Interactive Fly, Drosophila

NAP1 mediates chromatin assembly in conjunction with Drosophila CAF-1 and ATP. When histone H1 is included in the reaction medium prior to the assembly of chromatin, the estimated repeat length of nucleosomal arrays is 200 base pairs, compared with 161 bp in the absence of histone H1. Yeast NAP1 can replace the fly protein in an assembly reaction. In the absence of histones, Drosophila NAP1 alone appears to be multimeric. In the presence of core histones, NAP1 cosediments with all four core histones. In crude extracts H2A and H2B but not H3 coimmunoprecipitate with NAP1. It appears that NAP1 binds to all four core histones but with a higher affinity for H2A and H2B than with H3 or H4 (Ito, 1996a). NAP1 exhibits a higher affinity for core histones than does dNLP, the Drosophila Nucleoplasmin-like protein. NAP1 but not dNLP, is highly active for sperm chromatin decondensation (Ito, 1996b).

The assembly of chromatin by CAF-1, NAP1, purified histones, ATP, and DNA is a process that generates regularly spaced nucleosomal arrays with a repeat length that resembles that of bulk native Drosophila chromatin and is not obligatorily coupled to DNA replication. The assembly of chromatin by dCAF-1 and NAP1 is nearly complete within 10 min (Bulger, 1995).

A core histone-binding protein has been purifed and cloned from Drosophila melanogaster embryos. This protein resembles Xenopus laevis nucleoplasmin, and it has therefore been termed dNLP, for Drosophila nucleoplasmin-like protein. Nucleoplasmin has a 31% identity with Xenopus nucleoplasmin. NLP is a minor constituent of the dCAF-4 fraction from which Drosophila NAP1 was purified. dNLP is a nuclear protein that is present throughout development. Both purified native and recombinant dNLP bind to core histones and can function in the assembly of approximately regularly spaced nucleosomal arrays in a reaction that additionally requires DNA, purified core histones, ATP, and a partially purified fraction (containing at least one other assembly activity). The properties of an N-terminally truncated version of dNLP, termed dNLP-S were examined. The deletion of the N-terminal 31 residues of dNLP results in a loss of the specificity of the interaction of dNLP with core histones. When compared, dNLP and Drosophila nucleosome assembly protein-1 (dNAP-1) vary in their abilities to promote the decondensation of Xenopus sperm chromatin, a process that can be mediated by nucleoplasmin. dNAP-1, but not dNLP, is able to promote the decondensation of sperm chromatin, although another study (Crevel, 1997) does detect such an activity on the part of dNLP. These and other data collectively suggest that dNLP may participate in parallel with other histone-binding proteins such as dNAP-1 in the assembly of chromatin (Ito, 1996b).

CRP1, a Drosophila nuclear protein that can catalyze decondensation of demembranated Xenopus sperm chromatin was cloned and its primary structure was deduced from cDNA sequence. Alignment of deduced amino acid sequence with published sequences of other proteins reveals strong homologies to Xenopus nucleoplasmin and NO38. The protein is identical to dNLP, cloned by T. Ito (1996b). CRP1 is encoded by one or several closely related genes found at a single locus, position 99A on the right arm of chromosome 3. CRP1 mRNA is expressed throughout Drosophila development; it is highest during oogenesis and early embryogenesis. mRNA levels correlate closely with levels of protein expression measured previously. Results of chemical crosslinking indicate that CRP1 is either tetrameric or pentameric; similar ambiguity is revealed by direct visualization using scanning transmission electron microscopy. Consistent with previously published results, parallel crosslinking studies of Xenopus nucleoplasmin suggested a pentameric structure. Scanning transmission electron microscopic examination after negative staining reveal that CRP1 and Xenopus nucleoplasmin are morphologically similar. CRP1 is able to substitute for nucleoplasmin in Xenopus egg extract-mediated sperm chromatin decondensation. In vitro, CRP1-induced decondensation is accompanied by direct binding of CRP1 to chromatin (Crevel, 1997).

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).

SIR2 is required for polycomb silencing and is associated with an E(Z) histone methyltransferase complex

SIR2 was originally identified in S. cerevisiae for its role in epigenetic silencing through the creation of specialized chromatin domains. It is the most evolutionarily conserved protein deacetylase, with homologs in all kingdoms. SIR2 orthologs in multicellular eukaryotes have been implicated in lifespan determination and regulation of the activities of transcription factors and other proteins. Although SIR2 has not been widely implicated in epigenetic silencing outside yeast, Drosophila SIR2 mutations were recently shown to perturb position effect variegation, suggesting that the role of SIR2 in epigenetic silencing may not be restricted to yeast. Evidence is presented that Drosophila SIR2 is also involved in epigenetic silencing by the Polycomb group proteins. Sir2 mutations enhance the phenotypes of Polycomb group mutants and disrupt silencing of a mini-white reporter transgene mediated by a Polycomb response element. Consistent with this, SIR2 is physically associated with components of an E(Z) histone methyltransferase complex. SIR2 binds to many euchromatic sites on polytene chromosomes and colocalizes with E(Z) at most sites. It is concluded that SIR2 is involved in the epigenetic inheritance of silent chromatin states mediated by the Drosophila Polycomb group proteins and is physically associated with a complex containing the E(Z) histone methyltransferase (Furuyama, 2004).

