Gene name - ATP-utilizing chromatin assembly
Cytological map position -
Function - chromatin assembly
Symbol - Acf
FlyBase ID: FBgn0027620
Genetic map position -
Classification - PHD finger motifs and chromodomain
Cellular location - probably nuclear
|Recent literature||Börner, K., Jain, D., Vazquez-Pianzola, P., Vengadasalam, S., Steffen, N., Fyodorov, D.V., Tomancak, P., Konev, A., Suter, B. and Becker, P.B. (2016). A role for tuned levels of nucleosome remodeler subunit ACF1 during Drosophila oogenesis. Dev Biol [Epub ahead of print]. PubMed ID: 26851213
The Chromatin Accessibility Complex (CHRAC) consists of the ATPase ISWI, the large ACF subunit and a pair of small histone-like proteins, CHRAC-14 and CHRAC-16. CHRAC is a prototypical nucleosome sliding factor that mobilizes nucleosomes to improve the regularity and integrity of the chromatin fiber. This may facilitate the formation of repressive chromatin. This study explored roles for ACF1 during Drosophila oogenesis. ACF1 is expressed in somatic and germline cells, with notable enrichment in germline stem cells and oocytes. The asymmetrical localization of ACF1 to these cells depends on the transport of the Acf1 mRNA by the Bicaudal-D/Egalitarian complex. Loss of ACF1 function in the novel Acf17 allele leads to defective egg chambers and their elimination through apoptosis. In addition, a variety of unusual 16-cell cyst packaging phenotypes were found in the previously known Acf11 allele, with a striking prevalence of egg chambers with two functional oocytes at opposite poles. Surprisingly, Acf11 deletion - despite disruption of the Acf1 reading frame - expresses low levels of a PHD-bromodomain module from the C-terminus of ACF1 that becomes enriched in oocytes. Expression of this module from the Acf1 genomic locus leads to packaging defects in the absence of functional ACF1, suggesting competitive interactions with unknown target molecules. Remarkably, a two-fold overexpression of CHRAC (ACF1 and CHRAC-16) leads to increased apoptosis and packaging defects. Evidently, finely tuned CHRAC levels are required for proper oogenesis.
|Baldi, S., Jain, D. S., Harpprecht, L., Zabel, A., Scheibe, M., Butter, F., Straub, T. and Becker, P. B. (2018). Genome-wide rules of nucleosome phasing in Drosophila. Mol Cell 72(4): 661-672. PubMed ID: 30392927
Regular successions of positioned nucleosomes, or phased nucleosome arrays (PNAs), are predominantly known from transcriptional start sites (TSSs). It is unclear whether PNAs occur elsewhere in the genome. To generate a comprehensive inventory of PNAs for Drosophila, spectral analysis was applied to nucleosome maps and identified thousands of PNAs throughout the genome. About half of them are not near TSSs and are strongly enriched for an uncharacterized sequence motif. Through genome-wide reconstitution of physiological chromatin in Drosophila embryo extracts, the molecular basis of PNA formation was uncovered. Phaser, an unstudied zinc finger protein that positions nucleosomes flanking the motif, was uncovered. It also revealed how the global activity of the chromatin remodelers CHRAC/ACF, together with local barrier elements, generates islands of regular phasing throughout the genome. This work demonstrates the potential of chromatin assembly by embryo extracts as a powerful tool to reconstitute chromatin features on a global scale in vitro.
|Scacchetti, A., Brueckner, L., Jain, D., Schauer, T., Zhang, X., Schnorrer, F., van Steensel, B., Straub, T. and Becker, P. B. (2018). CHRAC/ACF contribute to the repressive ground state of chromatin. Life Sci Alliance 1(1): e201800024. PubMed ID: 30456345
The chromatin remodeling complexes chromatin accessibility complex and ATP-utilizing chromatin assembly and remodeling factor (ACF) combine the ATPase ISWI with the signature subunit ACF1. These enzymes catalyze well-studied nucleosome sliding reactions in vitro, but how their actions affect physiological gene expression remains unclear. This study explored the influence of Drosophila melanogaster chromatin accessibility complex/ACF on transcription by using complementary gain- and loss-of-function approaches. Targeting ACF1 to multiple reporter genes inserted at many different genomic locations revealed a context-dependent inactivation of poorly transcribed reporters in repressive chromatin. Accordingly, single-embryo transcriptome analysis of an Acf knock-out allele showed that only lowly expressed genes are derepressed in the absence of ACF1. Finally, the nucleosome arrays in Acf-deficient chromatin show loss of physiological regularity, particularly in transcriptionally inactive domains. Taken together, these results highlight that ACF1-containing remodeling factors contribute to the establishment of an inactive ground state of the genome through chromatin organization.
