Chromatin assembly factor 1 subunit
To ascertain the mechanism by which nucleosomes are assembled by factors derived from Drosophila
embryos, two proteins termed Drosophila chromatin assembly factors (CAFs) 1 and 4 (dCAF-1 and
dCAF-4) were fractionated and purified from a Drosophila embryo extract. The assembly of chromatin
by dCAF-1, dCAF-4, purified histones, ATP, and DNA is a process that generates regularly spaced
nucleosomal arrays with a repeat length resembling that of bulk native Drosophila chromatin and is
not obligatorily coupled to DNA replication. The assembly of chromatin by dCAF-1 and dCAF-4 is
nearly complete within 10 min. The dCAF-1 activity copurifies with the Drosophila version of
chromatin assembly factor-1 (CAF-1), a factor that has been found to be required for the assembly of
chromatin during large tumor (T) antigen-mediated, simian virus 40 (SV40) origin-dependent DNA
replication. The dCAF-4 activity copurifies with a 56-kDa core-histone-binding protein that was
purified to > 90% homogeneity (Bulger, 1995). The 56-kDa protein is now known to be NAP1.
To study the relationship between DNA replication and chromatin assembly, a factor
termed Drosophila chromatin assembly factor 1 (dCAF-1) has been purified to approximately 50% homogeneity from a
nuclear extract derived from embryos. dCAF-1 appears to consist of four polypeptides with molecular
masses of 180, 105, 75, and 55 kDa. dCAF-1 preferentially mediates chromatin assembly of newly
replicated DNA relative to unreplicated DNA during T-antigen-dependent simian virus 40 DNA
replication in vitro, as seen with human CAF-1. Analysis of the mechanism of DNA
replication-coupled chromatin assembly reveals that both dCAF-1 and human CAF-1 mediate
chromatin assembly preferentially with newly replicated DNA relative to unreplicated
DNA. The preferential assembly of the postreplicative DNA is observed at 30 min after
inhibition of DNA replication by aphidicolin, but this effect slowly diminished until it is no longer
apparent at 120 min after inhibition of replication. These findings suggest that the coupling between
DNA replication and chromatin assembly may not necessarily involve a direct interaction between the
replication and assembly factors at a replication fork (Kamakaka, 1996).
ACF, an ATP-utilizing chromatin assembly and
remodeling factor is a multisubunit factor that contains Imitation SWI protein and is distinct from NURF,
another ISWI-containing factor. ACF contains four polypeptides with apparent molecular masses of 47, 140, 170 and 185 kDa. ISWI is the 140 kDa component of ACF. In chromatin assembly, purified ACF combined with additional core histone chaperone
(such as NAP-1 or CAF-1) are sufficient for the ATP-dependent formation of periodic nucleosome
arrays. In chromatin remodeling, ACF is able to modulate the internucleosomal spacing of chromatin by
an ATP-dependent mechanism. ACF, acting with NAP-1 can mediate promoter-specific nucleosome
reconfiguration by Gal4-VP16 in an ATP-dependent manner. ACF can act catalytically by an ATP-dependent mechanism to modulate nucleosome spacing in the absence of a core histone chaperone. These results suggest that ACF acts
catalytically both in chromatin assembly and in the remodeling of nucleosomes that occurs during
transcriptional activation. The reaction mixture has a core histone octamer:ISWI ratio of about 90:1. This octamer:ISWI molar ratio of 90:1 reflects a minimal nucleosome:ACF ratio in the assembly reaction. The dNAP-1 polypeptide:ISWI polypeptide molar ratio is about 830:1, while the dNAP-1 polypeptide;histone polypeptide molar ratio is roughly 1:1. Thus these data suggest a catalytic function for ACF and a stoichiometric function for dNAP-1 as a core histone chaperone. It is concluded that ISWI is contained in two or more multi-protein complexes. ISWI and other closely related proteins are thought to function as ATP-driven, DNA-translocating motors that can displace histones from DNA. It is difficult, however, to envision a specific mechanism for a DNA-translocating motor in the deposition of nucleosomes. In this context, it is useful to consider a two-step mechanism for chromatin assembly, in which histone deposition by core histone chaperones (such as NAP-1 or CAF-1) initially occurs by an ATP-independent mechanism and is then followed by the ATP-dependent modulation of the internucleosomal spacing by ACF (Ito, 1997).
The high molecular component of ACF has now been cloned. ATP-utilizing chromatin assembly and remodeling factor (Acf)
encodes two varients, the 170- and 185-kD (p170 and p185) subunits of ACF (Note: the convention is to refer to the multiprotein complex as ACF and to the high molecular weight subunit as Acf). Purification of native Acf from Drosophila embryos leads to the isolation of ACF consisting of Acf (both p170 and p185 forms) and
subunits. Acf does not, however, copurify with components of NURF
or CHRAC, which are other chromatin remodeling complexes from
Drosophila that similarly contain an ISWI subunit. Studies of purified recombinant ACF reveal that the Acf and ISWI
subunits function synergistically in the ATP-dependent assembly of
nucleosome arrays. The purified reconstituted system requires ACF, core histones, DNA, ATP, and a histone chaperone.
NAP-1 and CAF-1 were each found to function as histone chaperones in conjunction with ACF (Ito, 1999).
Histone deacetylase activity specifically coimmunoprecipitates with affinity-purified antibodies against p55. The histone deacetylase activity in the anti-p55 pellet corresponds to approximately 10% of the total histone deacetylase activity in a crude nuclear extract. It thus appears that at least a fraction of the p55 in a crude nuclear extract is associated with a histone deacetylase. No interaction between p55 and Rb could be detected (Tyler, 1996).
dCAF-1 was immunopurified by using affinity-purified
antibodies against p55; the resulting dCAF-1 preparation possesses the four putative
subunits of dCAF-1 (p180, p105, p75, and p55) and is active for DNA
replication-coupled chromatin assembly. p55 is associated with the other subunits of dCAF-1 by coimmunoprecipitation analysis. Moreover, dCAF-1 activity, as measued using a SV40 DNA replication-chromatin assembly assay, is specifically
depleted with antibodies against p55. Thus, p55 is an integral component of dCAF-1.
p55 is localized to the nucleus and is present throughout Drosophila development.
Consistent with the homology between p55 and the HD1-associated RbAp48 protein,
histone deacetylase activity is observed to coimmunoprecipitate specifically with
p55 from a Drosophila nuclear extract. Furthermore, a fraction of the p55 protein
becomes associated with the newly assembled chromatin following DNA replication.
Collectively, these findings suggest that p55 may function as a link between DNA
replication-coupled chromatin assembly and histone modification (Tyler, 1996).
The Drosophila Polycomb Group (PcG) proteins are
required for stable long term transcriptional silencing of
the homeotic genes. Among PcG genes, esc is unique
in being critically required for establishment of PcG-mediated
silencing during early embryogenesis, but not for
its subsequent maintenance throughout development. Esc has been shown to be physically associated
with the PcG protein E(Z). Esc,
together with E(z), is present in a 600 kDa complex that is
distinct from complexes containing other PcG proteins. This Esc complex has been purified and it also
contains the histone deacetylase Rpd3 and the histone-binding
protein p55 (Chromatin assembly factor 1 subunit), which is also a component of the
chromatin remodeling complex NURF and the chromatin
assembly complex CAF-1. The association of Esc and
E(z) with p55 and Rpd3 is conserved in mammals. Rpd3 is required for silencing mediated by a
Polycomb response element (PRE) in vivo and E(z)
and Rpd3 are bound to the Ubx PRE in vivo, suggesting
that they act directly at the PRE. It is proposed that histone
deacetylation by this complex is a prerequisite for
establishment of stable long-term silencing by other
continuously required PcG complexes (Tie, 2001).
To test whether the association of Esc and E(z) with p55/Caf1 and
Rpd3 has been conserved in mammals, the
human complex containing the Esc homolog (EED) was examined for the
presence of Rpd3 and p55 homologs. Database searches
reveal that Drosophila Rpd3 is most closely related to
two human histone deacetylases, HDAC1 and HDAC2
(77% and 75% identical to Rpd3). Similarly, there are
two closely related p55 homologs in mammals,
RbAp48 and RbAp46 (91% and 86% identical to p55).
RbAp48 and RbAp46 have also been found together in
the SIN3 and Mi-2 deacetylase complexes, as have
HDAC1 and HDAC2. A test was performed to see
whether all four proteins are associated with the
human EED complex. A GST-ESC fusion
protein encoding full-length Esc can pull down full-length
in vitro translated Esc and a GST-ESC1-60 fusion
protein encoding just the N-terminal 60 residues of Esc
is sufficient to pull down full-length in vitro translated
Esc. Similarly, GST-EED1-81, which
contains the corresponding N-terminal region of EED, binds directly to in vitro
translated EED. In addition to FLAG-Esc, GST-ESC1-60 also
pulls down p55 and Rpd3 from Drosophila embryo
nuclear extract. This strongly suggests that GST-ESC1-
60 specifically pulls down the Esc complex. GST-EED1-81 pulls down HDAC1, HDAC2 and RbAp48
from HeLa cell nuclear extract.
RbAp46 has also been detected. Thus, the association of ESC with
p55 and Rpd3 is mirrored in the conserved association of
mammalian EED with RbAp48, RbAp46 and HDAC1 and
HDAC2. These results confirm the previously reported
association of EED with HDAC1 and HDAC2 (Tie, 2001).
The presence of p55 in the ESC complex provides a direct
molecular link to chromatin. The highly conserved mammalian
p55 homologs, RbAp48 and RbAp46, have been shown to bind
directly to histone H4 and possibly H2A, but not H2B or H3. The N- and C-terminal regions of RbAp48 that mediate
binding to histone H4 are virtually identical to the
corresponding regions of Drosophila p55,
strongly suggesting that p55 has the same histone-binding
specificity (Tie, 2001).
What, then, is the role of p55 in the Esc complex? It is
unlikely that p55 is responsible for the selective recruitment or
targeting of Esc and E(Z) to the ~100 specific chromosomal sites
at which they co-localize. The histone-binding
activity of p55 does not, by itself, suggest a mechanism for such
specificity and p55 binds to many more sites on the polytene
chromosomes than Esc and E(Z),
presumably reflecting its distribution in other complexes, such as
CAF1 and NURF. It seems more likely that p55 acts after the
Esc complex is recruited and serves to direct the deacetylase
activity of Rpd3 to local histone substrates. This is analogous to
the role proposed for RbAp46 in the heterodimeric HAT1
complex. RbAp46 greatly stimulates the acetyltransferase
activity of the non-histone-binding HAT1 catalytic subunit,
presumably by tethering it to its substrate via its histone-binding
activity. Similarly,
although recombinant Rpd3 can deacetylate histone H4 in vitro,
free Rpd3 does not bind to H4 when the two are co-expressed
in vivo and is unlikely to be able to
deacetylate nucleosomal histones. This suggests that p55 may
play a similar essential role in the Esc complex by targeting
Rpd3 to histone substrates for deacetylation (Tie, 2001).