The ability of Sir2 mutations to enhance PcG mutant phenotypes and perturb PRE-mediated silencing indicates that SIR2 plays a role in Polycomb silencing. However, like their yeast and C. elegans counterparts, Drosophila Sir2 mutants are viable under standard laboratory conditions, and they do not exhibit obvious PcG phenotypes. Mutations in several other genes that play a role in Polycomb silencing enhance the phenotypes of PcG mutants but do not themselves exhibit Polycomb phenotypes. These include E(Pc), Su(z)2, and the histone deacetylase Rpd3/HDAC1. Uncovering the role of Sir2 in Polycomb silencing required sensitive genetic assays. This could be due to functional redundancy; four other Drosophila genes encode conserved SIR2 paralogs, corresponding respectively to the mammalian SIRT2 (similar to yeast HST2), SIRT4, and the closely related SIRT6 and SIRT7. Although these SIR2 paralogs are likely to have physiological roles distinct from that of SIR2, in the absence of SIR2, one or more of them might at least partially compensate for the function of SIR2 in Polycomb silencing. In S. cerevisiae, the Sir2p paralog Hst1p, which normally functions as a gene-specific repressor, can rescue the silencing defects of Sir2 mutants when it is overexpressed or targeted to the mating-type locus. Another yeast Sir2p paralog, Hst2p, although not required for silencing, improves rDNA silencing when it is overexpressed almost as efficiently as overexpressed Sir2p itself, even though Hst2p remains exclusively cytoplasmic (Furuyama, 2004).

It is also possible that the another deacetylase, e.g., RPD3/HDAC1, which is also present in E(Z) complexes, may be able to at least partially substitute for the SIR2 function in these complexes. Indeed, Drosophila RPD3 and SIR2 appear to have similarly broad substrate specificities, at least in vitro. Alternatively, SIR2 may be more critically required for Polycomb silencing under particular environmental or nutritional conditions that differ from standard laboratory conditions. It was originally suggested that the NAD⁺ dependence of SIR2 deacetylase activity (or its inhibition by nicotinamide could serve to link SIR2 activity to environmental or nutritional conditions. Indeed, the yeast Sir2p paralog Hst1p has been shown to regulate genes involved in de novo NAD⁺ biosynthesis by functioning as a direct sensor of cellular NAD⁺ levels. Various stresses and nutritional conditions appear to regulate the expression or activity of mammalian SIRT1 as well as the association of SIRT1 with its protein substrates. By analogy, the requirement for SIR2 or its activity in Polycomb silencing may be modulated by environmental or nutritional conditions, perhaps so that the fidelity of Polycomb silencing and its epigenetic inheritance is maintained under unfavorable or stressful culture conditions during larval life (Furuyama, 2004).

The physical association of Drosophila SIR2 with E(Z), RPD3, and p55 is the first evidence that SIR2 is associated with proteins known to be involved in epigenetic silencing in multicellular eukaryotes. The association of SIR2 with E(Z) was only detected in post-embryonic extracts, despite the presence of SIR2 and E(Z) in embryos. This suggests that E(Z) complex(es) differ in their composition and possibly their physiological functions at different developmental stages. It also suggests that the role of Drosophila SIR2 in Polycomb silencing may be restricted to post-embryonic stages. The transition from embryonic to larval period upon hatching from the egg marks the onset of active feeding and concomitant exposure to fluctuations in nutrient sources and other environmental variables from which embryonic development may be relatively more insulated. The differential association of SIR2 with E(Z) complexes during the larval stages may serve to increase the fidelity of PcG silencing under stressful conditions, a function that might not be expected to be uncovered without sensitive genetic assays or knowledge of the conditions that would render SIR2 more critical for maintenance of PcG silencing (Furuyama, 2004).

The high degree of protein sequence conservation among SIR2 orthologs from divergent species suggests that their biological functions, including their roles in epigenetic silencing, are also likely to be generally conserved. The conserved NAD⁺-dependent histone deacetylase activity and chromosomal localization of the Drosophila SIR2 protein is further consistent with this. However, although other components of E(Z) complexes, including RPD3 and the histone binding protein p55, have been highly conserved among all eukaryotes during evolution, an unequivocal E(Z) ortholog is not identifiable in S. cerevisiae or S. pombe, despite the presence of E(Z) orthologs in plants and animals and the presence of SET domain-containing histone methyltransferases in yeast. Conversely, Drosophila and mammals contain no identifiable homologs of S. cerevisiae SIR3 and SIR4, two key proteins that collaborate with SIR2 in the creation of silent chromatin domains at the mating-type loci and telomeres. This suggests that the mechanisms underlying SIR2-dependent silencing in yeast and multicellular eukaryotes, although broadly similar, are likely to differ in additional mechanistic details. On the other hand, the conservation of PcG proteins between Drosophila and mammals suggests that the association of SIR2 with E(Z) complex(es) is also likely to be conserved in mammals (Furuyama, 2004).

At present it is not evident why both NAD⁺-dependent (SIR2) and NAD⁺-independent (RPD3) HDACs are associated with E(Z) in larval extracts, but this arrangement is not unique. The S. cerevisiae SET domain protein SET3 is also found in a complex that contains two HDACs, including Hos2p, an RPD3-related class I HDAC, and Hst1p, which is closely related to yeast and Drosophila SIR2. Drosophila Hairy also interacts with both Rpd3 and Sir2. Perhaps in such situations each HDAC functions in different contexts or deacetylates different substrates. Drosophila RPD3 is found in a complex with the SET domain protein SU(VAR)3-9 and appears to be required for SU(VAR)3-9 histone methyltransferase function in vivo. It remains to be determined whether SIR2 is required for or modulates the histone methyltransferase function of E(Z) in vivo. The developmentally regulated association of SIR2 with E(Z) raises the interesting possibility that SIR2 may alter the activity or substrate specificity of E(Z). Although the chromosomal association of Drosophila SIR2 suggests it could target histones, the identification of multiple transcription factors and other proteins as substrates of mammalian SIRT1 suggests that the SIR2 associated with E(Z) may also have other non-histone substrates that regulate transcriptional silencing, perhaps including proteins in the E(Z) complex itself (Furuyama, 2004).