Nucleosome assembly is a fundamental biological process that is required for the replication and maintenance of chromatin in the eukaryotic nucleus. In dividing cells newly synthesized DNA is assembled rapidly into chromatin by a process that appears to involve an indirect coupling between DNA replication and nucleosome assembly. In quiescent cells, nucleosome assembly is required to (1) replace histones that are lost in the course of histone turnover and to (2) regenerate chromatin upon DNA repair. In addition, there may be a requirement for chromatin assembly during events, such as transcription, that involves the disruption of nucleosomes by the passage of polymerases.
Biochemical studies have identified proteins that are able to mediate the reconstitution of core histones into nucleosomes. Nearly all of these factors are core histone-binding proteins that contain stretches of acidic amino acid residues. Some of these histone chaperones, such as nucleoplasmin and nucleosome assembly protein-1 (NAP-1), exhibit a preference for binding to histones H2A and H2B relative to histones H3 and H4. Other histone chaperones, which include chromatin assembly factor 1 (CAF-1), N1/N2, and Spt6, associate preferentially with H3 and H4. Interestingly, it has also been observed that newly synthesized histones are acetylated (such as at positions 5, 8, and 12 of histone H4) and then subsequently deacetylated after assembly into chromatin. Thus, factors that mediate histone acetylation or deacetylation may participate, perhaps indirectly by the covalent modification of histones, in the chromatin assembly process (Ito, 1999 and references).
It is useful to consider a two-step mechanism for chromatin assembly: 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 (Ito, 1997). Whereas the first step of nucleosome assembly is carried out by the histone chaperones, present in multiprotein complexes, the second step is carried out by an additonal multiprotein complex termed ACF (for ATP-utilizing chromatin assembly and remodeling factor), which has already been identified and purified (Ito, 1997). Purified ACF acts catalytically (at approximately one ACF protomer per 90 core histone octamers) in the deposition of histones to yield periodic nucleosome arrays in an ATP-dependent process. This chromatin assembly reaction, which can be carried out in a 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). The most purified preparations of ACF have been observed to consist of Imitation SWI (ISWI) protein (Elfring, 1994) in addition to three other polypeptides. ISWI, a component of multiple chromatin assembly machines, copurifies precisely with ACF. The other three polypeptides of 47, 170, and 185 kD were assigned tentatively as subunits of ACF (Ito, 1997).
The high molecular weight component of ACF has now been cloned. Acf1 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, the subject of this essay, as Acf1). Purification of native Acf1 from Drosophila embryos leads to the isolation of ACF consisting of Acf1 (both p170 and p185 forms) and subunits. Acf1 does not, however, copurify with components of NURF (Tsukiyama, 1995) or CHRAC (Varga-Weisz, 1997), which are other chromatin remodeling complexes from Drosophila that similarly contain an ISWI subunit. Studies of purified recombinant ACF reveal that the Acf1 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).
Synthesis and purification of the recombinant proteins was undertaken because native ACF appears to be made up of of Acf1 and ISWI. To this end, baculovirus expression vectors for full-length Acf1 protein as well as for full-length Acf1 with a carboxy-terminal Flag (Sigma) epitope tag (termed Acf-Flag) were constructed. A baculovirus vector that produces amino-terminally Flag-tagged Drosophila ISWI (Flag-ISWI) was also available for this project. Acf-Flag and Flag-ISWI were individually synthesized and purified. In addition, an ACF complex comprising both Acf1 and ISWI was obtained by cosynthesis of Acf1 and Flag-ISWI followed by affinity purification via the Flag epitope tag to yield ACF consisting of both Acf1 and Flag-ISWI (Ito, 1999).
Interestingly, both the p170 and p185 varients of Acf1 can be derived from the Acf1 cDNA. When Acf-Flag is affinity-purified via its carboxy-terminal Flag tag, only the p185 form of Acf1 is seen, even when a large excess of protein is analyzed by gel electrophoresis. In contrast, both the p185 and p170 forms of Acf1 copurify with Flag-ISWI. These data suggest that both p170 and p185 are produced from the Acf1 cDNA, but the structural relation between the p170 and p185 forms is not yet known (Ito, 1999).