Unlike mammals, which have two very closely
related Rpd3 orthologs (HDAC1 and HDAC2), both of which
are associated with mouse EED, the Drosophila genome
contains no equally closely related homolog of Rpd3. The next
most closely related Drosophila HDAC is an unequivocal
ortholog of mammalian HDAC3, which
is a class I HDAC like Rpd3. Interestingly, mouse HDAC3 has
been reported to interact with the mouse Esc homolog EED
in a yeast two-hybrid assay,
consistent with the possibility that Rpd3 function in the Esc
complex might be at least partially redundant. Further genetic
analysis of Rpd3 should help to clarify its role in the Esc
complex (Tie, 2001).
The 600 kDa Esc complex is distinct from complexes
containing PC and other PcG proteins. This suggests that the
Esc complex and other PcG complexes are likely to have
separate functions. Furthermore, in embryos lacking any
functional Esc protein, some weak residual Pc-dependent
silencing activity is still detected, also
supporting separate, if interdependent, functions. Similar
conclusions have been drawn for the homologous mammalian
PcG complexes, which have been reported to be expressed in temporally distinct stages of B cell
differentiation, further suggesting they have distinct functions. In Drosophila, derepression of
homeotic genes is detected slightly earlier in Esc mutants than
in other PcG mutants, raising the possibility
that Esc complex function might be required earlier than other
PcG complexes. However, unlike the apparent temporal
separation of the homologous complexes during mammalian B
cell development, both Esc- and PC-containing complexes are
present together throughout most of embryogenesis, before
Esc disappears, and E(z), like other PcG proteins, is required
continuously throughout development. The phenotypic
similarities between Esc, E(z) and other PcG mutants, the
genetic interactions among them and their common association
with PREs, suggests that their functions, however distinct at
the biochemical level, are interdependent (Tie, 2001).
The association of mammalian EED with the two closely
related HDACs and two histone-binding proteins could reflect
the existence of two separate EED complexes or some different
functionality of the EED complex compared with the Esc
complex. Consistent with this latter possibility, EED has
recently been shown to be required after embryogenesis for
aspects of adult hematopoietic development. Interestingly, analysis of the
complete Drosophila genome sequence
using the BLASTP and TBLASTN algorithms reveals that p55 has no other
closely related Drosophila homologs, strongly suggesting that
it is the functional counterpart of both RbAp48 and RbAp46
in Drosophila. Likewise, Rpd3 is the only Drosophila
counterpart of mammalian HDAC1 and HDAC2. Given the
remarkably high degree of similarity between RbAp48 and
RbAp46 and HDAC1 and HDAC2, it is not yet clear whether
each of these proteins has a distinct or redundant role in the
EED complex. Perhaps this situation reflects a greater degree
of functional specialization or versatility within the
mammalian EED complexes. Since HDAC1 and HDAC2 have
also been found together with RbAp48 and RbAp46 in other
co-repressor complexes, it is also possible that the EED and
Esc complexes represent specialized relatives of these
complexes, perhaps more dedicated to a specific subset of
genes (Tie, 2001).
Recombinant E(z) has no MTase activity in vitro but acquires this activity when combined with p55 and Esc. Both are typical WD40 proteins, containing seven WD40 repeats and thought to mediate protein-protein interactions. The p55 protein has been found associated with the Retinoblastoma protein, is a constituent of Chromatin Assembly Factor CAF-1, and is thought to mediate the interactions of CAF-1 with histones. WD40 domain proteins are also found in other repressive chromatin-modifying complexes, e.g., yeast Tup-1, fly and mammalian Groucho, and mammalian TBL1, and have been shown to interact with histones and particularly with H3. Esc and p55 are therefore likely to mediate the interaction of E(z) with histone H3 while the E(z) SET domain is responsible for the catalytic activity (Czermin, 2002 and references therein).
Chromatographic fractionation of Drosophila embryonic nuclear extracts reveals the presence of at least five distinct MTase activities, which have been named HIMalpha, ß, gamma, delta, and epsilon according to their elution from a resource Q column. HIMß contains the well-known MTase Su(var)3-9, which plays an important role in establishment and maintenance of methylated histones at and around the centromeric region. HIMalpha copurifies with E(z) and Esc proteins. Gel filtration chromatography indicates that the HIMalpha has an apparent size of 600- 650 kDa, in good agreement with the reported size of a E(z)/Esc complex found in embryos. By introducing a MonoQ fractionation prior to the Superdex200 column, it was possible to purify HIMalpha to apparent homogeneity. Judging from a silver-stained gel, HIMalpha consists of six components. Western blots with corresponding antibodies identify five of these as p55, Esc, Rpd3, E(z), and SU(Z)12 in order of increasing molecular weight. The sixth protein has a molecular weight of approximately 168 kDa and is not yet unambiguously identified. These results show that HIMalpha is likely to correspond to the MTase activity immunoprecipitated by the anti-Esc antibody (Czermin, 2002).
Recombinant human E(z) lacks MTase activity on H3 peptide substrates. To test whether the same is true for the Drosophila ortholog, Drosophila E(z) was expressed as an intein fusion protein in bacteria and the purified, recombinant E(z) was used in MTase assays with various substrates. As expected, no significant MTase function could be detected using the bacterially produced polypeptide. Both HIMalpha activity and endogenous E(z) reside in a high molecular weight complex containing several protein components. Histone-modifying enzymes often require other polypeptides, in addition to the catalytic subunit, to recognize their substrate. Human HAT1, for example, requires p48, a WD40-motif histone binding protein. The Drosophila ortholog of p48, called p55, has also been shown to interact with E(z), and Esc, another subunit of the E(z) complex, shares with p55 the WD40 domain structure, which is needed for histone binding. To test whether E(z) might require additional components to gain histone MTase activity, recombinant E(z), immobilized on chitin beads, was used to reconstitute a complex with either bacterially produced recombinant Esc, p55 produced in a baculoviral system, or both. While none of the components has appreciable activity individually, E(z) plus p55 has some MTase activity and the ternary complex of E(z), p55, and Esc displays even stronger ability to methylate histone H3. In contrast, when an E(z) protein mutated in the SET domain is used, the ternary complex has no MTase activity. This experiment demonstrates that the E(z) protein is responsible for the methylation and strongly suggests that the SET domain harbors the MTase catalytic function (Czermin, 2002).
The Polycomb Group proteins are required for stable long-term maintenance of transcriptionally repressed states. Two distinct Polycomb Group complexes have been identified, a 2-MDa PRC1 complex and a 600-kDa complex containing the ESC and E(Z) proteins together with the histone deacetylase RPD3 and the histone-binding protein p55. There are at least two embryonic ESC/E(Z) complexes that undergo dynamic changes during development and a third larval E(Z) complex that forms after disappearance of ESC. A larger embryonic ESC complex has been identified containing RPD3 and p55, along with E(Z), that is present only until mid-embryogenesis, while the previously identified 600-kDa ESC/E(Z) complex persists until the end of embryogenesis. Constitutive overexpression of ESC does not promote abnormal persistence of the larger or smaller embryonic complexes and does not delay a dissociation of E(Z) from the smaller ESC complex or delay appearance of the larval E(Z) complex, indicating that these changes are developmentally programmed and not regulated by the temporal profile of ESC itself. Genetic removal of ESC prevents appearance of E(Z) in the smaller embryonic complex, but does not appear to affect formation of the large embryonic ESC complex or the PRC1 complex. The ESC complex is already bound to chromosomes in preblastoderm embryos and genetic evidence is presented that ESC is required during this very early period (Furuyama, 2003).
The assembly of newly synthesized DNA into chromatin is essential for normal growth, development, and differentiation. To gain a better understanding of the assembly of chromatin during DNA synthesis, the Caf1-180 and Caf1-105 subunits of Drosophila chromatin assembly factor 1 (dCAF-1) have been identified, cloned, and characterized. The purified recombinant p180+p105+p55 dCAF-1 complex is active for DNA replication-coupled chromatin assembly. Furthermore, the putative 75-kDa polypeptide of dCAF-1 is a C-terminally truncated form of p105 that does not coexist in dCAF-1 complexes containing the p105 subunit. The analysis of native and recombinant dCAF-1 revealed an interaction between dCAF-1 and the Drosophila anti-silencing function 1 (dASF1) component of replication-coupling assembly factor (RCAF). The binding of dASF1 to dCAF-1 is mediated through the p105 subunit of dCAF-1. Consistent with the interaction between dCAF-1 p105 and dASF1 in vitro, dASF1 and dCAF-1 p105 colocalize in vivo in Drosophila polytene chromosomes. This interaction between dCAF-1 and dASF1 may be a key component of the functional synergy observed between RCAF and dCAF-1 during the assembly of newly synthesized DNA into chromatin (Tyler, 2001).
The analysis of factors that are required in addition to CAF-1 for DNA replication-coupled chromatin assembly led to the identification of RCAF (Tyler, 1999). RCAF comprises the Drosophila homolog of the yeast anti-silencing function 1 protein (dASF1) and histones H3 and H4. The specific acetylation pattern of
H3 and H4 in RCAF is identical to that of newly synthesized histones that are assembled onto newly replicated DNA. RCAF functions synergistically with
CAF-1 in the assembly of chromatin in DNA replication-chromatin assembly reactions. The study of yeast strains that are lacking CAF-1 and/or RCAF further
suggested that CAF-1 and RCAF have both common and unique functions in the cell (Tyler, 1999). RCAF-mediated chromatin assembly appears to be essential for normal
progression through the cell cycle, gene expression, DNA replication, and DNA repair. Furthermore, it appears that the checkpoint kinase Rad53 may regulate
the chromatin assembly function of ASF1 during DNA replication and repair (Tyler, 2001 and references therein).
To analyze the biochemical properties of dCAF-1, the p180, p105, and
p55 proteins were synthesized in Sf9 cells by using baculovirus expression vectors. The p180 subunit contained a C-terminal FLAG epitope tag and was thus designated as
p180-FLAG. The p105 subunit contained a C-terminal His6 tag and was therefore termed p105-His6. Different combinations of dCAF-1 subunits were synthesized and purified by either anti-FLAG or Ni(II) affinity chromatography. When p180-FLAG, p105-His6, and p55 were cosynthesized and subjected to anti-FLAG immunoaffinity chromatography, the purified p180+p105+p55 dCAF-1 complex was obtained. Similarly, cosynthesis of p180-FLAG with either p105-His6 or p55 yielded p180+p105 and p180+p55 subcomplexes. Although the three-subunit p180+p105+p55 complex can be purified by Ni(II) affinity chromatography via p105-His6, cosynthesis of p105-His6 and p55 and subsequent Ni(II) affinity chromatography yielded only p105. Hence, these findings indicate that dCAF-1 p180 interacts with both p105 and p55, but that p105 and p55 do not interact with one another (Tyler, 2001).