Distinct activities of CHD1 (and its cofactor chaperone NAP1) and ACF in ATP-dependent chromatin assembly

CHD1 is a chromodomain-containing protein in the SNF2-like family of ATPases. This study shows that CHD1 exists predominantly as a monomer and functions as an ATP-utilizing chromatin assembly factor. This reaction involves purified CHD1, NAP1 chaperone, core histones and relaxed DNA. CHD1 catalyzes the ATP-dependent transfer of histones from the NAP1 chaperone to the DNA by a processive mechanism that yields regularly spaced nucleosomes. The comparative analysis of CHD1 and ACF revealed that CHD1 assembles chromatin with a shorter nucleosome repeat length than ACF. In addition, ACF, but not CHD1, can assemble chromatin containing histone H1, which is involved in the formation of higher-order chromatin structure and transcriptional repression. These results suggest a role for CHD1 in the assembly of active chromatin and a function of ACF in the assembly of repressive chromatin (Lusser, 2005).

The packaging of DNA into chromatin is a critical step in the organization and utilization of the genome. In this process, DNA is assembled into arrays of nucleosomes, each of which contains an octamer of the core histone proteins. The nucleosome cores are joined together by linker DNA that is typically ~20-60 base pairs (bp) in length. In metazoans, an additional histone, termed histone H1, interacts with both the linker DNA and the nucleosome core, and promotes the higher-order folding of chromatin (Lusser, 2005).

Chromatin assembly involves the combined action of core histone chaperones and ATP-utilizing motor proteins. Several distinct histone chaperones, such as CAF-1 (chromatin assembly factor-1), Asf1 (anti-silencing function-1), NAP1 (nucleosome assembly protein-1), and HirA (histone regulatory protein A), participate in the deposition of the histones onto the DNA and/or mediate the translocation of core histones from the cytoplasm to the nucleus. The assembly of extended, periodic nucleosome arrays has thus far been observed in vitro with the ATP-utilizing factors ACF (ATP-utilizing chromatin assembly and remodeling factor), CHRAC (chromatin accessibility complex), and RSF (remodeling and spacing factor). ACF and CHRAC are nearly identical and seem to be conserved from yeast to humans. In Drosophila melanogaster, ACF and CHRAC each contain the Acf1 protein and the ISWI (imitation switch) ATPase, but CHRAC additionally comprises the small CHRAC-14 and CHRAC-16 polypeptides. In addition, recombinant ISWI polypeptide alone has ATP-dependent chromatin assembly. RSF has been studied in humans and consists of Rsf1 and hSNF2h, a human homolog of D. melanogaster ISWI. Thus, these ATP-utilizing chromatin assembly factors share an ATPase subunit that is a member of the ISWI/SNF2L subfamily of proteins (Lusser, 2005 and references therein).

In Drosophila, the ISWI ATPase has been found in four protein complexes: ACF, CHRAC, NURF (nucleosome remodeling factor), and TRF2 (TBP-related factor-2) complex. Aside from ISWI, the NURF and TRF2 complexes do not share any subunits with ACF or CHRAC, and a role for the NURF or TRF2 complex in chromatin assembly has not been described. Drosophila melanogaster ISWI is essential for viability, and the loss of ISWI function in vivo results in the misregulation of transcription and global alterations in chromosome structure (Lusser, 2005).

The function of the ACF and CHRAC chromatin assembly factors in vivo in D. melanogaster has been examined by the generation and characterization of acf1 null mutant flies. Unlike ISWI, the acf1 gene is not absolutely required for viability. The loss of Acf1 results in a decrease in nucleosome periodicity as well as a shorter nucleosome repeat length in chromatin derived from embryos. Extracts prepared from homozygous null acf1 embryos exhibit reduced yet detectable ATP-dependent chromatin assembly activity. These results support a role for Acf1 in chromatin assembly in vivo, and also suggest that D. melanogaster has at least one additional ATP-dependent chromatin assembly factor that is distinct from ACF or CHRAC (Lusser, 2005).

This work explored the function of ATPases other than ISWI in the ATP-dependent assembly of periodic nucleosome arrays. These studies led to the characterization of the CHD1 (chromo-ATPase/helicase-DNA-binding protein 1) protein. CHD1 was originally found as a chromodomain-helicase-DNA-binding domain-containing protein (Delmas, 1993). It is a member of the CHD1 subfamily of DNA-stimulated ATPases. Both the CHD1 and ISWI/SNF2L subfamilies are members of the SNF2-like family of proteins. One distinctive feature of proteins of the CHD1 subfamily is the presence of two chromodomains (Lusser, 2005).

The analysis of CHD1 has suggested that it functions in the elongation of transcription by RNA polymerase II as well as in chromatin dynamics. For example, CHD1 has been found to bind to DNA (Stokes, 1995), to localize to regions of decondensed chromatin (interbands) and high transcriptional activity (puffs) in D. melanogaster polytene chromosomes (Stokes, 1996), to participate in transcriptional termination (Stokes, 1996), and to interact with the FACT (facilitates chromatin transcription; mammalian SSRP1-Spt16 complex, or yeast Pob3-Spt16/Cdc68 complex; see Drosophila FACT), Rtf1 and Spt5 transcriptional elongation factors as well as with casein kinase II. In addition, studies in S. cerevisiae have revealed synthetic genetic interactions between ISW1 and ISW2 (which encode ISWI-related proteins) and CHD1 (Tsukiyama, 1999) as well as a partial loss of chromatin assembly activity in vitro by crude DEAE fractions derived from strains lacking either Asf1 or Chd1, but not Isw1, Isw2, Snf2, Swr1, NAP1 or CAC-1 (large subunit of CAF-1). Moreover, purified yeast CHD1 has been shown to remodel mononucleosomes in vitro (Lusser, 2005 and references therein).

This study has explored the function of CHD1 in chromatin assembly and found that the purified protein is an ATP-utilizing chromatin assembly factor that is distinct from the ISWI-containing factors. Moreover, comparison of the biochemical functions of CHD1 and ACF revealed differences in their properties that may reflect their participation in distinct biological processes in vivo (Lusser, 2005).

