Binding of Acf1 to DNA Involves a WAC Motif and Is Important for ACF-Mediated Chromatin Assembly (original) (raw)
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Genes & Development, 1999
The assembly of core histones and DNA into periodic nucleosome arrays is mediated by ACF, an ISWI-containing factor, and NAP-1, a core histone chaperone, in an ATP-dependent process. We describe the isolation of Drosophila acf1 cDNA, which encodes the p170 and p185 forms of the Acf1 protein in ACF. Acf1 is a novel protein that contains two PHD fingers, one bromodomain, and two new conserved regions. Human WSTF, which is encoded by one of multiple genes that is deleted in Williams syndrome individuals, is the only currently known mammalian protein with each of the conserved motifs in Acf1. Purification of the native form of Acf1 led to the isolation of ACF comprising Acf1 (both p170 and p185 forms) and ISWI. Native Acf1 did not copurify with components of NURF or CHRAC, which are other ISWI-containing complexes in Drosophila. Purified recombinant ACF, consisting of Acf1 (either p185 alone or both p170 and p185) and ISWI, catalyzes the deposition of histones into extended periodic nucleosome arrays. Notably, the Acf1 and ISWI subunits function synergistically in the assembly of chromatin. ISWI alone exhibits a weak activity that is ∼3% that of ACF. These results indicate that both Acf1 and ISWI participate in the chromatin assembly process and suggest further that the Acf1 subunit confers additional functionality to the general 'motor' activity of ISWI.
Genes & Development, 2004
Chromatin assembly is required for the duplication of chromosomes. ACF (ATP-utilizing chromatin assembly and remodeling factor) catalyzes the ATP-dependent assembly of periodic nucleosome arrays in vitro, and consists of Acf1 and the ISWI ATPase. Acf1 and ISWI are also subunits of CHRAC (chromatin accessibility complex), whose biochemical activities are similar to those of ACF. Here we investigate the in vivo function of the Acf1 subunit of ACF/CHRAC in Drosophila. Although most Acf1 null animals die during the larval-pupal transition, Acf1 is not absolutely required for viability. The loss of Acf1 results in a decrease in the periodicity of nucleosome arrays as well as a shorter nucleosomal repeat length in bulk chromatin in embryos. Biochemical experiments with Acf1-deficient embryo extracts further indicate that ACF/CHRAC is a major chromatin assembly factor in Drosophila. The phenotypes of flies lacking Acf1 suggest that ACF/CHRAC promotes the formation of repressive chromatin. The acf1 gene is involved in the establishment and/or maintenance of transcriptional silencing in pericentric heterochromatin and in the chromatin-dependent repression by Polycomb group genes. Moreover, cells in animals lacking Acf1 exhibit an acceleration of progression through S phase, which is consistent with a decrease in chromatin-mediated repression of DNA replication. In addition, acf1 genetically interacts with nap1, which encodes the NAP-1 nucleosome assembly protein. These findings collectively indicate that ACF/CHRAC functions in the assembly of periodic nucleosome arrays that contribute to the repression of genetic activity in the eukaryotic nucleus.
Dynamics of ATP-dependent chromatin assembly by ACF
Nature, 2002
The assembly of DNA into chromatin is a critical step in the replication and repair of the eukaryotic genome 1-8 . It has been known for nearly 20 years that chromatin assembly is an ATPdependent process 9 . ATP-dependent chromatin-assembly factor (ACF) uses the energy of ATP hydrolysis for the deposition of histones into periodic nucleosome arrays, and the ISWI subunit of ACF is an ATPase that is related to helicases 10,11 . Here we show that ACF becomes committed to the DNA template upon initiation of chromatin assembly. We also observed that ACF assembles nucleosomes in localized arrays, rather than randomly distributing them. By using a purified ACF-dependent system for chromatin assembly, we found that ACF hydrolyses about 2-4 molecules of ATP per base pair in the assembly of nucleosomes. This level of ATP hydrolysis is similar to that used by DNA helicases for the unwinding of DNA 12 . These results suggest that a tracking mechanism exists in which ACF assembles chromatin as an ATP-driven DNA-translocating motor. Moreover, this proposed mechanism for ACF may be relevant to the function of other chromatin-remodelling factors that contain ISWI subunits.