To test whether the p180, p105, and p55 subunits are required for
chromatin assembly, DNA replication-chromatin assembly reactions were performed with partial and complete (i.e., p180+p105+p55) dCAF-1 complexes.
These experiments revealed that the purified recombinant p180+p105+p55 dCAF-1 complex possesses a specific activity for DNA replication-coupled chromatin
assembly that is comparable to that of native dCAF-1, as demonstrated by plasmid supercoiling analysis. It was further confirmed that dCAF-1-mediated
plasmid supercoiling is a consequence of chromatin assembly by using micrococcal nuclease digestion analysis. In addition, the two-subunit
p180+p105 subcomplex is fully active for chromatin assembly. In contrast, neither the p180 subunit alone nor the p105 subunit alone is sufficient for chromatin
assembly. These results thus indicate that the p180 and p105 subunits are each essential for DNA replication-coupled chromatin assembly by dCAF-1 (Tyler, 2001).
It is relevant that the DNA replication extract used in these experiments contains significant amounts of hCAF-1 p60 and hCAF-1 p48 (also known as RbAp48), which are homologous to dCAF-1 p105 and dCAF-1 p55, respectively. Based on the requirement of dCAF-1 p105 for chromatin assembly, it appears that
the hCAF-1 p60 subunit cannot function with the Drosophila CAF-1 polypeptides. However, the lack of a requirement for dCAF-1 p55 may be due to the
ability of the hCAF-1 p48 subunit, which is about 87% identical to dCAF-1 p55, to function with the dCAF-1 p180 and p105 subunits in lieu of dCAF-1 p55.
It is also possible, however, that the dCAF-1 p180+p105 subcomplex has the intrinsic ability to mediate chromatin assembly. It has not been possible to
immunodeplete the hCAF-1 p48 protein from the DNA replication extract to differentiate between these possibilities. It is noteworthy, however, that the
Arabidopsis equivalent of dCAF-1 p55 is required for DNA replication-coupled chromatin assembly with the same assay (Tyler, 2001).
The assembly of newly replicated DNA into chromatin requires both dCAF-1 and the RCAF chromatin assembly factor, which comprises Drosophila ASF1 (dASF1) and specifically acetylates Histone H3 and Histone H4. To investigate this effect further, coimmunoprecipitation analyses was performed with a crude Drosophila embryo extract. In these experiments, it was observed that immunoprecipitation with anti-dASF1 results in the coimmunoprecipitation of dCAF1 p180, p105, and p55, but not dCAF-1 p75. Conversely, immunoprecipitation with anti-p105 or with anti-p55 results in the coimmunoprecipitation of dASF1. Thus, these findings indicate that native dASF1 interacts with the native p180+p105+p55 form of dCAF-1 but not with the p75-containing form of dCAF-1. Immunoprecipitation of dCAF-1
with anti-p105+p75 does not result in the coimmunoprecipitation of dASF1, which suggests that the anti-p105+p75 antibodies destabilize the interaction between
dASF1 and the p180+p105+p55 form of dCAF-1 (Tyler, 2001).
To summarize, the p75 subunit of dCAF-1 appears to be a C-terminally truncated form of p105 and there are distinct forms of dCAF-1 that contain either the p105 subunit or the p75 subunit. The p105-containing form of dCAF-1 comprises the p180, p105, and p55 proteins. The purified recombinant p180+p105+p55 dCAF-1 complex is as active for DNA replication-coupled chromatin assembly as native dCAF-1. Both the p180 and p105 subunits are essential for chromatin assembly. A preexisting interaction between dCAF-1 and the dASF1 chromatin assembly factor has been discovered in crude extracts. This dCAF-1-ASF1 interaction occurs via the
dCAF-1 p105 subunit, and this interaction appears to be direct. dASF1 and dCAF-1 p105 colocalize in vivo in Drosophila polytene chromosomes. These results suggest that there is physical cooperation between dCAF-1 and dASF1 during chromatin assembly. The p105 and p75 subunits of dCAF-1 are closely related, and
dCAF-1 is not a single four-subunit complex but rather a three-subunit p180+p105+p55 complex and a presumed p180+p75+p55 complex. Thus, the basic
three-subunit structure of CAF-1 is conserved among yeast, Drosophila, and humans. The presence of multiple forms of dCAF-1 is of particular interest. Because
dCAF-1 was isolated from whole embryos instead of a specific cell line, there is potential for considerable diversity in the range of functions that may be performed by the different forms of dCAF-1. It is possible, for instance, that the p105-containing form of dCAF-1 functions in ASF1-dependent processes, whereas the
p75-containing form of dCAF-1 may function in ASF1-independent processes. Alternatively, the activity of dCAF-1 may be regulated during embryogenesis by
processing the p105 polypeptide into p75 (Tyler, 2001).
This physical interaction between dCAF-1 and dASF1 may be a key component of the functional synergy observed between RCAF and dCAF-1 during the
assembly of newly synthesized DNA into chromatin. The coupling of DNA synthesis and chromatin assembly appears to require a specific interaction between
CAF-1 and PCNA. The results presented in this work further extend this model to include the binding of ASF1 to CAF-1. It is possible, for instance,
that a complex of RCAF and CAF-1 is recruited to sites of DNA synthesis via the interaction of CAF-1 with PCNA. In the future, it will be interesting to study how
RCAF and CAF-1 mediate the formation of nucleosomes in conjunction with the other components of the chromatin assembly machinery (Tyler, 2001).
Drosophila TATA-box-binding protein (TBP)-related factor 2 (TRF2) is a member of a family of TBP-related factors present in metazoan organisms. Recent evidence suggests that TRF2s are required for proper embryonic development and differentiation. However, true target promoters and the mechanisms by which TRF2 operates to control transcription remain elusive. A Drosophila TRF2-containing complex has been purified by antibody affinity; this complex contains components of the nucleosome remodelling factor (NURF) chromatin remodelling complex as well as the DNA replication-related element (DRE)-binding factor DREF. This latter finding leads to potential target genes containing TRF2-responsive promoters. A combination of in vitro and in vivo assays has been used to show that the DREF-containing TRF2 complex directs core promoter recognition of the proliferating cell nuclear antigen (PCNA) gene. Additional TRF2-responsive target genes involved in DNA replication and cell proliferation have also been identified. These data suggest that TRF2 functions as a core promoter-selectivity factor responsible for coordinating transcription of a subset of genes in Drosophila (Hochheimer, 2002).
In the absence of a functional assay allowing conventional purification of TRF2-associated factors a panel of monoclonal antibodies was generated directed against several domains of TRF2 to affinity purify TRF2 and its putative associated subunits. Approximately 3,500 hybridomas were screened and a clone was isolated that efficiently immunoprecipitated TRF2 and its associated factors from Drosophila embryo nuclear extract. A Sarkosyl-eluted complex containing TRF2 was analysed by SDS-polyacrylamide gradient gel electrophoresis (PAGE) that revealed a set of 18 polypeptides with relative molecular mass (Mr) ranging from 300K to 29K that co-immunoprecipitated consistently with TRF2 even under very stringent conditions (Hochheimer, 2002).
An 80K protein associated with TRF2 is identical to DREF1. DREF and its corresponding response element DRE have been well documented to be important for the regulation of cell-cycle and cell-proliferation genes in Drosophila (that is, genes for PCNA and the 180K and 73K subunits of DNA polymerase). The identification of the promoter-selective DNA-binding protein DREF was intriguing because Drosophila TRF2 thus far had failed to bind to canonical TATA-box elements, which suggests that TRF2 may cooperate with DREF to execute promoter specificity and perhaps operate like a metazoan sigma factor (Hochheimer, 2002).
The 140K protein associated with TRF2 is identical to Drosophila ISWI, which is the catalytic ATPase subunit of NURF, ACF and CHRAC chromatin remodelling complexes. Moreover, the 55K and 38K proteins associated with TRF2 turned out to be NURF-55/CAF-1 and NURF-38/inorganic pyrophosphatase, respectively. Notably, the peptide sequences obtained from the three largest (300K, 250K and 230K) proteins associated with TRF2 do not match NURF-301, suggesting that the presence of some NURF subunits is not merely a result of contaminating NURF in the TRF2 complex. However, analysis of the cDNAs encoding the 190K and 160K proteins associated with TRF2 revealed that both proteins contain conserved sequence motifs for 11 and 5 zinc finger motifs (C2H2), respectively; these smaller proteins thus resemble factors like the CCCTC-binding factor CTCF that has been implicated in mediating chromatin-dependent processes such as the regulation of insulator function. It is therefore possible that the TRF2 complex encompasses both promoter-selectivity functions and NURF-like components as well as other activities with distinct subunits and specificity (Hochheimer, 2002).
The retinoblastoma tumor suppressor protein (pRb) regulates gene transcription by binding E2F transcription factors. pRb can recruit several repressor complexes to E2F bound promoters; however, native pRb repressor complexes have not been isolated. E2F/RBF repressor complexes have been isolated from Drosophila embryo extracts and their roles in E2F regulation have been characterized. These complexes contain RBF, E2F, and Myb-interacting proteins that have previously been shown to control developmentally regulated patterns of DNA replication in follicle cells. The complexes localize to transcriptionally silent sites on polytene chromosomes and mediate stable repression of a specific set of E2F targets that have sex- and differentiation-specific expression patterns. Strikingly, seven of eight complex subunits are structurally and functionally related to C. elegans synMuv class B genes, which cooperate to control vulval differentiation in the worm. These results reveal an extensive evolutionary conservation of specific pRb repressor complexes that physically combine subunits with established roles in the regulation of transcription, DNA replication, and chromatin structure (Korenjak, 2004).
Identification of copurifying polypeptides revealed Twilight (also known as Mip130; from here on referred to as Mip130/TWIT), RBF1, RBF2, dMyb, dDP, dE2F2, CAF1p55, and Mip40. The identity of these polypeptides was confirmed by Western blot. Intriguingly, Mip130/TWIT, dMyb, CAF1p55, and Mip40 have recently been identified as components of a dMyb complex that regulates chorion gene amplification in follicle cells. The fifth subunit of the dMyb complex, Mip120, was apparently absent from the final preparation as judged by silver staining. However, Western analysis with Mip120-specific antibody has demonstrated that Mip120 coelutes with other complex subunits throughout the fractionation. The Mip120 signal became progressively weaker during purification but was still detectable in fractions eluting from the final gel filtration column, suggesting that Mip120 might have been progressively lost or degraded. Indeed, several results presented below suggest that Mip120 is a bona fide complex subunit. Since these complexes are a composite of known transcriptional regulators, they go by the acronym dREAM (Drosophila RBF, E2F, and Myb-interacting proteins) (Korenjak, 2004).