To investigate the biochemical activities of CHD1, Drosophila CHD1 was synthesized with a C-terminal Flag-tag by using a baculovirus expression system and then the protein was purified by Flag immunoaffinity chromatography. To determine whether the purified recombinant CHD1 was enzymatically active, ATPase assays nd were carried out and observed strong stimulation of ATPase activity by DNA or chromatin but not by free histones or RNA was observed, as seen previously with yeast CHD1 protein (Lusser, 2005).

Many proteins in the SNF2 like family are present in multi-subunit complexes. In addition, CHD1 proteins have been found to interact with factors involved in transcriptional elongation and with casein kinase II. Hence, before characterizing the purified recombinant CHD1 protein, whether native D. melanogaster CHD1 protein is a component of a multiprotein complex was investigated by carrying out glycerol gradient sedimentation experiments with crude nuclear extracts derived from Drosophila embryos. Western blot analysis of gradient fractions with an antibody against CHD1 revealed that bulk native CHD1 sediments with an apparent molecular mass of ~200 kDa, which corresponds closely to the calculated molecular mass of the CHD1 polypeptide (212 kDa). In parallel, it was also found that recombinant Flag-tagged CHD1 sediments with an apparent mass similar to that of native CHD1. These results indicate that native and recombinant CHD1 proteins are predominantly present as monomers (Lusser, 2005).

Thus, the majority of native D. melanogaster CHD1 seems to exist as a monomeric protein rather than as a subunit of a stable, multiprotein complex. This conclusion is also consistent with studies of yeast CHD1, which has been found to be a single polypeptide upon purification from a whole-cell extract. Hence, it is likely that subsequent analyses of purified recombinant CHD1 reflect the properties of the native form of the protein (Lusser, 2005).

To analyze the functional properties of CHD1, chromatin assembly assays were carried with a completely purified system consisting of recombinant Drosophila CHD1, recombinant D. melanogaster NAP1, native D. melanogaster core histones and relaxed circular plasmid DNA. Analysis of the reaction products by the micrococcal nuclease digestion assay revealed that CHD1 catalyzes the formation of extended arrays of regularly spaced nucleosomes. CHD1-mediated chromatin assembly requires ATP as well as the NAP1 chaperone. Therefore, CHD1 is an ATP-utilizing chromatin assembly factor. Hence, catalysis of the ATP-dependent assembly of periodic nucleosome arrays can be mediated by the CHD1 ATPase as well as by the ISWI ATPase (Lusser, 2005).

The generation of periodic nucleosome arrays by CHD1 could be achieved by different mechanisms. It is possible, for instance, that chromatin assembly by CHD1 occurs via a passive histone deposition mechanism in which the histones are randomly deposited onto the DNA by a chaperone such as NAP1 in an ATP-independent process, and then the resulting nucleosomes are redistributed into periodic arrays by CHD1 acting as an ATP-utilizing nucleosome mobilization factor. Alternatively, chromatin assembly may occur by an active histone deposition mechanism in which CHD1 function is involved in the transfer of histones to DNA as well as the formation of periodic arrays of nucleosomes. Thus, to clarify the mechanism of chromatin assembly by CHD1, its biochemical properties were further analyzed (Lusser, 2005).

Whether CHD1 can rearrange randomly distributed nucleosomes to give periodic arrays was tested, as in the second step of the passive deposition mechanism. To this end, chromatin was reconstituted by using salt dialysis, and the chromatin was purified from free histones and DNA by sucrose gradient sedimentation. In the salt dialysis reconstitution procedure, purified histones are combined with DNA in 2 M NaCl, and then, upon removal of the salt by dialysis, the histones are randomly deposited onto the DNA in an ATP-independent process. When subjected to micrococcal nuclease digestion analysis, the salt dialysis-reconstituted chromatin gave poorly defined bands with a repeat length of ~145 bp. Upon incubation with either CHD1 or ACF in the presence of ATP, the randomly distributed nucleosomes were converted into extended periodic arrays with a repeat length of ~160 bp. Therefore, CHD1, like ISWI-containing remodeling factors, is a nucleosome spacing factor. These results indicate that CHD1 can rearrange existing nucleosomes. This activity could be related to its participation in a passive deposition mechanism. To test this possibility, the function of CHD1 was further analyzed during the assembly of nucleosomes (Lusser, 2005).

Chromatin assembly reactions were carried out to determine whether or not CHD1 catalyzes the ATP-dependent transfer of histones onto the DNA. In an active transfer mechanism, CHD1 would facilitate histone deposition, whereas in a passive transfer mechanism, CHD1 would not affect the transfer of histones from NAP1 chaperone to the DNA. To distinguish between these mechanisms, nucleosome assembly was monitored by using the DNA supercoiling assay, which detects the formation of negative supercoiling in a circular template that results from the induction of approximately one negative supercoil for each nucleosome that is assembled. A series of reactions were carried out with relaxed circular DNA templates in the presence of purified topoisomerase I. These experiments revealed that CHD1 substantially enhances the extent of nucleosome assembly relative to that seen with either NAP1 alone or NAP1 with ATP (Lusser, 2005).

Hence, these results indicate that CHD1 catalyzes the transfer of histones from the NAP1 chaperone onto DNA in an ATP-dependent manner. This property of CHD1 suggests that it functions by an active rather than a passive histone deposition mechanism. In this respect, CHD1 is similar to ACF, which also mediates the transfer of histones onto DNA in an ATP-dependent manner. Moreover, in a related process, the SWR1 complex catalyzes histone H2A.Z-H2B exchange in conjunction with the NAP1 chaperone by an ATP-dependent mechanism. These findings collectively support an active role of ATP-utilizing motor proteins in nucleosome assembly and histone exchange (Lusser, 2005).