To determine whether the recombinant proteins can function in the assembly of nucleosome arrays, chromatin assembly reactions were carried out with purified core histones, purified dNAP-1, relaxed circular plasmid DNA, and ATP (with an ATP regenerating system) along with varying concentrations of purified recombinant ACF (cosynthesized Acf1 + Flag-ISWI). These experiments revealed significant chromatin assembly activity at a core histone octamer/ACF molar ratio of 150:1. An optimal chromatin assembly activity can be achieved at an octamer/ACF molar ratio of 50:1 (Ito, 1999). The ability of recombinant ACF to catalyze the assembly of nucleosome arrays at an octamer/ACF ratio of ~50:1 to 150:1 is consistent with the previously observed ability of native ACF to assemble chromatin at an estimated octamer/ACF ratio of 90:1 (Ito, 1997).
An important feature of ACF is its ability to facilitate the deposition of histones onto DNA (Ito, 1997). The ability of recombinant ACF to catalyze the formation of nucleosomes by was analyzed using the DNA supercoiling assay. There is inefficient deposition of histones by purified dNAP-1 in the absence of ACF. Once ACF is added, there is a significant increase in the extent of nucleosome assembly. Thus, recombinant ACF functions as a nucleosome assembly factor that facilitates the deposition of histones onto DNA by a process that yields periodic nucleosome arrays. Hence, the recombinant ACF, which comprises Acf1 and ISWI, possesses chromatin assembly activity that is comparable to that of native ACF (Ito, 1999).
To investigate the role of Acf1 in nucleosome assembly by ACF, standard chromatin assembly reactions were carried out with recombinant Acf1 alone, ISWI alone, or ACF. These experiments revealed no detectable chromatin assembly with either Acf1 alone or ISWI alone. In contrast, chromatin assembly activity was seen with ACF and is dependent on the presence of ATP. The finding that 1 unit of ACF yields greater chromatin assembly activity than 10 units of either subunit alone indicates that the Acf1 and ISWI subunits of ACF function cooperatively in the assembly of periodic nucleosome arrays. It is estimated that ACF assembles chromatin ~30-fold more efficiently than ISWI alone. Because the Acf1 and ISWI subunits are both required for full ACF activity, purified preparations of the separate subunits were tested to determine if they could be combined to yield active ACF. Chromatin assembly reactions were carried with Acf-Flag and Flag-ISWI that had been combined after synthesis and purification (postsynthetic) as well as with Acf1 + Flag-ISWI that had been cosynthesized and purified as the ACF complex (cosynthesis). The activity of the postsynthetically combined ACF subunits is essentially identical to that of the cosynthesized subunits. Therefore, cosynthesis of the Acf1 and ISWI subunits is not necessary to obtain fully active ACF. In addition, full ACF activity can be achieved with the p185 form of Acf1 along with ISWI (Ito, 1999).
The finding that Acf1 can significantly increase the activity of the ISWI protein indicates that the proposed motor-like activity of ISWI (as well as related proteins, such as SNF2/SWI2, STH2, Brahma, hSNF2h, hBRM, and BRG1, which are in other chromatin remodeling complexes) can be programmed for specific functions by other components of the remodeling complexes. Thus far, three ISWI-containing complexes, termed NURF, CHRAC, and ACF, have been identified in Drosophila; one ISWI (hSNF2h)-containing complex termed RSF (remodeling and spacing factor) has been found in humans (LeRoy, 1998), and two complexes termed ISW1 and ISW2, which each contain a distinct ISWI-related protein, have been purified from S. cerevisiae (Tsukiyama, 1999). It seems likely that some of the non-ISWI subunits in NURF, CHRAC, RSF, and the ISW1 and ISW2 complexes in yeast will similarly confer additional functionality to the ISWI motor in a manner analogous to Acf1. In the future, it will be interesting to investigate the nature of Acf1 function, as well as to compare the properties of Acf1 with those of related proteins in other chromatin remodeling complexes (Ito, 1999).
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).
acf1 is a novel gene that encodes a protein of 1476 amino acid residues and has a calculated molecular mass of 170,350 daltons. Acf1 protein has two PHD finger motifs, one bromodomain, and two novel conserved regions termed WAC (WSTF/Acf1/cbp146) and WAKZ (WSTF/Acf1/KIAA0314/ZK783.4) motifs. An analysis of sequence databases reveals only one protein, human WSTF (Williams syndrome transcription factor; Lu, 1998), which possesses both WAC and WAKZ motifs as well as a PHD finger and a bromodomain at the carboxyl terminus. WSTF has been identified as a gene that is deleted in Williams syndrome (WS) individuals (Lu, 1998). WS is a developmental disorder that results from hemizygous deletion of multiple contiguous genes at chromosome 7q11.23. WSTF has been presumed to be a transcription factor because it contains a PHD finger and a bromodomain, but its biochemical function is not known. Given the structural relation between Acf1 and WSTF, it is possible that the analysis of ACF may provide information that is relevant to WS (Ito, 1999).