The mechanism of E2F regulation provided by dREAM appears to be highly conserved during evolution. Strikingly, with the exception of dMyb, all components of dREAM are either homologs of previously described C. elegans synMuv class B genes (mip130/twit/lin-9, rbf1 and rbf2/lin-35, de2f2/efl-1, ddp/dpl-1, and caf1p55/lin-53), contain regions of sequence conservation (Mip40/lin-37), or produce a synMuv phenotype when the corresponding C. elegans gene is inactivated (Mip120/JC8.6). Genetic studies have shown that synMuv class B genes are required for development of the worm's male and female reproductive systems, and it has been suggested that some encode subunits of a hypothetical complex that represses vulva-specific gene transcription; however, the precise transcriptional changes underlying the synMuv phenotype are unknown (Ceol, 2001). The discovery of dREAM complexes suggests an intriguing model for synMuv class B gene function: it is proposed that at least seven synMuv class B gene products physically associate to form a complex that, like its Drosophila counterpart, represses sex-related targets and that misexpression of these genes causes a change in cell fate. Given the vast differences between C. elegans and Drosophila embryogenesis, it is considered unlikely that REAM complexes will regulate the exact same set of genes in both species. However, it is proposed that, in both organisms, REAM complexes control transcriptional programs required for development of the reproductive system. In agreement with this model, dE2F2 has been shown to be needed to repress genes like vasa and spn-E that are important for Drosophila gametogenesis (Dimova, 2003) and that dE2F2 mutants have both male and female fertility defects (Korenjak, 2004).
Do mammalian cells contain similar complexes? Mammalian homologs exist for all dREAM subunits. Intriguingly, B-Myb associates with the N terminus of p107. RbAp48/p46, human orthologs of CAF1p55, were first isolated through their ability to bind a pRb-affinity column but are now known as components of several chromatin-associated complexes, including a putative pRb-histone deacetylase and the NuRD complex. Human homologs of Mip130/TWIT, Mip120, and Mip40 had not previously been linked to pRb. All three interact with pRb in vitro. Furthermore, endogenous hMip130/TWIT associates specifically with pRb, p107, and p130 fusion proteins. In agreement with these results, interaction has been demonstrated between pRb and Mip130/TWIT in human cells in vivo (S. Gaubatz, personal communication to Korenjak, 2004). Clearly, further studies are needed to define the properties and biological roles of pRb/hMip complexes. Nevertheless, these preliminary findings suggest that such complexes may well exist in mammalian cells, and, if studies in C. elegans and Drosophila are a guide, it might be expected that they function in developmentally regulated aspects of E2F/pRB function (Korenjak, 2004).
The ESC-E(Z) complex of Drosophila Polycomb group (PcG) repressors is a histone H3 methyltransferase (HMTase). This complex silences fly Hox genes, and related HMTases control germ line development in worms, flowering in plants, and X inactivation in mammals. The fly complex contains a catalytic SET domain subunit, E(Z), plus three noncatalytic subunits, SU(Z)12, ESC, and NURF-55/CAF-1. The four-subunit complex is >1,000-fold more active than E(Z) alone. ESC and SU(Z)12 play key roles in potentiating E(Z) HMTase activity. Loss of ESC disrupts global methylation of histone H3-lysine 27 in fly embryos. Subunit mutations identify domains required for catalytic activity and/or binding to specific partners. Missense mutations are described in surface loops of ESC, in the CXC domain of E(Z), and in the conserved VEFS domain of SU(Z)12, which each disrupt HMTase activity but preserve complex assembly. Thus, the E(Z) SET domain requires multiple partner inputs to produce active HMTase. A recombinant worm complex containing the E(Z) homolog, MES-2, has robust HMTase activity, which depends upon both MES-6, an ESC homolog, and MES-3, a pioneer protein. Thus, although the fly and mammalian PcG complexes absolutely require SU(Z)12, the worm complex generates HMTase activity from a distinct partner set (Ketel, 2005).
In vitro and in vivo data indicate that the noncatalytic ESC subunit makes a critical contribution to HMTase function of the ESC-E(Z) complex. In particular, since global levels of H3-K27 methylation are similarly reduced by genetic loss of ESC or E(Z), ESC appears to be an obligate functional partner for E(Z) HMTase activity (Ketel, 2005).
Two main molecular explanations are envisioned for the ESC requirement. (1) ESC could potentiate HMTase activity through direct interaction with E(Z). ESC binding could trigger a conformational change in E(Z) that improves catalytic efficiency, and/or ESC residues could directly interact with and influence the E(Z) active site. (2) Alternatively, the main role of ESC could be to bind nucleosomes. In this scenario, ESC would boost HMTase activity by facilitating interaction of the enzyme complex with its substrate. Based on several lines of evidence, a mechanism is favored that works through direct ESC-E(Z) contact. (1) The ESC M236K and V289M mutations, which significantly reduce HMTase activity, are located in surface loops previously shown to mediate direct ESC contact with E(Z). Furthermore, M236K displays dominant-negative properties in vivo. This genetic behavior is consistent with an enzyme complex that assembles normally but is compromised in catalytic function. (2) A recent report documents that ESC lacks nucleosome-binding activity on its own and that addition of ESC to a trimeric NURF-55/SU(Z)12/E(Z) complex has little additive effect on ability to bind nucleosomes. (3) ESC potentiation through direct E(Z) binding is supported by evolutionary considerations. Every organism examined that has an E(Z) homolog, ranging from plants to worms, flies, and humans, has at least one ESC homolog. In addition, 28 residues within the ESC surface loops implicated in E(Z) binding are identical from flies to humans. This conservation may reflect a tight functional requirement wherein direct ESC-E(Z) partnership, combining to produce HMTase activity, is maintained by evolutionary pressure. Future studies will be needed to define the precise biochemical mechanism by which ESC potentiates HMTase activity, including tests for binding-induced conformational changes in E(Z) (Ketel, 2005).
Studies on the mammalian homolog of ESC, called EED, have also highlighted its important role as a regulatory subunit. In human cells, multiple EED isoforms are expressed, which differ in the extents of their N-terminal tails. These isoforms are generated by alternative start codon usage of the same EED mRNA. Intriguingly, incorporation of particular EED isoforms into EZH2 complexes can shift the enzyme specificity so that K26 of histone H1 is methylated in addition to H3-K27. Thus, it appears that the ESC/EED subunit can influence both catalytic efficiency and lysine substrate preference. In the fly system, ESC isoforms produced from the same mRNA have not been detected. Instead, alternative ESC isoforms could be supplied by an esc-related gene (CG5202) located about 150 kb proximal to esc. Since mutations in this second esc gene, called esc-like (escl), have not yet been reported, its in vivo contributions remain to be assessed. However, since genetic loss of ESC alone dramatically reduces global methylation of H3-K27 in fly embryos, it is concluded that ESC is the predominant functional E(Z) partner during embryonic stages (Ketel, 2005).
Studies on recombinant complexes show that fly SU(Z)12 is absolutely required for HMTase activity of the ESC-E(Z) complex. A key requirement for SU(Z)12 in mammalian EZH2 complexes has also been established based upon in vitro tests and loss-of-function studies in vivo. How does SU(Z)12 contribute molecularly to HMTase activity? Again, two main possibilities are envisioned: influence through direct contact with E(Z) or by mediating nucleosome binding. To address this, it is instructive to consider the SU(Z)12 mutants affecting the conserved VEFS domain. Deletion of the entire VEFS domain eliminates assembly of the fly complex by disrupting SU(Z)12-E(Z) binding. Pairwise binding assays with mammalian SU(Z)12 have similarly shown that the VEFS domain is needed for binding to EZH2 in vitro. Thus, a conserved function of this domain is to contact E(Z). However, missense mutations within the VEFS domain, D546A and E550A, preserve full complex assembly yet have reduced levels of HMTase. Taken together, these results implicate the VEFS domain in both binding to E(Z) and potentiating its enzyme activity, which suggests a connection between these two functions. In contrast, a recent report provides evidence that SU(Z)12 contributes to affinity for nucleosomes. Although SU(Z)12 cannot bind to nucleosomes by itself, the SU(Z)12/NURF-55 dimer has nucleosome-binding properties that are similar to those of the four-subunit complex. Thus, as also suggested for the human PRC2 complex, at least one role of SU(Z)12 is to mediate nucleosome binding. Further work will be needed to define the SU(Z)12 functional domains required for interactions with NURF-55 and with nucleosomes. Based on the available data, SU(Z)12 potentiation of E(Z) HMTase activity may involve both direct E(Z) contact and facilitated binding to nucleosome substrate (Ketel, 2005).
Although the SET domain is the most well-characterized functional domain of E(Z), the adjacent cysteine-rich CXC domain is also remarkably conserved from flies to humans. To address CXC function, in vitro properties of two missense mutants, C545Y and C603Y were analyzed. Both mutations correspond to E(z) loss-of-function alleles; in particular, in vivo effects of the E(z)61 mutation (C603Y) have been well documented. This mutation disrupts global H3-K27 methylation in embryos and causes loss of methyl-H3-K27 from a Hox target gene in imaginal discs. Mutant complexes bearing E(Z)-C603Y can assemble normally but show an approximately 10-fold reduction in HMTase levels. C545Y causes a more modest HMTase reduction, which parallels results obtained with the analogous substitution (C588Y) in human EZH2. These results suggest that the CXC domain interfaces with the SET domain to produce robust HMTase activity. In this regard, the CXC domain could be considered similar to cysteine-rich "preSET" domains required for robust HMTase activity in other SET domain proteins. Another effect of these CXC mutations in vivo is that they dislodge E(Z) from target sites in chromatin. Although the molecular basis for this dissociation is not known, in vitro assembly results suggest that it is not due to wholesale destabilization of the ESC-E(Z) complex. The dissociation may reflect another proposed role for the CXC domain, which is to interact with the PcG targeting factor PHO (Ketel, 2005).
In vitro tests were performed to investigate the role of E(Z) domain II. Both the complete domain deletion and the C363Y missense mutation show that domain II is required for stable association of E(Z) with SU(Z)12. Thus, the composite domain organization of E(Z) reflects division of labor among catalytic functions and requirements for complex assembly. In addition, it appears that none of the E(Z) domains are specifically built for nucleosome interactions; E(Z) plays little or no role itself in stable binding of the complex to nucleosomes (Ketel, 2005).
