Dynamics of ATP-dependent chromatin assembly by ACF (original) (raw)
Related papers
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.
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.
The chromatin remodeller ACF acts as a dimeric motor to space nucleosomes
Nature, 2009
Evenly spaced nucleosomes directly correlate with condensed chromatin and gene silencing. The ATP-dependent chromatin assembly factor (ACF) forms such structures in vitro and is required for silencing in vivo. ACF generates and maintains nucleosome spacing by constantly moving a nucleosome towards the longer flanking DNA faster than the shorter flanking DNA. But how the enzyme rapidly moves back and forth between both sides of a nucleosome to accomplish bidirectional movement is unknown. We show that nucleosome movement depends cooperatively on two ACF molecules, suggesting that ACF functions as a dimer of ATPases. Further, the nucleotide state determines whether the dimer closely engages one vs. both sides of the nucleosome. Three-dimensional reconstruction by single particle electron microscopy of the ATPase-nucleosome complex in an activated ATP state reveals a dimer architecture in which the two ATPases face each other. Our results suggest a model in which the two ATPases work in a coordinated manner, taking turns to engage either side of a nucleosome, thereby allowing processive bidirectional movement. This novel dimeric motor mechanism differs from that of dimeric motors such as kinesin and dimeric helicases that processively translocate unidirectionally and reflects the unique challenges faced by motors that move nucleosomes.
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 ...
Journal of Biological Chemistry, 1998
To study the mechanisms of replication and transcription on chromatin, we have been using the adenovirus DNA complexed with viral basic core proteins, called Ad core. We have identified template activating factor (TAF)-I from uninfected HeLa cells as the factor that stimulates replication and transcription from the Ad core. The nuclease sensitivity assays have revealed that TAF-I remodels the Ad core, thereby making transcription and replication apparatus accessible to the template DNA. To examine whether TAF-I remodels the chromatin consisting of histones, the chromatin structure was reconstituted on the DNA fragment with core histones by the salt dialysis method. The transcription from the reconstituted chromatin was completely repressed, while TAF-I remodeled the chromatin and stimulated the transcription. TAF-I was found to interact with histones. Furthermore, it was shown that TAF-I is capable not only of disrupting the chromatin structure but also of preventing the formation of DNA-histone aggregation and transferring histones to naked DNA. The possible function of TAF-I in conjunction with a histone chaperone activity is discussed.
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.
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.