Purification and Characterization of a Human Factor That Assembles and Remodels Chromatin
Journal of Biological Chemistry, 2000
We have previously reported the isolation and characterization of a nucleosome remodeling and spacing factor, RSF. One of the RSF subunits is hSNF2h, a SNF2 homologue. Here we set out to isolate and characterize other hSNF2h-containing complexes. We have identified a novel hSNF2h complex that facilitates ATP-dependent chromatin assembly with the histone chaperone NAP-1. The complex possesses ATPase activity that is DNA-dependent and nucleosome-stimulated. This complex is capable of facilitating ATP-dependent nucleosome remodeling and transcription initiation from chromatin templates. In addition to hSNF2h, this complex also contains a 190-kDa protein encoded by the BAZ1A gene. Since both subunits are homologues of the Drosophila ACF complex (ATP-utilizing chromatin assembly and remodeling factor), we have named this factor human ACF or hACF.
Effects of nucleosome remodeling factor ACF1 on in vivo chromatin organization
2015
Eukaryotic genomes make use of nucleosomes to considerably reduce their packaging volumes. As a consequence, the underlying DNA is rendered inaccessible. Cells make use of ATP-dependent remodeling factors to disrupt histone-DNA contacts and bring about access to the DNA. ACF1 is the largest regulatory subunit of two nucleosome remodeling factors, namely ACF and CHRAC. These complexes assemble, slide or evenly space nucleosomes on DNA with an ability to sense the linker lengths. However, roles of ACF1 in organizing nucleosomes in vivo and their physiological consequences are largely unclear. To understand the roles of ACF1 on chromatin organization, I compared nucleosome occupancy and transcription profiles in wild-type and ACF1-deficient Drosophila embryos. To further investigate and corroborate these chromatin changes, I performed genomewide mapping of ACF1 using chromatin immunoprecipitation. Nucleosome occupancy was mapped by subjecting DNA obtained from MNase-digested chromatin ...
Chromatin Assembly In Vitro with Purified Recombinant ACF and NAP1
Methods in Enzymology, 2003
To study eukaryotic transcriptional mechanisms in vitro, it is important to analyze gene regulatory sequences in the context of chromatin. Here we describe the ATP-dependent assembly of chromatin by using completely purified components. This system uses chromatin assembly factors ACF and NAP-1 in conjunction with purified core histones for the assembly of extended periodic arrays of nucleosomes. We additionally describe the assembly of chromatin that contains the linker histone H1. This histone H1-containing chromatin resembles bulk native chromatin in metazoans.
Role of Nucleosome Remodeling Factor NURF in Transcriptional Activation of Chromatin
Molecular Cell, 1997
studies show that the yeast and human SWI/SNF complex can directly assist site-specific binding of GAL4 The Drosophila nucleosome remodeling factor (NURF) derivatives in an ATP-dependent manner and can assist is a protein complex of four subunits that assists in local reconfiguration within an array of preassembled transcription factor-mediated perturbation of nucleonucleosomes (Cote et al., 1994; Imbalzano et al., 1994, somes in an ATP-dependent manner. We have investi-1996; Kwon et al., 1994; Brown et al., 1996; Owengated the role of NURF in activating transcription from Hughes et al., 1996; Wang et al., 1996b). RSC is an a preassembled chromatin template and have found essential and abundant multisubunit complex that conthat NURF is able to facilitate transcription mediated tains a subunit related to SWI2/SNF2 and has DNAby a GAL4 derivative carrying both a DNA binding and dependent ATPase activity and the ability to alter an activator domain. Interestingly, once nucleosome nucleosome structure in vitro (Cairns et al., 1996). remodeling by the DNA binding factor is accom-CHD-1, another member of the SWI2/SNF2 family, is plished, a high level of NURF activity is not continulocalized to the decondensed interbands of polytene ously required for recruitment of the general transcripchromosomes and to a number of active chromosome tional machinery and transcription for at least 100 puffs (Stokes et al., 1996). Another activity has been nucleotides. Our results provide direct evidence that identified in Drosophila embryo extracts, which in-NURF is able to assist gene activation in a chromatin creases accessibility of nucleosomal arrays to restriccontext, and identify a stage of NURF dependence tion enzymes and can render mobile an entire nucleosoearly in the process leading to transcriptional initimal array in an ATP-dependent manner (Varga-Weisz et ation. al., 1995). In previous studies, we purified the nucleosome re
ACF1 improves the effectiveness of nucleosome mobilization by ISWI through PHD–histone contacts
Embo Journal, 2004
The nucleosome remodelling ATPase ISWI resides in several distinct protein complexes whose subunit composition reflects their functional specialization. Association of ISWI with ACF1, the largest subunit of CHRAC and ACF complexes, improves the efficiency of ISWI-induced nucleosome mobilization by an order of magnitude and also modulates the reaction qualitatively. In order to understand the principle by which ACF1 improves the efficiency of ISWI, we mapped their mutual interaction requirements and generated a series of ACF complexes lacking conserved ACF1 domains. Deletion of the C-terminal PHD finger modules of ACF1 or their disruption by zinc chelation profoundly affected the nucleosome mobilization capability of associated ISWI in trans. Interactions of the PHD fingers with the central domains of core histones contribute significantly to the binding of ACF to the nucleosome substrate, suggesting a novel role for PHD modules as nucleosome interaction determinants. Connecting ACF to histones may be prerequisite for efficient conversion of ATP-dependent conformational changes of ISWI into translocation of DNA relative to the histones during nucleosome mobilization.