The mechanism of CHD1 function was further investigated by testing whether it acts processively in the assembly of chromatin. To this end, chromatin assembly reactions were carried out at a substoichiometric amount of CHD1 relative to DNA templates (approximately one CHD1 molecule per five DNA templates), and then the early reaction products were analyzed by two-dimensional DNA supercoiling analysis. Under these conditions, if CHD1 functions by a processive mechanism, then CHD1 would assemble multiple nucleosomes on a subset of the templates, and two distinct populations of reaction products would be observed: partially assembled chromatin and naked DNA. Alternatively, if CHD1 functions in a nonprocessive manner, then a single normal distribution of supercoiled DNA species would be seen (Lusser, 2005).

In the assembly of chromatin by CHD1, two populations of reaction products were seen: partially assembled chromatin templates with a peak of nine nucleosomes, and naked DNA templates with a peak of zero nucleosomes. Micrococcal nuclease digestion analysis also revealed short arrays of nucleosomes, which indicate local clustering of nucleosomes. These results indicate that CHD1 assembles chromatin processively, as seen with ACF. Moreover, the processivity of CHD1 supports an active histone deposition mechanism (Lusser, 2005).

Activities of CHD1 that may be distinct from those of ACF were examined, and therefore a comparative analysis of the two factors was carried out. A notable difference between CHD1 and ACF was revealed upon examination of the nucleosome repeat lengths of chromatin assembled by each of these factors. Under identical reaction conditions, except for the presence of CHD1 or ACF, CHD1 assembled chromatin with a repeat length of ~162 bp, whereas ACF assembled chromatin with a substantially longer repeat length of ~175 bp. These results indicate that the nucleosome repeat length is dictated by the ATP-driven factor that assembles the chromatin (Lusser, 2005).

In metazoans, bulk native chromatin contains approximately one molecule of the linker histone H1 (and/or H5) per nucleosome. H1 histones have a broad range of effects on chromatin compaction and organization as well as the regulation of gene expression. Whether CHD1 can catalyze the assembly of histone H1-containing chromatin was examined. To this end, chromatin assembly reactions were carried out in the absence or presence of purified histone H1 (at a 1:1 molar ratio of H1/core histone octamers) with either CHD1 or ACF. In the absence of H1, nucleosome repeat lengths of ~162 bp were seen with CHD1 and 172 bp with ACF, consistent with the results described above. Upon addition of H1 to ACF assembly reactions, a distinct micrococcal nuclease digestion pattern was observed that reveals an increase in the nucleosome repeat length from ~172 bp in the absence of H1 to ~200 bp in the presence of histone H1. This alteration in the repeat length is a consequence of the incorporation of histone H1. In contrast, under identical conditions, a dispersed, smeary micrococcal nuclease digestion pattern was observed upon inclusion of H1 in CHD1 assembly reactions, indicating a disruption of the periodicity of the nucleosomes that may be due to random association of the free H1 with nucleosomes. Further attempts were made to assemble H1-containing chromatin with CHD1 under a variety of other reaction conditions, but incorporation of histone H1 was not observed. It therefore seems that histone H1-containing chromatin can be assembled with ACF but not with CHD1 (Lusser, 2005).

The incorporation of histone H1 into chromatin was further characterized by native nucleoprotein gel electrophoresis of mononucleosome species that are generated upon extensive micrococcal nuclease digestion of chromatin. With chromatin assembled by either CHD1 or ACF in the absence of histone H1, core particles as well as some dinucleosomes were detected. Upon inclusion of histone H1 in the assembly reactions, the formation of chromatosomes (mononucleosomes containing histone H1) was observed with ACF but not with CHD1. CHD1 assembly reactions with histone H1 yielded a heterogeneous mixture of mononucleosome species that migrated faster than the chromatosomes. It is possible that nonspecific interactions of free histone H1 with the core particles result in a retardation of their migration through the nondenaturing gel. The addition of excess free competitor DNA after chromatin assembly but before micrococcal nuclease digestion did not affect the efficiency of formation of the chromatosome species (Lusser, 2005).

The DNA fragments present in the mononucleosome preparations were additionally analyzed by deproteinization of the micrococcal nuclease-digested chromatin followed by agarose gel electrophoresis. These experiments revealed that the mononucleosomal DNA fragments derived from ACF-assembled chromatin exhibited a ~25-bp increase in length upon addition of H1 to the chromatin assembly reactions, whereas mononucleosomal DNA fragments derived from CHD1-assembled chromatin were found to be nearly the same length whether or not H1 was present in the assembly reactions. Hence, these results, combined with the observation of chromatosome species with ACF but not with CHD1, provide further evidence for the incorporation of histone H1 into chromatin by ACF but not by CHD1 (Lusser, 2005).

It was also of interest to testing whether the ISWI ATPase subunit of ACF is sufficient for the assembly of histone H1-containing chromatin. Therefore, assembly reactions were carried out with purified ISWI polypeptide, and it was observed that ISWI alone can catalyze the formation of histone H1-containing chromatin. The quality of the H1-containing chromatin generated with ISWI was, however, consistently lower than the quality of that assembled with ACF. Notably, the smearing between the micrococcal nuclease bands, which reflects irregularities in the nucleosome arrays, is more prominent with ISWI-assembled H1-containing chromatin than with ACF-assembled chromatin. Thus, ISWI can catalyze the assembly of histone H1-containing chromatin, but is less effective than ACF in the assembly of periodic arrays of H1-containing nucleosomes. These findings indicate that the difference in the abilities of ACF and CHD1 to assemble H1-containing chromatin is due, at least in part, to the activities of the ISWI versus the CHD1 ATPases (Lusser, 2005).

Thus, chromatin assembly by CHD1 can be carried out with a completely purified system that consists of recombinant CHD1, recombinant NAP1, native core histones and DNA. Therefore, the ATP-dependent assembly of periodic nucleosome arrays can be mediated by CHD1 as well as by ISWI-containing proteins. In contrast, the catalysis of chromatin assembly by other related ATPases such as BRG-1 and Rad54 was not observed. Hence, chromatin assembly is not a general property of members of the SNF2-like family of ATPases (Lusser, 2005).

