This latter reaction takes place in the absence of CAF-1 (Krude, 1995 and references). Thus the current data suggest that NAP-1 interacts preferentially with H2A and H2B (Ito, 1996) and CAF-1 binds to H3 and H4 (Kaufmann, 1995). To make matters even more complex two other histone chaperones have been identified, Nucleoplasmin, which like NAP-1 prefers H2A and N1/N2 which binds preferntially to H3 and H4 (Ito, 1996b and references).

Sequences of the two largest subunits of human CAF-1 reveal one protein of 150 kD containing large clusters of charged residues, and a second protein of 60 kD, containing WD repeats. Recombinant p150 and p60 are both required for chromatin assembly in a p60-depleted extract, but only p150 is required in an undepleted extract, and both proteins produce assembled chromatin in replicating DNA. P150 and p60 form complexes in cell extracts with newly synthesized histone H3 and acetylated H4, suggesting that such complexes are intermediates beween histone synthesis and chromatin that has been assembled into replicating DNA. Cells use acetylation to target newly synthesized histones for replication-linked chromatin assembly. It is possible that acetylation plays a role in regulating transcription factor access to nascent chromatin (Kaufman, 1995). Drosophila CAF-1 appears to comprise four subunits of 180, 105, 75 and 55 kDa. The smallest subunit of the Drosophila CAF-1 fraction, p55, also known as dCAF-1 protein, is homologous to a mammalian RbAp48 protein which is associated with the HD1 histone deacetylase. p55 contains seven WD repeat motifs. A fraction of p55 becomes associated with the newly assembled chromatin following DNA replication. CAF-1 protein is a core histone chaperone that delivers newly synthesized and acetylated histones to other components of the chromatin assembly machinery at the replication fork (Tyler, 1996).

The third component of human CAF-1 is RbAp48, so named because it was isolated in association with the Retinoblastoma protein and has a molecular weight of 48 kD. RbAp48 associates with histone deacetylase (HD1). The two proteins show sequence homology to yeast Hat2p, a component of the yeast Histone acetyltransferase complex of the B type (HAT B). The p48 family members are conserved subfamily of WD-repeat proteins. Acetylation of newly synthesized H4 is catalyzed by cytoplasmic HAT Bs, while nuclear histone acetylation is carried out by another complex, HAT A. The yeast transcriptional adaptor protein Gcn5p, functions as the catalytic subunit of a HAT A activity. Gcn5p is highly similar in sequence to a ciliate HAT A complex protein. The initial histone acetylation may be required to neutralize its high positive charge, allowing it to be assembled into chromatin. Deacetylation of histones carried out by histone deacetylase, could be a prerequisite to maturation of chromatin. In any case, it is now clear that chromatin assembly and maturation involves histone acetylation and that this process begins in cytoplasm and is subsequently transferred to the nucleus (Roth, 1996 and references).

In Drosophila, NAP1 and CAF-1 fraction (dCAF-1) act cooperatively to assemble chromatin. Drosophila NAP1 was purified and cloned from Drosophila CAF-4 (dCAF-4) fraction. The CAF-4 fraction interacts with the CAF-1 fraction to assemble regularly spaced nucleosome arrays on a DNA template (Bulger, 1995). Purified recombinant Drosophila NAP1 functions in a cooperative manner with the active component(s) in the dCAF-1 fraction to mediate the ATP-facilitated assembly of nucleosomal arrays. NAP1 acts as a histone chaperone, binding newly synthesized histones in the cytoplasm, and shuttling the histones to the nucleus where they are used to assemble chromatin on newly synthesized DNA. Purified yeast NAP1, while fulfiling the same function, can substitute for Drosophila NAP1 in mediating the efficient assembly of extended nucleosomal arrays. NAP1 alone (from yeast or flies) yields chromatin products that exhibit a significantly shorter repeat length than do the nucleosome arrays assembled in the presence of both NAP1 and dCAF-1 (Ito, 1996a).

One model for the role of core histone chaperones in chromatin assembly suggests CAF-1 binds to newly synthesized H3 and (acetylated) H4 and mediates the formation of the H3-H4 tetramer into newly replicated DNA. Histones H2A-H2B are then thought to be subsequently incorporated with the assistance of other histone chaperones, such as nucleoplasmin or NAP1, to give the complete histone octamer (Tyler, 1996).

Two other NAP1 like proteins have been characterized in Drosophila. dNLP or CRP1 (Ito, 1996b and Crevel, 1997) is a Nucleoplasmin-like protein in Drosophila purified as a minor component of the dCAF-4 fraction, the same fraction from which NAP1 was purified. dNLP, like NAP1 acts in the ATP-facilitated assembly of chromatin, and like NAP1, preferentially interacts with histones H2A and H2B, but with a lower affinity. An additional protein has been cloned on the basis of homology to the NAP/SET family of proteins. Yeast NAP1 of the NAP/SET family, interacts specifically with B-type cyclins and can participate in the remodeling of chromatin. In addition, the yeast protein acts with mitotic cyclins to perform mitotic functions and suppress polar bud growth in budding yeast (Kellogg, 1995a and b).

The different processes used to assemble chromatin are still far from being well understood. Nevertheless, it appears that the machine-like protein puzzles that assemble and restructure chromatin are now being solved, and a better understanding of the process is within reach.

GENE STRUCTURE

Bases in 5' UTR - 63

Bases in 3' UTR - 145

PROTEIN STRUCTURE

Amino Acids - 370

Structural Domains

NAP1 contains a highly acidic segment near the C-terminus that resembles the C-terminal acidic region of nucleoplasmin: this region may be involved in binding histones. There is also a stretch of amino acid residues (amino acids 240-258) that has characteristics of nuclear localization signals, as well as a segment at positions 57-65 that resembles a nuclear export signal. Drosophila NAP1 is 47% identical to the human protein and 31% identical to the yeast protein. There are three highly conserved motifs in positions 207-215, 340-439 and 373-394 that are important for the function (Ito, 1996a).

date revised: 15 MAY 97 

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