CHRAC/ACF contribute to the repressive ground state of chromatin
Life Science Alliance
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. Here, we 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 d...
Molecular Cell, 2001
taining complexes (SWI/SNF complexes) are most extensively studied and have been demonstrated to play a role in transcription in vivo and in vitro (Armstrong and Emerson. Although the SWI2/SNF2 family protein complexes display similar levels of ATP Summary utilization and generally increase chromatin accessibility , each family member appears to NURF is an ISWI complex of four proteins that uses recognize different aspects of the chromatin substrate the energy of ATP hydrolysis to catalyze nucleosome and can produce different outcomes in the process of sliding. Three NURF components have been identified chromatin remodeling. previously. We have cloned cDNA encoding the largest For instance, the ATPase activity of SWI/SNF is stimu-NURF subunit, revealing a 301 kDa polypeptide lated by either free or nucleosomal DNA, while the ISWI (NURF301) that shares structural motifs with ACF1. and Mi-2 proteins or complexes are stimulated only by We have reconstituted full and partial NURF comnucleosomes (Cote et al., 1994; Tsukiyama and Wu, plexes from recombinant proteins and show that 1995; Corona et al., 1999; Brehm et al., 2000; Guschin NURF301 and the ISWI ATPase are necessary and sufet al., 2000a). ISWI proteins or complexes require the ficient for accurate and efficient nucleosome sliding. flexible N-terminal histone tails, particularly the H4 tail, An HMGA/HMGI(Y)-like domain of NURF301 that facilifor stimulating ATPase activity and inducing nucleotates nucleosome sliding indicates the importance of some mobility (Georgel et al., 1997; Clapier et al., 2001), DNA conformational changes in the sliding mechabut histone tails have no apparent role in activating nism. NURF301 also shows interactions with sethe Mi-2 proteins (Brehm et al., 2000). ISWI complexes quence-specific transcription factors, providing a bamediate nucleosome "sliding," the movement of histone sis for targeted recruitment of the NURF complex to octamers in cis without permanent diplacement from specific genes. DNA (Hamiche et al., 1999; Langst et al., 1999). By contrast, the SWI/SNF or RSC complexes can mobilize Introduction nucleosomes (Whitehouse et al., 1999; Peterson, 2000), create a stably remodeled nucleosome intermediate The compaction of the eukaryotic genome in nucleo-(Cote et al., 1998; Schnitzler et al., 1998; Bazett-Jones somes limits the access of DNA to regulatory proteins et al., 1999), or transfer a histone octamer from one and the enzymes that process genetic information. To DNA fragment to another (Lorch et al., 1999). These overcome this constraint, cells employ a variety of stratdifferences indicate that there are diverse mechanisms egies to disrupt locally the organization of nucleosomes by which nucleosome structure can be altered to in-(Kornberg and Lorch, 1999; Wu and Grunstein, 2000). crease nucleosome dynamics and DNA accessibility. Prominent among these are several distinct classes of Biochemical assays for activities that can disrupt or enzymes that covalently modify specific residues of the enhance the periodic organization of nucleosome arrays N-terminal histone tails, thereby affecting nucleosome have been used to identify several chromatin remodeling stability or higher order nucleosome interactions (Strahl complexes that contain the ISWI ATPase. These include and Allis, 2000). A second group of multisubunit protein NURF, ACF, CHRAC, RSF, WCRF, ISWI-B, and ISWI-D complexes uses the energy of ATP hydrolysis to alter complexes in metazoa and the Isw1 and Isw2 complexes chromatin structure and mobilize nucleosomes (Vignali in budding yeast (Tsukiyama and Wu, 1995; Tsuet al., 2000). kiyama et al., 1995, 1999; Ito et al., 1997; Varga-Weisz To date, four classes of ATP-dependent chromatin et al., 1997; LeRoy et al., 1998, 2000; Bochar et al., 2000; remodeling complexes, each containing the SWI2/ Corona et al., 2000; Poot et al., 2000; Guschin et al., 2000b). SNF2, ISWI, CHD/Mi-2, and INO80 ATPases, or their Although the native ISWI complexes and the recombihighly related paralogs, have been characterized (Eisen nant ISWI protein have provided substantial insights, et al., 1995; Guschin and Wolffe, 1999; Peterson, 2000;