The NURF-55 subunit is distinct from the other three subunits in several ways. (1) It makes only minimal contributions to in vitro HMTase activity in both the fly and mammalian complexes. (2) Whereas the other three subunits appear dedicated to PcG function, NURF-55 is present in diverse chromatin-modifying complexes, including NURF, chromatin assembly factor 1 (CAF-1), and histone deacetylase complexes. The ability of the mammalian NURF-55 homologs RbAp46 and RbAp48 to bind to free histone H4 has led to the suggestion that NURF-55 may help chromatin complexes interact with substrate. Indeed, the absence of a NURF-55-related protein from the trimeric worm MES complex could help explain its inability to methylate free histones. The free histone-binding property of NURF-55 has also prompted the intriguing suggestion that silencing by the ESC-E(Z) complex in vivo could involve methylation of histones prior to nucleosome assembly. Since NURF-55 loss-of-function alleles have not been described in flies, many questions about roles of NURF-55 remain to be addressed. Even with alleles available, the multiplicity of NURF-55-containing complexes will likely complicate in vivo dissection of its PcG functions (Ketel, 2005).
The basic enzymatic function of the ESC-E(Z) complex, to methylate H3-K27, is shared between the worm, fly, and mammalian versions. Another similarity revealed from this study of recombinant worm complexes is that robust HMTase activity depends critically upon the two noncatalytic subunits. Although MES-6 and MES-3 can each individually bind to the catalytic subunit, MES-2, all three subunits are required together to produce enzyme activity. Since worm MES-6 is a WD repeat protein related to fly ESC, it seems likely that MES-6 and ESC potentiate HMTase activity through similar mechanisms. It is suggested that this mechanism entails direct subunit interactions rather than an influence upon affinity for nucleosomes. However, a major puzzle is presented by the dissimilarity between worm MES-3 and fly SU(Z)12. Though each is required for HMTase activity in their respective complexes, no relatedness was recognized between these two proteins in primary sequence or predicted secondary structure arrangement. From an evolutionary standpoint, it appears that SU(Z)12 represents the more ancient partner, since it is functionally conserved across plant and animal kingdoms. MES-3, a novel protein, may have evolved more recently to replace SU(Z)12 in the worm complex. Since a molecular role attributed to SU(Z)12 in the fly complex is nucleosome binding, it is speculated that MES-3 may supply this function for the worm complex. There are many strategies for building nucleosome contact domains, as represented among divergent chromatin proteins, so MES-3 could have acquired functional similarity without overt sequence similarity to SU(Z)12. In this view, MES-3 function in the worm complex would require, at minimum, affinity for nucleosomes and ability to bind MES-2. In this regard, it is interesting that MES-2 appears to lack domain II, which is needed in E(Z) for stable binding to SU(Z)12. Presumably, MES-2 has instead acquired a site for stable MES-3 interaction. In summary, it is suggested that the E(Z)/ESC and MES-2/MES-6 dimers have been conserved as core subunits of the HMTase complex, whereas the additional required partners in each complex, SU(Z)12 and MES-3, have been allowed to diverge. Future studies will be needed, including functional tests of chimeric worm and fly proteins, to address such a model (Ketel, 2005).
The Drosophila Polycomb group protein E(z) is a histone methyltransferase (HMTase) that is essential for maintaining HOX gene silencing during development. E(z) exists in a multiprotein complex called Polycomb repressive complex 2 (PRC2) that also contains Su(z)12, Esc and Nurf55 (Caf1). Reconstituted recombinant PRC2 methylates nucleosomes in vitro, but recombinant E(z) on its own shows only poor HMTase activity on nucleosomes. This study investigated the function of the PRC2 subunits. It was shown that PRC2 binds to nucleosomes in vitro but that individual PRC2 subunits alone do not bind to nucleosomes. By analysing PRC2 subcomplexes, it was shown that Su(z)12-Nurf55 is the minimal nucleosome-binding module of PRC2 and that Esc contributes to high-affinity binding of PRC2 nucleosomes. Nucleosome binding of PRC2 is not sufficient for histone methylation and only complexes that contain Esc protein show robust HMTase activity. These observations suggest that different subunits provide mechanistically distinct functions within the PRC2 HMTase: the nucleosome-binding subunits Su(z)12 and Nurf55 anchor the E(z) enzyme on chromatin substrates, whereas Esc is needed to boost enzymatic activity (Nekrasov, 2005).
Baculovirus expression vectors have been used to reconstitute and purify recombinant tetrameric PRC2 from Sf9 cells. To identify intermolecular interactions within this complex, tests were performed for reconstitution of PRC2 subcomplexes on coexpression of two or more subunits in Sf9 cells. In each case, the Flag-epitope tag present on one of the subunits was used for affinity purification from Sf9 cell extracts. The following stable dimeric complexes could be purfied: Esc-E(z), E(z)-Su(z)12, E(z)-Nurf55 and Su(z)12-Nurf55. In contrast, purification either from cells expressing Flag-Esc and Nurf55 or from cells expressing Flag-Esc and Su(z)12 resulted in the isolation of Flag-Esc protein only, suggesting that these proteins do not bind directly to each other. Purification from Sf9 cells expressing three subunits allowed the isolation of trimeric Esc-E(z)-Su(z)12 and E(z)-Su(z)12-Nurf55 complexes. Finally, tetrameric PRC2 was isolated, using either Flag-E(z) or Flag-Su(z)12 for affinity purification. Importantly, each of these complexes was stable in buffers containing up to 1 M KCl. Taken together, these data suggest that E(z) binds tightly to Su(z)12, Esc and Nurf55 and that Su(z)12 also binds to Nurf55. The failure to isolate dimeric complexes that contain Esc and Su(z)12 or those that contain Esc and Nurf55 indicates that E(z), Su(z)12 and Nurf55 form a trimeric core complex to which Esc binds through interaction with E(z). The observation that E(z) forms stable dimeric complexes with either Esc or Nurf55 in this reconstitution assay is consistent with earlier studies that reported physical interactions between these proteins in glutathione-S-transferase (GST) pull-down assays (Nekrasov, 2005).
It is noted that the molecular architecture of mammalian PRC2 is unclear at present; conflicting data on intermolecular interactions between subunits have been reported. Specifically, it has been reported that human EZH2, SU(Z)12 and RbAp48 all bind to EED, the Esc homologue, and that EZH2 does not interact with SU(Z)12 or RbAp48 in GST pull-down assays; it has been proposed that EED is the core component of the complex and EZH2 associates with other components through EED. It has also been reported that EZH2 binds to SUZ12 in GST pull-down assays, consistent with the finding that Drosophila E(z) and Su(z)12 form a stable complex (Nekrasov, 2005).
The HMTase activity of recombinant E(z) protein is significantly lower than the activity observed with recombinant tetrameric PRC2. A simple mechanistic explanation would be that one or several PRC2 subunits are needed for nucleosome binding to facilitate interaction of the E(z) HMTase with its substrate, the histone H3 tail. Since it is not known whether any of the PRC2 subunits binds to nucleosomes, tests were performed to see whether complex components alone or in combination could form stable complexes with mononucleosomes, in a bandshift assay. To this end, mononucleosomes were reconstituted with recombinant core histones that were expressed in E. coli and a 201 base pairs (bp) long radioactively labelled DNA template that contained a strong nucleosome-binding sequence called '601'. When recombinant tetrameric PRC2 was incubated with such mononucleosomes and the reaction mixture was resolved on a polyacrylamide gel, distinct, slowly migrating complexes were observed that appeared in a concentration-dependent manner. In contrast, when PRC2 was incubated with naked 601 DNA template, it was not possible to resolve specific protein-DNA complexes. Together, these observations suggest that PRC2 binds to mononucleosomes and that these protein-nucleosome complexes remain stably associated under electrophoretic conditions. Individual PRC2 subunits were tested for nucleosome binding, but no formation of protein-nucleosome complexes was detected with any of the four proteins. This suggests that more than one subunit is needed for nucleosome binding and therefore the different di- and trimeric PRC2 subcomplexes were tested. Among the different subcomplexes, only incubation with the Su(z)12-Nurf55 or E(z)-Su(z)12-Nurf55 complexes results in the appearance of distinct, slowly migrating protein-nucleosome complexes. These protein-nucleosome complexes migrate similarly to the complexes observed with tetrameric PRC2, but two- to threefold higher concentrations of the Su(z)12-Nurf55 or E(z)-Su(z)12-Nurf55 complex are needed to shift all of the nucleosome probe. Thus, the presence of Esc in PRC2 increases the affinity of the complex for nucleosomes or allows the complex to bind more stably under the experimental conditions compared with PRC2 subcomplexes that lack Esc. In contrast to the distinct protein-nucleosome complexes observed with the Su(z)12-Nurf55 or E(z)-Su(z)12-Nurf55 complex, no specific protein-nucleosome complexes are formed if nucleosomes are incubated with the E(z)-Su(z)12, E(z)-Nurf55, Esc-E(z) or Esc-E(z)-Su(z)12 complexes. However, incubation with high concentrations of the trimeric Esc-E(z)-Su(z)12 complex also shifts almost all of the nucleosome probe, and much of the probe is retained in the well of the gel. Thus, it seems that the Esc-E(z)-Su(z)12 complex also binds to nucleosomes but that it binds in a manner distinct from the other PRC2 (sub)complexes. Taken together, these binding assays suggest that several subunits need to cooperate for nucleosome binding of PRC2, as follows: (1) Su(z)12 is essential for nucleosome binding because only complexes containing Su(z)12 bind; (2) the minimal nucleosome-binding complex contains Su(z)12 and Nurf55, and only complexes that contain these two proteins give rise to distinct, slowly migrating protein-nucleosome complexes; Su(z)12 and Nurf55 together thus form the minimal nucleosome-binding module of PRC2; (3) as discussed above, Esc also contributes to nucleosome binding because (a.) tetrameric PRC2 binds more strongly than the E(z)-Su(z)12-Nurf55 complex and (b.) in the absence of Nurf55, that is, in the Esc-E(z)-Su(z)12 complex, Esc seems to cooperate with Su(z)12 to cause retention of the nucleosome probe. E(z) is thus the only subunit for which no contribution to nucleosome binding was detected. Note that comparable concentrations of the Su(z)12-Nurf55 or E(z)-Su(z)12-Nurf55 complex are required to shift 50% of the nucleosome probe (Nekrasov, 2005).
Nurf55 and Esc are both WD40 repeat proteins. RbAp46 and RbAp48, the mammalian homologues of Nurf55, have been reported to bind directly to helix 1 of histone H4, a portion of H4 that is thought to be inaccessible within the nucleosome, and, consistent with this, RbAp46 and RbAp48 are unable to bind to H4 in nucleosomal templates. As shown in this study, Drosophila Nurf55 or Esc alone are not able to bind to mononucleosomes but they bind in combination with Su(z)12, which, by itself, also does not bind to nucleosomes. It is possible that the combination of Su(z)12 and Nurf55 or Esc is needed to create the necessary surface for stable nucleosome binding. Alternatively, it could be that these proteins act in a cooperative manner to disrupt histone-DNA contacts locally to expose the histone core (i.e. H4) for binding by Nurf55 or Esc (Nekrasov, 2005).
