Nucleosome assembly protein 1

Protein Interactions

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).

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).

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