CHD1 catalyzes the transfer of histones from a chaperone to the DNA template by a processive mechanism that yields periodic nucleosome arrays. CHD1 can also convert randomly distributed nucleosomes into periodic nucleosome arrays. Notably, CHD1 and ACF catalyze the assembly of chromatin with different internucleosomal spacing. In addition, ACF, but not CHD1, can assemble histone H1-containing chromatin. Hence, CHD1 is an ATP-utilizing chromatin assembly and remodeling factor with activities that are distinct from those of ACF (Lusser, 2005).

Transcriptionally active chromatin generally has a shorter nucleosome repeat length than transcriptionally repressed chromatin. Thus, the ability of CHD1 to mediate the reconstitution of chromatin with relatively short internucleosomal spacing might reflect its proposed involvement in active transcription in vivo. The lack of H1 assembly activity by CHD1 supports this model and, in addition, suggests a mechanistic link between these two activities. It is relevant to note that bulk chromatin in Drosophila lacking the Acf1 subunit of ACF and CHRAC has a shorter repeat length than chromatin in wild-type flies. This reduction in the nucleosome repeat length could be due to the loss of ACF (and CHRAC), which catalyzes the assembly of chromatin with a longer repeat length than CHD1-assembled chromatin. Moreover, the shorter repeat length of chromatin in the Acf1-deficient flies could also be due to a defect in the incorporation of histone H1 into the chromatin upon loss of ACF (and CHRAC) (Lusser, 2005).

From a broader perspective, the findings from this study contribute to the current understanding of the biological roles of CHD1 and ACF. ACF seems to promote the assembly of repressive chromatin. For instance, Drosophila Acf1 contributes to heterochromatic repression, such as that seen in position-effect variegation. In addition, Drosophila ISWI is mostly associated with nontranscribed regions of polytene chromosomes. Consistent with these observations, ACF can assemble transcriptionally repressive histone H1-containing chromatin. In contrast, CHD1 associates with factors that promote transcriptional elongation, such as FACT, Rtf1 and Spt5, and localizes to transcriptionally active regions of the genome. The function of CHD1 as a chromatin assembly factor fits well with its proposed role in the reassembly of nucleosomes subsequent to their disruption during transcription. Moreover, the ability of CHD1 to assemble H1-deficient chromatin, but not H1-containing chromatin, is consistent with a function in the assembly of transcriptionally active DNA into chromatin. It was also observed that the majority of native Drosophila CHD1 seems to exist as a monomer. These results further suggest that CHD1 and the elongation factors interact transiently rather than as components of a stable complex, or that only a small fraction of CHD1 is present in a multiprotein complex. Future studies may address whether there are other ATP-utilizing chromatin assembly factors and examine their shared and unique functions (Lusser, 2005).

Heterochromatin protein 2 interacts with Nap-1 and NURF: a link between heterochromatin-induced gene silencing and the chromatin remodeling machinery in Drosophila

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)

Histone chaperones ASF1 and NAP1 differentially modulate removal of active histone marks by LID-RPD3 complexes during NOTCH silencing

Histone chaperones are involved in a variety of chromatin transactions. By a proteomics survey, the interaction networks of histone chaperones ASF1 (Anti-silencing factor 1), CAF1, HIRA, and NAP1 were identified. This study analyzed the cooperation of H3/H4 chaperone ASF1 and H2A/H2B chaperone NAP1 with two closely related silencing complexes: LAF and RLAF. NAP1 binds RPD3 and LID-associated factors (RLAF) comprising histone deacetylase RPD3, histone H3K4 demethylase LID/KDM5, SIN3A, PF1, EMSY, and MRG15. ASF1 binds LAF, a similar complex lacking RPD3. ASF1 and NAP1 link, respectively, LAF and RLAF to the DNA-binding Su(H)/Hairless complex, which targets the E(spl) Notch-regulated genes. ASF1 facilitates gene-selective removal of the H3K4me3 mark by LAF but has no effect on H3 deacetylation. NAP1 directs high nucleosome density near E(spl) control elements and mediates both H3 deacetylation and H3K4me3 demethylation by RLAF. It is concluded that histone chaperones ASF1 and NAP1 differentially modulate local chromatin structure during gene-selective silencing (Moshkin, 2009).

Regulated modulation of the chromatin structure is essential for the transmission, maintenance, and expression of the eukaryotic genome. The combined actions of ATP-dependent chromatin-remodeling factors (remodelers), histone chaperones, and histone-modifying enzymes drive chromatin dynamics. Histones are subjected to a wide range of reversible posttranslational modifications, including acetylation, phosphorylation, methylation, and ubiquitylation. Histone modifications, in turn, can promote the recruitment of selective regulatory factors and modulate chromatin accessibility. Chromatin remodelers control DNA accessibility by mediating nucleosome mobilization either through sliding or by nucleosome (dis)assembly (Moshkin, 2009).

Whereas originally considered mainly as mere chaperones, it has become clear that histone chaperones play diverse roles during chromatin transactions. Histone chaperones bind selective histones and include the highly conserved H3/H4 chaperones ASF1, CAF1, HIRA, and Spt6 and the H2A/H2B chaperones NAP1, Nucleoplasmin, and FACT. Although their biochemical activity, binding and release of histones, appears rather mundane, in conjunction with other factors, histone chaperones participate in a variety of chromatin transactions and other cellular tasks. For example, yeast NAP1 participates in an extensive interaction network including a diverse set of transcription initiation/elongation factors, chromatin remodelers, RNA-processing factors, cell-cycle regulators, and other proteins (Moshkin, 2009).

