BIOLOGICAL OVERVIEW

Understanding the pathway by which histones are incorporated into replicating chromosomes is a primary goal of biologists, since the structuring of histones in chromosomes regulates gene activation. The pathway is complex: histones are modified by acetylation (Sobel, 1994 and 1995) in the cytoplasm, and then assembled into nucleosomes, piecemeal on DNA in the nucleus (Smith, 1991 and Kaufmann, 1995). Histones are subsequently modified again in a way that reflects the activation status of the gene with which they associate. The literature on chromatin assembly suggests a multi-component, still not completely solved puzzle of factors is responsible, some cytoplasmic and others nuclear, and still others found in both locations.

One protein, the smallest subunit of a protein complex called Chromatin assembly factor (Caf1) (Kaufmann, 1996), appears to have both cytoplasmic and nuclear functions. p55, a protein of molecular mass of 55 kDa, is the Drosophila version of this subunit, while RbAp48 (p48) serves a similar function in vertebrates. Both are considered histone chaperones; proteins that accompany histones on their journey from the cytoplasm to the nucleus. Both proteins are members of an evolutionarily conserved subfamily of WD-repeat proteins. In the cytoplasm, p48 family members are found to associate with Histone H4 and with an enzyme called histone acetyltransferase (HAT) (Parthun, 1996). In the nucleus, p48 family members are found associated with histones H3 and H4 and with other proteins constituting a complex called Chromatin assembly factor 1 (Verreault, 1996). Chromatin assembly factor 1 is associated with the DNA replication fork (Krude, 1995). Also in the nucleus and associated with a p48 family member (RbAp48) and histones H3 and H4 is another enzyme, histone deacetylase (Taunton, 1996). Thus p48 family members are found in three different contexts. Since p48 family members are found associated with histones in each of these contexts, p48 proteins are called histone chaperones, proteins that accompany histones through the chromatin maturation process (Roth, 1996 and references).

A sequenced process for histone maturation is suggested by these findings. In the cytoplasm, Histones H3 and H4, accompanied by their p48 chaperone (Caf1 subunit in Drosophila), are acetylated by histone acetyltransferase. Histone acetyltransferase is subsequently removed and CAF1 components are substituted, transferring p48 and its histones from the cytoplasm to the nucleus, where histones are assembled into nucleosomes at the DNA replication fork. Acetylation of histones reduces the histone electrical charge, promoting the ability of histones to associate with DNA. Subsequently histone deacetylase removes deposition-related acetyl groups from Histone H3 and H4. Again, p48 family members partner their histones in interaction with histone deacetylase (Roth, 1996).

What of two other histones constituting the core nucleosome, Histones H2A and H2B? Another chaperone, Nucleosome assembly protein 1 or NAP1, is associated with these histones, and this histone companion has both a cytoplasmic and nuclear location. NAP1 is really one of several proteins found in Drosophila with an affinity for histones H2A and H2B. These two histones are assembled into chromatin after Histones H3 and H4. H2A and H2B to not require acetylation for assembly into chromatin.

Drosophila p55 has several characteristics that distinguish it from its mammalian and yeast homologs. While a mammalian homolog does associate with Rb, a cell cycle regulatory protein, p55 does not. Whereas the mammalian protein is found in a CAF-1 complex with two other proteins, the Drosophila CAF-1 associates with three other proteins. While there are at least four members of the p48 family in mammals, in Drosophila there is apparently only one. The functional differences between mammalian and Drosophila CAFs still await elucidation, regarding the functions of their accompanying subunits (Tyler, 1996).

Caf1 is found in a second protein complex, Drosophila nucleosome remodeling factor (NURF), a protein complex of four distinct subunits that assists transcription factor-mediated chromatin remodeling. One NURF subunit, ISWI, is related to the transcriptional regulators Drosophila Brahma and yeast SWI2/SNF2. A second integral subunit of NURF (the 55-kDa subunit) has been termed p55 and is generally associated with polytene chromosomes. In general, p55 is not found to be focused at specific polytene chromosome loci as has been frequently observed for sequence specific transcription factors. The predicted sequence of p55 reveals a WD repeat protein that is identical with the 55-kDa subunit of the Drosophila chromatin assembly factor (Caf1). Caf1 but not NURF, associates with histone acetyltransferase (HAT), and is found in a complex with active HAT enzyme(s) in nuclear extracts. Given that WD repeat proteins related to p55 are associated with histone deacetylase and histone acetyltransferase, these findings suggest that p55 and its homologs may function as a common platform for the assembly of protein complexes involved in chromatin metabolism (Martinez-Balbas, 1998).

GENE STRUCTURE

Bases in 5' UTR - 291

Bases in 3' UTR - 221

PROTEIN STRUCTURE

Amino Acids - 430

Structural Domains

To gain a better understanding of DNA replication-coupled chromatin assembly, the smallest subunit (apparent molecular mass, 55 kDa and termed p55) of Drosophila melanogaster chromatin assembly factor 1 (dCAF-1) was characterized. CAF1 is a multisubunit protein that is required for the assembly of nucleosomes onto newly replicated DNA in vitro. The p55 polypeptide is homologous to the mammalian RbAp48 protein, which is associated with the HD1 histone deacetylase. There is an 87% amino acid identity between p55 and either mouse or human RbAp48, while there is 84% identity between p55 and either mouse or human RbAp46. p55 is related to two proteins in S. cerevisiae: a protein designated Ye1056p (34% identity) and the protein encoded by the MSI1 gene (26% identity).

p55, with seven WD repeat motifs, is a member of the WD repeat family of proteins. Members of this diverse group of protein have 4 to 10 copies of the WD repeat, which is a motif of approximately 40 amino acid residues that includes a conserved Trp-Asp dipeptide, the WD of the repeat sequence. The structures of the WD repeat motifs form a beta-propeller structure, wherein each of the seven "blades" of the propeller contains the residues of one WD repeat unit. Other conserved residues also contribute to the structure (Tyler, 1996 and references).

date revised: 6 MAY 97 

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