Other proteins possess either a WAC or a WAKZ motif. A WAC motif is present in mouse cbp146 and in two Saccharomyces cerevisiae open reading frames, YGL133w and YPL216w. Unlike Acf1, neither YGL133w nor YPL216w proteins possess a WAKZ motif, a PHD finger, or a bromodomain. Because the cbp146 cDNA is incomplete, it is not known whether it shares other features of Acf1. A WAKZ motif is immediately amino-terminal to the first PHD finger of Acf1 and is also found in human cDNA KIAA0314 and Caenorhabditis elegans ZK783.4. KIAA0314 was identified in a screen for novel human cDNAs and encodes a protein of unknown function. It appears to be identical to TTF-I interacting peptide 5, which was found in a yeast two-hybrid screen for proteins that can bind to TTF-I. ZK783.4 was identified in the C. elegans genome sequence. Notably, both KIAA0314 and ZK783.4 have one WAKZ motif, one PHD finger, and one bromodomain in an arrangement similar to that of Acf1 and WSTF. Also, the bromodomain motifs of Acf1, WSTF, KIAA0314, and ZK783.4 are closely related. Thus, it is possible that WSTF, KIAA0314, and ZK783.4 are subunits of factors that are related to ACF (Ito, 1999).
Chromatin remodelling complexes containing the nucleosome-dependent ATPase ISWI were first isolated from Drosophila embryos (NURF, CHRAC and ACF). ISWI was the only common component reported in these complexes. Purification of human CHRAC (HuCHRAC) shows that ISWI chromatin remodelling complexes can have a conserved subunit composition in completely different cell types, suggesting a conserved function of ISWI. The human homologs of two novel putative histone-fold proteins in Drosophila CHRAC (CHRAC-14 and CHRAC-16) are present in HuCHRAC. The two human histone-fold proteins form a stable complex that binds naked DNA but not nucleosomes. HuCHRAC also contains human ACF1 (hACF1), the homolog of Acf1, a subunit of Drosophila ACF. The N-terminus of mouse ACF1 was reported as a heterochromatin-targeting domain. hACF1 is a member of a family of proteins with a related domain structure that all may target heterochromatin. A possible function for HuCHRAC in heterochromatin dynamics is discussed. HuCHRAC does not contain topoisomerase II, which was reported earlier as a subunit of Drosophila CHRAC (Poot, 2000).
The histone fold is a structural motif with which two related proteins interact and is found in complexes involved in wrapping DNA, the nucleosome, and transcriptional regulation, as in the transcriptional regulator NC2. A novel function for histone-fold proteins has been revealed: facilitation of nucleosome remodeling. ACF1-ISWI complex (TP-dependent chromatin assembly and remodeling actor [ACF]) associates with histone-fold proteins (CHRAC-15 and CHRAC-17 in the human chomatin accessibility complex [CHRAC]) whose functional relevance has been unclear. These histone-fold proteins facilitate ATP-dependent nucleosome sliding by ACF. Direct interaction of the CHRAC-15/17 complex with the ACF1 subunit is essential for this process. CHRAC-17 interacts with another histone-fold protein, p12, in DNA polymerase epsilon, but CHRAC-15 is essential for interaction with ACF and enhancement of nucleosome sliding. Surprisingly, CHRAC-15/17, p12/CHRAC-17, and NC2 complexes facilitate ACF-mediated chromatin assembly by a mechanism different from nucleosome sliding enhancement, suggesting a general activity of H2A/H2B type histone-fold complexes in chromatin assembly (Kukimoto, 2005).
Possible biological roles of the CHRAC-15/17 complex are suggested. SNF2H and hACF1 are targeted to replication foci in pericentromeric heterochromatin and these proteins have a role in DNA replication through condensed chromatin. In light of these findings, it is interesting that CHRAC-17 has also been identified as a subunit of the DNA polymerase epsilon. There is evidence that DNA polymerase epsilon is specifically targeted to replication foci in heterochromatin in HeLa cells. It is tempting to speculate that CHRAC-17 may coordinate some interaction between DNA polymerase epsilon and CHRAC. The enhanced nucleosome assembly supplied by CHRAC-15/17 may suggest a role of CHRAC in the formation of regular nucleosome arrays in heterochromatin after replication. Alternatively, the observation that CHRAC-15/17 facilitates nucleosome sliding under conditions where ACF alone is not efficient may point to a role for these proteins in areas of chromatin, where conditions for nucleosome sliding are not favorable. Nucleosome remodeling within heterochromatin, which is highly compact and less accessible than euchromatin, may require the accessory help of these histone-fold proteins (Kukimoto, 2005).
date revised: 1 December 2007
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
Society for Developmental Biology's Web server.