These results suggest that Su(z)12 together with Nurf55 or Esc tethers the complex to nucleosomes, whereas E(z), the catalytic subunit of the complex, contributes little to nucleosome binding. Next the HMTase activity of the different PRC2 (sub)complexes were analyzed. As substrates for these reactions, non-radiolabelled mononucleosomes were used identical to those used in the bandshift assay. Recombinant tetrameric PRC2 methylates H3 in mononucleosomes. In contrast, E(z) protein alone, the dimeric Esc-E(z), and the Su(z)12-E(z) or E(z)-Nurf55 complexes do not detectably methylate mononucleosomes. Strikingly, the Su(z)12-E(z)-Nurf55 complex also shows no detectable HMTase activity, whereas the Esc-E(z)-Su(z)12 complex methylates H3 in mononucleosomes with efficacy similar to tetrameric PRC2. Thus, no straightforward correlation is observed between nucleosome binding in bandshift assays and HMTase activity. In particular, the Su(z)12-E(z)-Nurf55 complex seems to bind to nucleosomes with only two- to threefold lower affinity than tetrameric PRC2, but shows markedly reduced HMTase activity. In contrast, the Esc-E(z)-Su(z)12 complex, which shows almost no nucleosome binding at the concentration used in the HMTase assay, shows HMTase activity comparable with PRC2. Together, these data suggest that nucleosome binding is not sufficient for HMTase activity and that Esc has a crucial role in boosting the enzymatic activity of E(z). It is possible that Esc is required to dock the complex in a specific orientation on the nucleosome that presents the H3 tail in a particularly favourable position to the E(z) enzyme. Alternatively, Esc could directly increase the catalytic activity of E(z) by inducing a conformational change in the enzyme. However, it is important to note that the Esc-E(z) complex shows no detectable HMTase activity on mononucleosomes and the presence of Su(z)12 is thus essential for HMTase activity of the Esc-E(z)-Su(z)12 complex. The bandshift data show that Su(z)12 is strictly needed for nucleosome binding of PRC2. At the low complex concentrations used in the HMTase assay, there is probably very little nucleosome binding of the Esc-E(z)-Su(z)12 complex; nevertheless, it seems probable that the nucleosome interactions that Su(z)12 shows in cooperation with Esc contribute to the HMTase activity of the Esc-E(z)-Su(z)12 complex. Finally, it is puzzling that the absence of Nurf55 from the complex (i.e. in the Esc-E(z)-Su(z)12 complex) does not seem to diminish HMTase activity because Nurf55 is important for the formation of stable PRC2-nucleosome complexes in bandshift assays. It is possible that Nurf55 is not needed for HMTase activity under the assay conditions that were used but that it is important for methylation of chromatin in vivo (Nekrasov, 2005).
Recent studies have shown that SU(Z)12 is crucial for HMTase activity of mammalian PRC2 in vitro, but the molecular basis for this requirement has remained unclear. This study reports two main findings: (1) Su(z)12 has a crucial role in nucleosome binding of Drosophila PRC2; (2) PRC2 subcomplexes that bind to nucleosomes but lack Esc are poorly active; Esc thus has an important role in boosting HMTase activity of the complex (Nekrasov, 2005).
The requirement of Su(z)12 for HMTase activity can for the most part be explained by its ability to tether PRC2 to nucleosomes, and the data suggest that nucleosome binding requires Su(z)12 in combination with either Nurf55 or Esc. Although both Nurf55 and Esc contribute to nucleosome binding of the complex, only inclusion of Esc leads to an increase in HMTase activity. The contribution of Esc to HMTase activity thus goes beyond the activity that one would expect if Esc were required only for nucleosome binding. In summary, these data suggest that the Su(z)12, Nurf55 and Esc subunits all contribute to nucleosome binding of PRC2 but that these three subunits make distinct contributions to the activation of the E(z) HMTase (Nekrasov, 2005).
The findings reported here imply that E(z) HMTase activity in vivo could be regulated at the level of chromatin binding and/or enzyme activity by modulating the abundance or activity of different PRC2 subunits. It is important to discuss the results reported here in the context of the in vivo requirement for different PRC2 subunits. Genetic studies have shown that Su(z)12 and E(z) are required throughout development to maintain silencing of HOX genes in Drosophila and that this process requires the enzymatic activity of E(z). In contrast, Esc protein is required early in development and then becomes to a large extent, although not completely, dispensable for maintenance of HOX gene silencing during postembryonic development. There are two possible explanations for the paradoxical observation that Esc is required for strong HMTase activity in vitro but that the protein seems to be largely dispensable for the HMTase activity of E(z) that is needed to maintain HOX gene silencing during larval development. (1) It is possible that strong HMTase activity of PRC2 is required primarily early in embryogenesis and that, once it is established, H3-K27 methylation can be maintained with a catalytically less active form of the complex (i.e., lacking Esc). (2) It is possible that another protein substitutes for Esc at later developmental stages. It is noted that the Drosophila genome encodes a second Esc-like protein (CG5202) but, at present, it is not known whether this protein is required for HOX gene silencing (Nekrasov, 2005).
Heterochromatin protein 2 (HP2) is a nonhistone chromosomal protein from Drosophila melanogaster that binds to heterochromatin protein 1 (HP1) and has been implicated in heterochromatin-induced gene silencing. Heretofore, HP1 has been the only known binding partner of HP2, a large protein devoid of sequence motifs other than a pair of AT hooks. In an effort to identify proteins that interact with HP2 and assign functions to its various domains, nuclear proteins were fractionated under nondenaturing conditions. On separation of nuclear proteins, nucleosome assembly protein 1 (Nap-1) has an overlapping elution profile with HP2 (assayed by Western blot) and has been identified by mass spectrometry in fractions with HP2. Upon probing fractions in which HP2 and Nap-1 are both present, this study found that the nucleosome remodeling factor (NURF), an ISWI-dependent chromatin remodeling complex, is also present. Results from coimmunoprecipitation experiments suggest that HP2 interacts with Nap-1 as well as with NURF; NURF appears to interact directly with both HP2 and Nap-1. Three distinct domains within HP2 mediate the interaction with NURF, allowing assignment of NURF binding domains in addition to the AT hooks and HP1 binding domains already mapped in HP2. Mutations in Nap-1 are shown to suppress position effect variegation, suggesting that Nap-1 functions to help to assemble chromatin into a closed form, as does HP2. On the basis of these interactions, it is speculated that HP2 may cooperate with these factors in the remodeling of chromatin for silencing (Stephens, 2005)
Every eukaryotic chromosome requires a centromere for attachment to spindle microtubules for chromosome segregation. Although centromeric DNA sequences vary greatly among species, centromeres are universally marked by the presence of a centromeric histone variant, centromeric histone 3 (CenH3/CID), which replaces canonical histone H3 in centromeric nucleosomes. Conventional chromatin is maintained in part by histone chaperone complexes, which deposit the S phase-limited (H3) and constitutive (H3.3) forms of histone 3. However, the mechanism that deposits CenH3 specifically at centromeres and faithfully maintains its chromosome location through mitosis and meiosis is unknown. To address this problem, a soluble assembly complex has been biochemically purified that targets tagged CenH3 to centromeres in Drosophila cells. Two different affinity procedures led to purification of the same complex, which consists of CenH3, histone H4, and a single protein chaperone, RbAp48, a highly abundant component of various chromatin assembly, remodeling, and modification complexes. The corresponding CenH3 assembly complex reconstituted in vitro is sufficient for chromatin assembly activity, without requiring additional components. The simple CenH3 assembly complex is in contrast to the multisubunit complexes previously described for H3 and H3.3, suggesting that centromeres are maintained by a passive mechanism that involves exclusion of the complexes that deposit canonical H3s during replication and transcription (Furuyama, 2006a; full text of article).
RbAp48 is sufficient for centromeric chromatin assembly in vitro, but is it necessary for this process in vivo? RbAp48 is found in various chromatin-associated protein complexes, where it is thought to play a common role in mediating their interactions with histones. Although no mutations have been reported to eliminate Drosophila RbAp48 (NURFp55), mutations in other components of RbAp48-associated complexes are lethal [Nurf-38, E(z), sin3, and many others]; therefore, it would be expected that removal of RbAp48 would have pleiotropic effects. Indeed, knock-down of RbAp48 by RNAi in Drosophila S2 cells results in S phase arrest and derepression of various Rb/E2F target genes. These pleiotropic effects caused by reduction in RbAp48 levels would mask any centromere defect, and, in any case, such a defect would not be expected to occur immediately, because disruption of fission yeast RbAp48 did not affect chromosome segregation until the second round of mitosis (Furuyama, 2006a).
The single chaperone purified by using tagged CID contrasts with the multiple subunits found in purified chaperone complexes using tagged H3.1 and H3.3. The H3.1-specific replication-coupled assembly complex contains more than seven nonhistone subunits, and the H3.3-specific replication-independent complex contains at least five. Furthermore, H3.1- and H3.3-specific assembly reactions were performed in the presence of crude lysates, suggesting requirements for additional components that might restrict deposition to polymerase-driven processes. In contrast, both purified and reconstituted CID/H4-RbAp48 are sufficient for chromatin assembly in the absence of any other processes (Furuyama, 2006a).
The formation of chromatin from histones and DNA is a thermodynamically favorable reaction, and it is thought that histone chaperones are needed to prevent nonproductive aggregation between highly positively charged histones and highly negatively charged DNA in a dense protein environment. Both replication-coupled assembly of H3.1/H4 and transcription-coupled assembly of H3.3/H4 take place in the highly dynamic context of multisubunit polymerase transit, and assembly in both cases might require a large number of subunits to facilitate tethering of assembly complexes for rapid histone deposition. However, the basic assembly reaction appears to have minimal requirements, and conventional nucleosomes can be assembled in the presence of the NAP1 protein chaperone, polyglutamate, or high concentration of salt. It is suggested that the simplicity of CID/H4-RbAp48 reflects a simple in vivo situation in which assembly occurs in the absence of rapidly transiting polymerases and associated factors. Although both H3.1- and H3.3-specific complexes also contain RbAp48 and RbAp48 alone can assemble H3 nucleosomes, other components in these complexes might prevent spontaneous deposition at gaps in chromatin due to steric hindrance, whereas the much simpler CID/H4-RbAp48 would gain access to these chromatin gaps without impediment. In other words, H3- and H3.3-specific chromatin assembly complexes may have evolved to strictly couple their activities to replication and transcription, respectively, to increase the efficiency of these cellular processes, and to delineate assembly pathways of different histone 3 variants. There is precedence for such a variant-dependent exclusion mechanism: H3 appears to be prevented from assembling by replication-independent deposition anywhere in the genome, whereas H3.3 appears to deposit anywhere except at centromeres. When overproduced, CID deposits in a euchromatic pattern that is similar to that seen for H3.3, suggesting that CenH3s have fewer constraints than either H3 or H3.3 and that other chaperones in these complexes are the best candidates for mediating differential exclusion. Any CenH3 that incorporates in euchromatin at transient gaps created by transcription would be continuously replaced by transcription-coupled assembly of H3.3; in this way, CenH3 would be passively retained at centromeres but actively removed from transcriptionally active regions (Furuyama, 2006a).
