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).

The enhancer of trithorax and polycomb gene Caf1/p55 is essential for cell survival and patterning in Drosophila development

In vitro data suggest that the human RbAp46 and RbAp48 genes encode proteins involved in multiple chromatin remodeling complexes and are likely to play important roles in development and tumor suppression. However, to date, understanding of the role of RbAp46/RbAp48 and its homologs in metazoan development and disease has been hampered by a lack of insect and mammalian mutant models, as well as redundancy due to multiple orthologs in most organisms studied. This study reports the first mutations in the single Drosophila RbAp46/RbAp48 homolog Caf1, identified as strong suppressors of a senseless overexpression phenotype. Reduced levels of Caf1 expression result in flies with phenotypes reminiscent of Hox gene misregulation. Additionally, analysis of Caf1 mutant tissue suggests that Caf1 plays important roles in cell survival and segment identity, and loss of Caf1 is associated with a reduction in the Polycomb Repressive Complex 2 (PRC2)-specific histone methylation mark H3K27me3. Taken together, these results suggest suppression of senseless overexpression by mutations in Caf1 is mediated by participation of Caf1 in PRC2-mediated silencing. More importantly, the mutant phenotypes confirm that Caf1-mediated silencing is vital to Drosophila development. These studies underscore the importance of Caf1 and its mammalian homologs in development and disease (Anderson, 2011).

Several lines of evidence suggest that the participation of Caf1 in PcG complexes may account for many of the phenotypes observed in flies with altered expression of Caf1. First, Caf1 loss-of-function clones in the eye have phenotypes ranging from slight disorganization and bristle defects to almost complete loss of homozygous tissue in adults, and incomplete rescue of Caf1 results in adult eyes that are small and disorganized. Clones of many PcG genes have similar phenotypes in the eye. Loss of E(z) or Pc causes mild defects in differentiation in the third instar disc, but clones fail to survive in adults. An analogous situation occurs in Caf1^short clones, where expression of Elav, which marks differentiating neurons, is present in Caf1 clones at third instar but Caf1^short tissue is largely missing in the adult. Derepression of Hox genes could account for these phenotypes, as ectopic expression of many Hox genes in the eye field causes small disorganized eyes in adults (Anderson, 2011).

Second, flies with incomplete rescue of Caf1 display a range of homeotic phenotypes, notably transformation of arista to leg. Similar homeotic transformations, including antenna-to-leg transformations, are a hallmark of mutations in PcG genes. Also a genetic interaction was observe between Caf1 and the PRC1 gene Pc, since mutations in Caf1 are able to dominantly suppress the homeotic transformation of second or third leg to first leg in Pc¹⁵/+ males (Anderson, 2011).

Third, the disrupted patterning of Caf1^short mutant heads may also result from PcG dysfunction. It is possible that these patterning defects are non-cell autonomous and may be an indirect result of widespread apoptosis in the eye disc. Normally, when an imaginal disc is injured, remaining cells proliferate and assume correct identities, leading to a perfectly patterned adult structure. This type of regeneration requires that some determined cells must change their fates and involves substantial chromatin remodeling. Under specific circumstances, the disc can regenerate with incorrect patterning, leading to duplication, deletion or transformation of structures, a phenomenon referred to as transdetermination. Levels of many PcG transcripts are increased in transdetermining imaginal discs, and heterozygous mutations in PcG genes can enhance transdetermination in regenerating imaginal discs. Therefore, one interpretation of the patterning defects in Caf1^short mutant discs is that under the stress of widespread apoptosis, the remaining heterozygous tissue is haploinsufficient for the chromatin remodeling activity required to properly regenerate and pattern the injured disc. Consistent with this interpretation, no extra or missing appendages are observed in flies with Caf1^long clones, which show less active Caspase 3 staining at third instar (Anderson, 2011).

Finally, in Caf1 mutant tissue, a reduction is observed in levels of the H3K27me3 mark, which is associated with inactive chromatin and PRC2 activity. These data are consistent with a disruption of PRC2 function as a result of loss of Caf1 and represent the first in vivo evidence that Caf1 is an essential member of this chromatin remodeling complex in an animal model (Anderson, 2011).

One obvious question arises from the current study: why were multiple Caf1 alleles identified in a screen for modifiers of the sens overexpression phenotype? Moreover, it is surprising that mutations in Caf1 were not identified in previous Drosophila modifier screens involving PcG or Rb pathway members. It is proposed that the link between sens and Caf1 is due to the role of Caf1 in PcG-mediated silencing (Anderson, 2011).