ASF1 is one of the major H3/H4 chaperones, and through association with other proteins, it contributes to diverse chromatin transactions. (1) In conjunction with CAF1 and the MCM2-7 DNA helicase, ASF1 participates in replication-coupled chromatin assembly. (2) When associated with HIRA, ASF1 participates in replication-independent chromatin assembly and histone replacement. (3) DNA-repair-associated chromatin assembly requires the cooperation between ASF1 and the H3K56 acetyltransferase Rtt109. (4) ASF1 functionally cooperates with the Drosophila BRM chromatin remodeler, and (5) interaction of ASF1 with transcription activators stimulates histone eviction from promoter areas and facilitates recruitment of chromatin-specific coactivator complexes. (6) ASF1 itself is one of the targets of Tousled-like kinase (TLK), which controls cell-cycle progression and chromatin dynamics. (7) Finally, ASF1 is involved in developmental gene expression control by mediating transcriptional repression of Notch target genes. ASF1 is recruited to E(spl) genes by the sequence-specific DNA-binding protein Su(H) and its associated corepressor complex, harboring Hairless (H) and SKIP (Moshkin, 2009).

Notch is the central component of a highly conserved developmental signaling pathway that is present in all metazoans. Notch is a single-pass transmembrane protein that is activated through ligand binding, resulting in the release of the Notch intracellular domain (Nicd), which is targeted to the nucleus to activate gene expression. The CSL (CBF1, Su(H), and Lag1) family of sequence-specific DNA-binding proteins is the key targeting factor of Nicd and coactivators and, in the absence of Nicd, corepressors. The repression of Notch target genes involves multiple chromatin-modifying activities including histone deacetylases, H3K9 methyltransferases, CtBP, NcoR/SMRT, and Goucho (GRO). In the absence of the Nicd, loss of ASF1 leads to derepression of the E(spl) genes, revealing its essential role in silencing (Moshkin, 2009).

The molecular mechanism by which ASF1 achieves gene-specific transcription repression and the potential roles of other histone chaperones in developmental gene regulation remains largely unknown. To address these issues, a proteomics survey was performed of the protein interaction networks of ASF1, CAF1, HIRA, and NAP1 in Drosophila embryos. This analysis revealed that ASF1 and NAP1 interact with two related but distinct corepressor complexes: LAF and RLAF. LAF, comprising LID/KDM5 SIN3A, PF1, EMSY, and MRG15, associates with ASF1 (forming LAF-A). RLAF, comprising LAF plus RPD3, interacts with NAP1 (forming RLAF-N). Through a combination of biochemistry and developmental genetics, it was established that LAF-A and RLAF-N are tethered to Notch target genes by the Su(H)/H complex and mediate gene-selective silencing. Both ASF1 and NAP1 are required for the targeted removal of the positive H3K4me3 mark by facilitating LID/KDM5 recruitment to chromatin. Furthermore, NAP1 mediates nucleosome assembly at regulatory elements of Notch target genes and histone deacetylation by RLAF. These results uncover extensive crosstalk between distinct histone chaperones and histone-modifying enzymes in developmental gene regulation (Moshkin, 2009).

These results emphasize that, rather than generic, redundant factors, histone chaperones play highly specialized roles in gene-specific regulation. This study has dissected the molecular mechanism underpinning coordinate silencing of Notch target genes by the histone H3/H4 chaperone ASF1 and the H2A/H2B chaperone NAP1. ASF1 interacts with LAF, comprising SIN3A, PF1, EMSY, MRG15, and the histone H3K4me2/3 demethylase LID/KDM5, forming LAF-A. A closely related complex, RLAF that includes the deacetylase RPD3, does not bind ASF1. Instead, RLAF associates with NAP1, forming RLAF-N. The chaperones ASF1 and NAP1 link, respectively, LAF and RLAF to the Su(H)/H DNA-binding complex, tethering them to the E(spl) genes. Both ASF1 and NAP1 bind the SKIP subunit of the Su(H)/H complex (Goodfellow, 2007). Thus, at least in part, ASF1 and NAP1 facilitate H3K4me3 demethylation activity at the E(spl) genes through LID recruitment. Other LAFs might provide additional links to the Su(H)/H complex by contacting GRO and CtBP, which themselves associate with the Su(H)/H complex. For example, mammalian PF1, MRG15, and SIN3A have been reported to bind GRO. This study identified CtBP in LID, PF1, and NAP1 immunopurifications, providing an additional contact between the Su(H)/H complex and (R)LAF (Moshkin, 2009).

ASF1 does not bind RLAF and has no effect on histone H3 deacetylation by RPD3. In contrast, NAP1 does associate with RLAF and stimulates both H3K4 demethylation by LID and H3 deacetylation by RPD3. SIN3A had a mild effect, but the other LAF subunits played no apparent role in deacetylation. Finally, NAP1 depletion caused a dramatic loss of histones at the E(spl) regulatory elements, whereas ASF1 depletion had no effect on local histone density (Moshkin, 2009).

ASF1 has been proposed to function in chromatin assembly by acting as a donor that hands off the H3/H4 tetramer to either CAF1 or HIRA (De Koning, 2007). Because LAF-A does not associate with either CAF1 or HIRA, this might explain that ASF1 does not modulate nucleosome density at the E(spl) genes. In conclusion, the H3/H4 chaperone ASF1 mediates silencing of Notch target genes by (1) providing a connection between LAF and the Su(H)/H tether and (2) facilitating H3K4 demethylation by LID. The H2A/H2B chaperone NAP1 participates in E(spl) silencing by (1) linking RLAF to Su(H)/H, (2) facilitating H3K4 demethylation by LID, (3) facilitating H3 deacetylation by RPD3, and (4) directing high nucleosome density at repressed loci. The functioning of the H2A/H2B chaperone NAP1 in demethylation and deacetylation of histone H3 provides an example of trans-histone regulation (Moshkin, 2009).