Exclusion of H3 and H3.3 but not CenH3 from centromeric chromatin, such as by steric hindrance or RNA-mediated targeting, might help account for the deposition of CenH3s at a wide variety of sequences within a genome, including human neocentromeres, nematode holocentromeres, and gene-rich rice centromeres. Furthermore, budding yeast CenH3 (Cse4p) can localize properly to human centromeres and rescue a CENP-A depletion phenotype. Because of the high degree of divergence between Cse4p and CENP-A relative to the near invariance of H3, it is unlikely that a protein complex that normally recognizes CENP-A can associate with Cse4p and deposit it only at the centromeres. Rather, assembly of CenH3-H4 into centromeric chromatin in other organisms might be achieved by a simple H4-binding chaperone, such as RbAp48. Perhaps what distinguishes a CenH3 from a canonical H3 is that it is not accepted by H3- or H3.3-specific chaperone complexes (Furuyama, 2006a).
The efficient propagation of centromeric chromatin domains during every cell cycle requires the correct localization of CenH3s. The robustness and precision of this process is extraordinary; for example, the location of centromeres have not changed in this lineage for 30 million years. It has been proposed that the compact structure of the CENP-A/H4 protein tetramer leads to the perpetuation of correct CENP-A localization, but it is not clear how compactness by itself can facilitate the faithful recruitment of additional CENP-A/H4 protein tetramers during every cell division. The apparent simplicity of CenH3 assembly can provide a mechanism to delineate this assembly pathway from that of H3 and H3.3. Torsional stress induced at centromeres at anaphase may be an efficient mechanism to clear H3 or H3.3 from centromeres and to create gaps for CenH3 deposition. Thus, the assembly of centromeric nucleosomes at gaps, which are created by the very process that requires CenH3, would provide a robust self-enforcing mechanism to maintain centromeres indefinitely (Furuyama, 2006a).
Centromeres are chromosomal sites of microtubule binding that ensure correct mitotic segregation of chromosomes to daughter cells. This process is mediated by a special centromere-specific histone H3 variant (CenH3), which packages centromeric chromatin and epigenetically maintains the centromere at a distinct chromosomal location. However, CenH3 is present at low abundance relative to canonical histones, presenting a challenge for the isolation and characterization of the chaperone machinery that assembles CenH3 into nucleosomes at centromeres. To address this challenge, controlled overexpression of Drosophila CenH3 (CID) and an efficient biochemical purification strategy offered by in vivo biotinylation of CID was used to successfully purify and characterize the soluble CID nucleosome assembly complex. It consists of a single chaperone protein, RbAp48, complexed with CID and histone H4. RbAp48 is also found in protein complexes that assemble canonical histone H3 and replacement histone H3.3. This study highlights the benefits of the improved biotin-mediated purification method, and addresses the question of how the simple CID/H4-RbAp48 chaperone complex can mediate nucleosome assembly specifically at centromeres (Furuyama, 2006b).
The Drosophila Myb-Muv B (MMB)/dREAM complex regulates gene expression and DNA replication site-specifically, but its activities in vivo have not been thoroughly explored. In ovarian amplification-stage follicle cell nuclei, the largest subunit, Mip130, is a negative regulator of replication, whereas another subunit, Myb, is a positive regulator. A mutation has been identified in mip40, and a mutation has been generated in mip120, two additional MMB subunits. Both mutants were viable, but mip120 mutants had many complex phenotypes including shortened longevity and severe eye defects. mip40 mutant females had severely reduced fertility, whereas mip120 mutant females were sterile, substantiating ovarian regulatory role(s) for MMB. Myb accumulation and binding to polytene chromosomes was dependent on the core factors of the MMB complex. In contrast to the documented mip130 mutant phenotypes, both mip40 and mip120 mutant males were sterile. Mip40-containing complexes were purified from testis nuclear extracts and tMAC, a new testis-specific meiotic arrest complex was identified that contains Mip40, Caf1/p55, the Mip130 family member, Always early (Aly), and a Mip120 family member, Tombola (Tomb). Together, these data demonstrate that MMB serves diverse roles in different developmental pathways, and members of MMB can be found in alternative, noninteracting complexes in different cell types (Beall, 2007).
Coordinating developmentally regulated transcription and replication patterns in metazoans is critical for differentiation of tissue-specific cell types. Central to these processes is the modification and/or remodeling of chromatin by multisubunit complexes through association of site-specific DNA-binding proteins. A multisubunit complex in Drosophila, the Myb-Muv B (MMB) or dREAM complex contains the previously identified five-subunit Myb complex (containing Myb, Caf1/p55, Mip40, Mip120, and Mip130), in addition to E2f2, Rbf1 or Rbf2, DP, and Lin-52. Curiously, the complex contains both activator (Myb) and repressor (Mip130, Rbf1, Rbf2, and E2f2/DP) proteins. That MMB is widely expressed in different tissues has led to the idea that MMB may function both as an activator or repressor at specific chromosomal locations. Depending on the developmental pathways in particular tissues or cell types, different signals might regulate switching of function at a particular site. It has been suggested that at sites known to be repressed by MMB, Myb is a silent member not participating in the transcriptional repression, even though Myb itself is present at the cis-acting site, and that activation of MMB at a subsequent time would depend on Myb function (Beall, 2007).
The finding that Lethal (3) Malignant Brain Tumor [L(3)MBT], NURF, and the histone deacetylase Rpd3 associate with MMB suggests that histone binding and/or modification are possible mechanisms by which MMB acts to repress transcription and/or replication. Thus, MMB and individual subunits are poised to change their measured role by switching off repression (or activation) in a given cell lineage by post-transcriptional modifications or association of coactivators (or repressors) (Beall, 2007).
The genes regulated by MMB in Drosophila tissue culture cells are primarily differentiation and development-specific genes, and most often, MMB is a transcriptional repressor. Recent genomic profiling in Kc cells of five MMB members (Mip130, Mip120, Myb, E2F2, and Lin-52) showed that these proteins were bound together at thousands of chromosome sites, and RNA interference (RNAi) experiments revealed that MMB participated in either transcriptional repression or activation for many genes. In cell culture and in vivo, the accumulation of Myb and E2F2 proteins, but not mRNAs, depends on the integrity of MMB: Loss of Mip130 dramatically affects the levels of both proteins. These data, together with the biochemical finding that essentially all of Myb is found in complex with MMB, led to the proposal that most if not all of the phenotypes previously identified as Myb-specific (or E2F2-specific) must be evaluated in terms of loss of MMB function in either myb or e2f2 mutants (Beall, 2007).
myb is an essential gene in Drosophila. However, mutations in the largest subunit of MMB, mip130, are viable and suppress myb lethality. Furthermore, homozygous mip130 mutant females have drastically reduced fecundity. Cytological and developmental studies of egg chambers from several MMB subunit mutants were critical in building a heuristic model for MMB function. Normally in the ovary, a developmentally controlled replication program occurs in the somatic follicle cell nuclei surrounding the developing oocyte. In these nuclei, overall genomic replication ceases at stage 9 during egg chamber development and is followed at stage 10 by specific DNA replication at four loci that results in amplification of the genes critical for egg shell formation. Myb binds to the well-defined enhancer for one such amplicon in vivo. When myb is removed by genetic manipulation, replication no longer occurs at the four foci, demonstrating a direct and positive role for Myb in replication at these sites. In contrast, mip130 mutant ovaries display global genomic replication in amplification-stage follicle cell nuclei, indicating a negative role for mip130 in replication at sites other than at the chorion origins. Based on these observations, it has been suggested that MMB functions as either an activator or repressor of chromosomal functions depending on the chromosomal and developmental context. Critically in this model, the essential function of Myb is to activate a repressive complex to which it belongs. In its absence, this unchecked repressive activity by the partial MMB complex is lethal. The presumption was that animals lacking MMB (as in mip130 mutants) maintained expression (or repression) of normal target genes through less robust or redundant mechanisms, resulting in viability of these mutants. Furthermore, a critical, but previously untested prediction of this model is that an MMB complex devoid of Myb could still be targeted to chromosomes (Beall, 2007).
In order to gain further insight into the role(s) for MMB in vivo, a mutation was generated in the second largest subunit, mip120. In addition, a P-element-induced allele was identified in another subunit, mip40. As with mip130, it was found that mip40 and mip120 mutants were viable and displayed either sterility (mip120) or reduced fecundity (mip40) of mutant females. Moreover, mip40 and mip120 suppressed myb lethality, again suggesting that the essential function of Myb in vivo is to counter a repressive activity of MMB. Immunostaining of polytene chromosomes revealed that the association of Myb with specific chromosomal sites was dependent on Mip120, reinforcing the idea that Myb needs MMB for chromatin binding. Conversely, Mip120 and Mip130 did not require Myb for polytene chromosome binding (Beall, 2007).
Unanticipated and in contrast to mip130 mutants, it was found that mip40 and mip120 mutant males were sterile, thus defining a new role for these proteins in male fertility. Given that Mip130 has a paralog in the testis called Always early (Aly), the possibility was investigated that Mip40 and/or Mip120 might either function in a testis-specific Aly complex or that one or both might have testis-specific paralogs (Beall, 2007).
A combination of affinity, ion-exchange, and gel filtration chromatography was used to isolate Mip40-containing complexes from testis nuclear extracts. In addition to MMB, tMAC, a new testis-specific meiotic arrest complex in which the only MMB subunits found were Mip40 and Caf1/p55, was identified, in addition to the testis-specific proteins Aly, Cookie monster (Comr), Matotopetli (Topi), and Tomb. It is suggested is that MMB functions as a cell-type- and developmental-stage-specific regulator of transcription and replication with various subunits contributing to a 'Swiss Army Knife' type of versatility: the ability to interact specifically with numerous cis-elements, and to interact with numerous coactivators or corepressors as determined by context. This versatility extends beyond MMB itself, as some subunits are part of other tissue-specific complexes involved in gene expression (Beall, 2007).