Recent evidence suggests that Sens and Hox proteins can compete for binding at overlapping sites at an enhancer of the rhomboid (rho) locus (Li-Kroeger, 2008). When the Hox protein Abdominal-A (Abd-A) binds, transcription of rho is activated, whereas binding by Sens leads to repression of rho. In the embryo, this mechanism acts as a molecular switch to allow differentiation of either chordotonal organs (under control of Sens) or hepatocyte-like cells called oenocytes (by the action of Abd-A). It is proposed that a similar mechanism underlies the suppression of the Sens overexpression phenotype. It is hypothesized that one or more targets of Sens in the eye contain similar overlapping sites that can be bound by either Sens or a Hox protein. During normal development, these loci are bound by neither Sens nor Hox in undifferentiated cells posterior to the furrow, as no Hox genes are known to be widely expressed in the eye field. In the absence of both types of factors, these loci are transcriptionally active, and are necessary to ultimately attain the proper fates of the cells in which they are expressed. When Sens is overexpressed, as in ls, Sens binds to its recognition site in the downstream loci, repressing transcription. Repression of these genes initiates a cascade leading to a change in cell fate; for example, some of the cells that would normally become secondary or tertiary pigment cells now become bristle precursors, giving rise to the extra bristles of ls. However, when one copy of Caf1 is lost, a slight derepression of the Hox genes occurs due to loss of PcG activity. Hox proteins are now able to compete with Sens for the overlapping binding sites, tipping the balance towards activation of downstream genes and attainment of normal cell fate - effectively suppressing ls. The ability of ectopic expression of pb and Antp in the eye to suppress ls is consistent with this hypothesis. Suppression of ls by Hox proteins is particularly significant given that ectopic expression of Antp alone in the eye field leads to a small and disorganized eye. As Sens activity is exquisitely sensitive to Hox proteins, especially in the eye, the screen for modifiers of a sens overexpression phenotype was therefore ideal for identifying mutations in Caf1 (Anderson, 2011).

Previous studies have explored pro-apoptotic roles of Hox proteins and anti-apoptotic roles of Sens. It is therefore possible that one effect of Hox gene derepression in ls eyes suppressed by Caf1 may be restoration of an apoptotic fate in cells that would otherwise form bristle precursors due to ectopic Sens. Abd-A expression in the abdomen during normal third instar larval development leads to apoptosis of proliferating neuroblasts of the central nervous system, and ectopic expression of other Hox genes can also cause neuroblast apoptosis. Accordingly, survival of neuroblasts is dependent on PcG activity to repress Hox gene expression. Furthermore, expression of Sens is necessary in the Drosophila embryonic salivary gland to prevent apoptosis. Thus, one possible mechanism for suppression of ls by Caf1 mutations is that in the ls eye, Sens may promote the ectopic bristle fate partly by repressing apoptotic genes in cells normally fated to die, whereas in the ls eye suppressed by mutations in Caf1, ectopic Hox proteins may promote apoptosis and prevent bristle formation. Increased Ubx expression was not detected by antibody staining; however, a very small increase in one or more Hox proteins may be all that is necessary to change the transcriptional state of downstream loci and prevent the ectopic bristles and other defects in the highly sensitized ls eye - especially in the eye field, where no Hox genes are known to be highly expressed. Furthermore, the fact that multiple Hox proteins can recognize the same DNA binding site offers the possibility that the competitive effect of each Hox protein type on genes with overlapping Sens/Hox binding sites would be additive. Therefore, although loss of one copy of Caf1 may only cause a small derepression of any one Hox gene, mild derepression of many Hox genes collectively can lead to strong repression of the ls phenotype (Anderson, 2011).

Biochemical evidence suggests that Caf1 is a member of multiple complexes that effect gene regulation through chromatin remodeling, suggesting that it is a vital component of the cell's arsenal of chromatin modifying factors. Although the results suggest that disruption of PRC2 function may be the most important consequence of Caf1 gain- or loss-of-function, many phenotypes that were observed in Caf1 mutant tissue are also reminiscent of mutations in members of other complexes previously shown to contain Caf1. It is not surprising that all three alleles of Caf1 in the current study are homozygous lethal, and that Caf1^short cells have poor viability, considering that Caf1 has been found in the NURF and CAF-1 complexes, which have fundamental roles in nucleosome assembly and spacing. The apoptosis observed in eyes with Caf1^short clones is also consistent with a role for Caf1 in the dREAM (Drosophila Rbf, E2F2, and Myb-interacting proteins) complex. Members of the E2f family of transcription factors can complex with Dp proteins and bind short recognition sites to activate transcription. When Rb binds the E2f-Dp complex, transcription is repressed. Like Caf1 homozygotes, homozygous rbf1 null flies die in early larval development. Fully rbf1-deficient embryos display increased apoptosis, a phenotype reminiscent of the increased active Caspase-3 staining seen anterior to the morphogenetic furrow in eye discs with Caf1^short clones (Anderson, 2011).

The mammalian homologs of sens, Growth Factor Independence 1 (Gfi1) and Gfi1b are essential to the development of multiple cell types and have been implicated as oncogenes. Therefore, the possibility that Caf1 links Sens with the activity of PcG complexes through parallel, competing pathways has implications for both Drosophila development and the activity of Gfi1 family members in human development and disease, and warrants additional study beyond the scope of the present work. The results underscore the importance of Caf1 to diverse processes, including cell survival and tissue identity, and highlight the participation of Caf1 in multiple chromatin remodeling complexes. Further studies are needed to fully assess the importance of Caf1 in Drosophila development, as well as its developmental role in other chromatin remodeling complexes (Anderson, 2011).

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: 1 June 2024

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