LID and its interacting factors appear to work in a context-dependent manner. For example, LID facilitates activation of dMYC target genes in a manner independent of its demethylase activity. Suggestively, this study observed a genetic interaction between ASF1 and dMYC, indicating a potential role for LAF-A. Recently, it has been suggested that selective RLAF subunits could interact with a homolog of GATA zinc-finger domain-containing protein 1 to facilitate expression of targets by inhibition of RPD3 activity. In mammalian cells, LID homolog RBP2 and MRG15 have been implicated in transcription elongation by restricting H3K4me3 levels within transcribed regions. Identification of SIN3A as a LAF and RLAF subunit provides a molecular explanation for the recent observation that SIN3A is involved in genome-wide removal of both H3K4 methyl and acetyl marks. Collectively, these findings suggest that LID and RPD3 enzymatic activities can be modulated through association with specific partners. The proteomics analysis of the LID, PF1, and EMSY interaction networks further emphasizes the diverse involvement of LAFs in regulation of chromatin dynamics (Moshkin, 2009).

In conclusion, these results emphasize the close interconnectivity between distinct chromatin transactions and reveal cooperation between histone chaperones and targeted histone modifications during developmental gene control. The proteomic survey of ASF1, CAF1, HIRA, and NAP1 provides a starting point for the functional analysis of the regulatory networks in which these chaperones participate. As illustrated by the analysis of LAF-A and RLAF-N, specific protein-protein associations and gene targeting provide an intricate network of combinatorial gene expression control (Moshkin, 2009).

Histone Chaperone NAP1 Mediates Sister Chromatid Resolution by Counteracting Protein Phosphatase 2A

Chromosome duplication and transmission into daughter cells requires the precisely orchestrated binding and release of cohesin. This study found that the Drosophila histone chaperone NAP1 is required for cohesin release and sister chromatid resolution during mitosis. Genome-wide surveys revealed that NAP1 and cohesin co-localize at multiple genomic loci. Proteomic and biochemical analysis established that NAP1 associates with the full cohesin complex, but it also forms a separate complex with the cohesin subunit stromalin (SA). NAP1 binding to cohesin is cell-cycle regulated and increases during G2/M phase. This causes the dissociation of protein phosphatase 2A (PP2A) from cohesin, increased phosphorylation of SA and cohesin removal in early mitosis. PP2A depletion led to a loss of centromeric cohesion. The distinct mitotic phenotypes caused by the loss of either PP2A or NAP1, were both rescued by their concomitant depletion. In is concluded that the balanced antagonism between NAP1 and PP2A controls cohesin dissociation during mitosis (Moshkin, 2013).

As reflected by their name, a major activity of histone chaperones is to prevent illicit liaisons and guide newly synthesized histones to sites of chromatin assembly. This study describes a mitotic function for the canonical histone chaperone NAP1 that is unrelated to nucleosome assembly. NAP1 was found to bind cohesin and block dephosphorylation of SA by PP2A, thereby promoting cohesin dissociation from the chromosome arms. Consequently, chromosomal binding of cohesin during mitosis is controlled by the balance between the opposing activities of NAP1 and PP2A (Moshkin, 2013).

NAP1 is part of a large assemblage including the full cohesin complex and PP2A. In addition, NAP1 and SA form a subcomplex, which lacks the other cohesin subunits and PP2A. An attractive scenario is that the NAP1-SA module or NAP1 alone competes with PP2A-bound SA within the full cohesion complex. PP2A displacement by NAP1 allows stable phosphorylation of cohesin and its dissociation during early mitosis. NAP1 might also act as a direct inhibitor of PP2A catalytic activity, because a mammalian NAP1 homolog, SET, has been identified as a potent PP2A inhibitor, which promotes sister chromatid segregation during mouse oocyte miosis (Qi, 2013; Chambon, 2012). In addition, NAP1 might help cohesin phosphorylation by tethering Polo kinase to cohesin. In fact, a potential association between NAP1 and Polo kinase was detected. However, the dramatic chromosome condensation defects after Polo kinase depletion precluded a functional evaluation of a possible role of NAP1 in its function. Nevertheless, although additional NAP1 activities cannot be excluded, functional experiments established that blockage of PP2A suffices to explain the crucial role of NAP1 during sister chromatid resolution (Moshkin, 2013).

NAP1 not only regulates the chromosomal distribution of cohesin and PP2A, but also that of MeiS332, a fly homolog of Sgo. The function of MeiS332 and PP2A appears to be largely conserved from mammals to flies because they bind each other and depletion of either factor causes a loss of centromeric cohesion. Either knockdown of NAP1 or over-expression of PP2A caused spreading of MeiS332 onto the arms of mitotic chromosomes, accompanying the loss of sister chromatid resolution. Thus, the balanced antagonism between NAP1 and PP2A controls chromosomal association of both cohesin and MeiS332 during mitosis (Moshkin, 2013).

One level of regulation involves changes in NAP1's subcellular localization and chromatin binding through the cell cycle. At prophase there is a strong increase in the level of nuclear NAP1, but by metaphase, NAP1 and cohesin have dissociated from the chromosomes. Thus, the dynamic behavior of NAP1 correlates well with its function in promoting cohesin release at early mitosis. Regulation of NAP1 localization may involve cyclin B-cdc2/cdk1 kinase complexes. Previously it was found that yeast and vertebrate NAP1 are phosphorylated by cyclin B-cdc2 and that yeast cyclin B requires NAP1 for its full range of mitotic functions (Moshkin, 2013).

It is suggested that histone chaperones are at the hubs of specialized protein networks that perform a wide variety of tasks in chromosome biology. Through association with distinct partners, NAP1 is able to perform different functions. By acting as a histone chaperone, NAP1 mediates chromatin assembly. Through recruitment of the histone H3 deacetylase and H3K4 demethylase complex RLAF, NAP1 controls gene-selective silencing at developmental loci. Finally, by binding cohesin and blocking SA dephosphorylation by PP2A, NAP1 mediates sister chromatid resolution during mitosis. These results emphasize the surprisingly diverse- and specific regulatory functions of histone chaperones in chromosome biology (Moshkin, 2013).

Nucleosome assembly protein 1: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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