These genetic findings that clearly separate developmental functions for Mip40 and Mip120 do not provide mechanistic insights into how the pleiotropic effects are manifested. For example, partial MMB complexes, resulting from loss-of-function alleles, may assume neomorphic activity. It has been argued that myb lethality in Drosophila is a consequence of such effects. The model that rationalizes 'silent subunits' present at a given location to promote switching from repression to activation (or vice versa) adds genetic complexity to the timing of critical execution functions for different MMB factors. This model anticipates that loss-of-function alleles of genes for different MMB subunits would manifest different arrest points. The possibility was considered that MMB subunits do not always function as a unit. The finding that mip40 mutant males displayed a staining pattern for Mip40 protein in testes quite distinct from Mip130 or Mip120 was curious. Epitope masking of one or another protein in MMB, due to changing coactivator or corepressor association, might explain such staining patterns. However, further work led to the search for a putative testis-specific complex that contained Mip40, where it reasonably would act in a distinct way from its functions in MMB (Beall, 2007).
A complex was purified from testis nuclear extracts that contains MMB members Mip40 and Caf1/p55, in addition to the testis-specific meiotic arrest proteins Aly, Comr, Topi, and Tomb. This complex was named tMAC, because aly, comr, topi, and mip40 mutants all display the same testis phenotype: an arrest at the primary spermatocyte stage of development, consistent with the notion that they are all acting together in a complex to promote differentiation and meiotic cell cycle progression. It seems likely that other proteins might interact with tMAC to aid in the regulation of testis-specific transcripts. This idea stems from what is already known about MMB, where proteins such as Rpd3 and L(3)MBT physically associate with MMB only during early steps in the biochemical purification process and are critical for function at different DNA sites. To date, no other alternative or subcomplexes containing members of MMB have been isolated from either embryo or tissue culture nuclear extracts. Furthermore, genomic profiling in KC cells of key MMB members (Myb, E2F2, Lin-52, Mip120, and Mip130) substantiates the hypothesis that these proteins work together as a group rather than as isolated factors on DNA. Had testis-enriched starting material had not been examined, it would have been impossible to identify tMAC. Thus, despite the present data showing that the MMB core factors always work as an ensemble, it is possible that some of the pleiotropic phenotypes observed for different MMB subunit mutants could reflect the activity of a subunit functioning outside of MMB (Beall, 2007).
It is striking that both tMAC and MMB contain proteins, other than Mip40 and Caf/p55, that are similar to each other in domain architecture: Aly (tMAC) or Mip130 (MMB) and Tomb (tMAC) or Mip120 (MMB). Given that MMB and tMAC contain multiple site-specific DNA-binding proteins (Myb, E2F2/DP, Mip120, Mip130 in MMB; and Tomb and Topi in tMAC), a potentially large number of genes may be regulated by tMAC as is now know is true for MMB (Beall, 2007).
Antisera against the MMB subunit, Lin-52, failed to coimmunoprecipitate Comr or Aly; therefore, it is not likely a tMAC subunit. However, it is interesting to note that there is another Lin-52 family member in Drosophila (CG12442). The adult Drosophila gene expression database indicates that this gene is, indeed, highly expressed in testis relative to other tissues. Given that this protein is extremely small (predicted molecular weight of 16 kDa), it is possible that it was not present in sufficient quantities to be detected in the mass spectrometry analysis and may in fact be part of tMAC. If so, that would be the third MMB subunit to have an alternative 'testis-specific' form present in tMAC (Beall, 2007).
Gonad-specific forms of proteins that are ubiquitously expressed and generally found in complexes that regulate transcription may, indeed, be a common theme. For example, gonad-specific components of the basal RNA polymerase II transcription machinery are crucial for developmentally regulated gene expression programs in these tissues. Five testis-specific TATA-binding protein-associated factors (TAFs) have been identified in Drosophila (encoded by the can, sa, mia, nht, and rye genes). All are required in spermatocytes for the normal transcription of target genes involved in post-meiotic spermatid differentiation (the so-called can class of genes). It is thought that these testis-specific TAFs may associate with some of the general TAF subunits to create a testis-specific TFIID (tTFIID) that carries out the developmentally regulated transcriptional program in spermatocytes. The mip40-null allele is also in the can class, suggesting that tMAC may interact with tTFIID at can class gene promoters. It will be interesting to explore the possibility that tMAC is a testis-specific coactivator with tTFIID. Other tMAC subunits fall into the aly class and might be what is expected for a large complex paralogous to MMB, where one or another subunit may be silent and subsequently required at a later stage or developmental pathway (Beall, 2007).
Mip130 family members, such as Aly, share a domain that is called a 'DIRP' (domain in Rb-related pathway) domain that is thought to be responsible for interaction with Rb. The DIRP domain of human-Lin-9 (Mip130) is necessary for association with Rb; however, the interaction between hLin-9 and Rb may be indirect as hLin-9 may exist in a complex with other proteins that directly touch Rb. Neither of the two Drosophila Rb proteins was in tMAC-containing fractions. When alignments were made with Mip130 family members, a region was noticed within the DIRP domain that was conserved between all family members except Aly. It is possible that this divergent region within the DIRP domain is critical for Rb interaction in other family members and has been lost in Aly. Although no direct understanding is available of how Aly works for transcriptional activation, it is possible that tMAC contains both activating and repressing components similar to MMB and that repression at particular loci does not require E2F/Rb (Beall, 2007).
When examined for replication profiles in mutant ovaries, an absence of amplification-stage egg chambers was found in mip120 mutants, and widespread BrdU incorporation and Orc2 staining in mip40 mutant amplification-stage follicle cell nuclei. The mip40 egg chamber phenotype is similar to that of mip130 and is consistent with a negative regulatory role for these proteins in genome-wide replication at these stages. It is suggested that both Mip40 and Mip120 are functioning in complex with MMB in ovaries and that both are required for normal patterns of replication in amplification-stage follicle cell nuclei. It is speculated, based in part on unpublished studies of the intricate regulatory network of MMB in Kc cells, that the different mutant phenotypes may simply reflect differences in gene expression profiles that result when individual MMB complex members are missing. A key role for Mip120 in the stability of chromatin-bound MMB might, therefore, explain the more severe phenotype of mip120 mutants. More specifically, MMB may regulate the expression of genes critical for amplification-stage egg chamber development either directly or indirectly, and Mip120 is required for targeting MMB to these gene promoters at a particular developmental stage prior to amplification stages. In contrast to Mip120, Myb, and E2F2/DP, Mip40 has no direct DNA-binding ability. Mip40 may be required for repression or activation only after MMB is targeted to chromosomal sites (Beall, 2007).
As with myb; mip130 mutants, it was found that myb; mip40 and myb; mip120 double mutants were viable, further demonstrating that function(s) of MMB without Myb are responsible for myb lethality. Myb protein was no longer associated with chromatin in mip120 and mip130 mutant polytene spreads. However, staining of polytene chromosomes demonstrated that Mip120 and Mip130 proteins were still bound to chromatin in myb mutants in such a way that may prove lethal in the absence of myb. Together, these data support a model in which Myb is critically dependent on members of the MMB complex for both stability and association with chromatin (Beall, 2007).
It is suggested that the presence of MMB at the replication enhancer ACE3 in stage 7-9 egg chambers may actively repress DNA replication here and at other sites in the genome. MMB at ACE3 at these early stages seems poised to await signals for initiation of amplification. The conversion of a repressive MMB complex into an activating complex may require cyclin E/Cdk2 activity, which is required for amplification. In this context it is likely that Rbf association with MMB will persist during amplification as Rbf association with MMB remains unchanged even after saturating hyperphosphorylation by Cdk:cyclin E in vitro. In the future, determining the cell-type-specific signals that target MMB at well-defined cis-regulatory elements at both the follicle cell amplicons and in other tissues will help unravel how MMB functions in vivo (Beall, 2007).
p55 is a common component of many chromatin-modifying complexes and has been shown to bind to histones. This study presents a crystal structure of Drosophila p55 bound to a histone H4 peptide. p55, a predicted WD40 repeat protein, recognizes the first helix of histone H4 via a binding pocket located on the side of a β-propeller structure. The pocket cannot accommodate the histone fold of H4, which must be altered to allow p55 binding. Reconstitution experiments show that the binding pocket is important to the function of p55-containing complexes. These data demonstrate that WD40 repeat proteins use various surfaces to direct the modification of histones (Song, 2008).
Since p55 is a common subunit of many complexes, whether disrupting the binding pocket in p55 affects the activity of two of these complexes was examined. The human Hat1 complex is composed of the Hat1p catalytic subunit and RbAp48 (the human ortholog of p55), and acetylates free but not nucleosomal histone H4 at Lys5 and Lys12. Histone acetyltransferase (HAT) activity was compared with Hat1 complexes containing wildtype or H4-binding pocket mutants of RbAp48. Two mutants of RbAp48, equivalent to the p55 mutants, were constructed based on sequence alignment between RbAp48 and p55. Leu31 in the N-terminal helix and Asp358 and Asp361 in the binding loop of RbAp48 were mutated to alanines. Hat1 complexes were expressed and copurified with either wild-type or one of the mutant RbAp48 subunits. Wild-type and mutant RbAp48 form equivalently stable complexes with the Hat1 subunit, and all complexes behaved similarly during gel filtration chromatography, indicating that these mutations do not disrupt folding or complex formation. The Hat1 complexes containing mutant RbAp48 show significantly less HAT activity than the wild-type Hat1 complex. This supports a role for the H4-binding pocket of p55 in the acetyltransferase activity of the Hat1 complex (Song, 2008).
p55 binding to a buried region of histone H4 is consistent with the function of some, but not all, p55-containing complexes. The Hat1 and CAF-1 complexes are involved in histone assembly; the p55 subunit in these complexes most likely binds to free histone H4. p55 is also found in the ATP-dependent chromatin remodeling complexes NURF and NuRD. It is tantalizing to propose that the first helix of histone H4 becomes accessible to p55 during the ATP-dependent chromatin remodeling process where substantial structural changes in the nucleosome might occur. Another p55-containing complex, PRC2, methylates histone H3 at Lys27 in the intact nucleosome where the first helix of histone H4 is buried. It is therefore of interest to examine the function of p55 in this complex (Song, 2008).
To examine this, attempts were made to form PRC2 complexes that contained wild-type and mutant forms of p55. However, mutant forms of p55 would not stably associate with the remaining subunits of PRC2. This suggests that the binding pocket of p55 might be interacting with other subunits of PRC2 rather than with histone H4 within this complex. This does not appear to be caused by a general defect in the ability of these mutants to form interactions, as these same mutants are able to form a stable complex with hHAT complex and bind to histone H3. This observation implies an interesting function of the histone H4-binding pocket of p55; it might be utilized in different ways depending upon the role for p55 in the complex. Elucidating the functional role for p55 in PRC2 will require further biochemical and structural studies (Song, 2008).
The results presented in this study reveal the molecular basis for histone H4 recognition by p55. The structure of p55 bound to the first helix of histone H4 suggests that the canonical histone fold has to be altered upon p55 binding. Moreover, the histone H4-binding pocket of p55 plays a critical role in the function of p55-containing complexes. Together these data suggest that p55 might serve as a multifunctional protein interaction platform within the many p55-containing complexes (Song, 2008).
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