ATP-dependent Chromatin Assembly Factor Research Articles

Abstract 5713: Alternative splicing of the chromatin modifier gene BAZ1A in colorectal cancer

Abstract Bromodomain Adjacent to Zinc finger Domain 1A (BAZ1A) is a non-catalytic subunit of the ATP-dependent chromatin assembly factor that regulates key cellular processes such as DNA replication, chromatin assembly, DNA repair, and transcriptional gene regulation (1). Defective DNA repair is a hallmark of cancer etiology, and BAZ1A is one of the deregulated proteins in colorectal cancer. The BAZ1A transcript exhibits full-length and truncated forms, generated via alternative splicing of exon-13. We observed that the histone deacetylase inhibitor sulforaphane (SFN) modulated alternative splicing of BAZ1A in human colon cancer cells by increasing the relative expression of the short vs. the long transcript. Cell Cycle and Apoptosis Regulator 2 (CCAR2) and Zinc finger protein 326 (ZIRD) are components of the DBIRD complex that integrates alternative splicing and the rate of transcript elongation (2). Knockdown of DBIRD members, including ZIRD and splicing factor hnRNPA1, downregulated expression of BAZ1A long form, whereas the BAZ1A short form was unaltered. Previous studies in SFN-treated colon cancer cells revealed that the BAZ1A bromodomain recognized a novel K97 acetylation site in CCAR2 (3). Co-immunoprecipitation analyses revealed that CCAR2 and BAZ1A interacted with each other in the presence and absence of SFN. On investigating the involvement of CCAR2 in BAZ1A alternative splicing, SFN treatment increased the expression of both BAZ1A long and short forms in CCAR2-/- HCT116 colon cancer cells. Although the total expression of BAZ1A was influenced by CCAR2 loss, alternative splicing of BAZ1A was independent of DBIRD complex-mediated regulation. Pursuing the role of p53 and p21 in BAZ1A alternative splicing, SFN treatment increased the expression of BAZ1A short form in p21-/- HCT116 cells, whereas this was impaired in p53-/- HCT116 cells. Overall, this investigation provided mechanistic insights into the regulation of alternative splicing in SFN-treated colon cancer cells, and BAZ1A functionalities that might be targeted via next-generation combinatorial epigenetic agents (4). Supported in part by NCI grant CA122959, by the John S. Dunn Foundation, and by a Chancellor’s Research Initiative.

A Nucleotide-Driven Switch Regulates Flanking DNA Length Sensing by a Dimeric Chromatin Remodeler

The ATP-dependent chromatin assembly factor (ACF) spaces nucleosomes to promote formation of silent chromatin. Two copies of its ATPase subunit SNF2h bind opposite sides of a nucleosome, but how these protomers avoid competition is unknown. SNF2h senses the length of DNA flanking a nucleosome via its HAND-SANT-SLIDE (HSS) domain, yet it is unclear how this interaction enhances remodeling. Using covalently connected SNF2h dimers we show that dimerization accelerates remodeling and that the HSS contributes to communication between protomers. We further identify a nucleotide-dependent conformational change in SNF2h. In one conformation the HSS binds flanking DNA, and in another conformation the HSS engages the nucleosome core. Based on these results, we propose a model in which DNA length sensing and translocation are performed by two distinct conformational states of SNF2h. Such separation of function suggests that these activities could be independently regulated to affect remodeling outcomes.

The Chromatin-Remodeling Complex ACF Functions as a Dimeric Motor to Space Nucleosomes

The chromatin structure at a given locus is a key determinant of its transcriptional state. Evenly spaced nucleosomes directly correlate with condensed chromatin structures and gene silencing. The ATP-dependent chromatin assembly factor (ACF) generates such structures in vitro and is required for transcriptional silencing in vivo. ACF generates and dynamically 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 such bidirectional movement is not known. Using FRET to follow disruption of histone-DNA interactions in real time we show that nucleosome movement depends cooperatively on two ACF molecules, suggesting that ACF functions as a dimer of ATPases. Employing Electron Paramagnetic Resonance (EPR) to resolve different populations of the nucleosome-ATPase complex, we find that the nucleotide state determines whether the dimer closely engages one vs. both sides of the nucleosome. Furthermore 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 bind facing 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. Such a mechanism would allow rapid sampling of both sides of the nucleosome and allow bidirectional movement without dissociation. This novel dimeric motor mechanism differs from that of other dimeric motors such as kinesin and dimeric helicases that processively translocate in one direction and reflects the unique challenges faced by motors that move nucleosomes.

The chromatin remodeller ACF acts as a dimeric motor to space nucleosomes

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.

Mechanism of the ATP‐dependent chromatin remodeling complex ACF

Silent loci are often characterized by regularly spaced nucleosomes. The ATP-dependent chromatin assembly factor ACF generates these structures, yet how it measures out equal spacing between nucleosomes is not known. We have used fluorescence resonance energy transfer to follow nucleosome movement in real time. We show that ACF senses the length of flanking DNA and transduces this information in an ATP-dependent manner to regulate the rate of nucleosome movement. Our data also suggests a mechanism to explain previous results indicating a role for the histone H4 tail in the ACF reaction.

The chromatin-remodeling enzyme ACF is an ATP-dependent DNA length sensor that regulates nucleosome spacing

Arrays of regularly spaced nucleosomes directly correlate with closed chromatin structures at silenced loci. The ATP-dependent chromatin-assembly factor (ACF) generates such arrays in vitro and is required for transcriptional silencing in vivo. A key unresolved question is how ACF 'measures' equal spacing between nucleosomes. We show that ACF senses flanking DNA length and transduces length information in an ATP-dependent manner to regulate the rate of nucleosome movement. Using fluorescence resonance energy transfer to follow nucleosome movement, we find that ACF can rapidly sample DNA on either side of a nucleosome and moves the longer flanking DNA across the nucleosome faster than the shorter flanking DNA. This generates a dynamic equilibrium in which nucleosomes having equal DNA on either side accumulate. Our results indicate that ACF generates the characteristic 50- to 60-base-pair internucleosomal spacing in silent chromatin by kinetically discriminating against shorter linker DNAs.

The Histone Chaperone TAF-I/SET/INHAT Is Required for Transcription In Vitro of Chromatin Templates

To uncover factors required for transcription by RNA polymerase II on chromatin, we fractionated a mammalian cell nuclear extract. We identified the histone chaperone TAF-I (also known as INHAT [inhibitor of histone acetyltransferase]), which was previously proposed to repress transcription, as a potent activator of chromatin transcription responsive to the vitamin D3 receptor or to Gal4-VP16. TAF-I associates with chromatin in vitro and can substitute for the related protein NAP-1 in assembling chromatin onto cloned DNA templates in cooperation with the remodeling enzyme ATP-dependent chromatin assembly factor (ACF). The chromatin assembly and transcriptional activation functions are distinct, however, and can be dissociated temporally. Efficient transcription of chromatin assembled with TAF-I still requires the presence of TAF-I during the polymerization reaction. Conversely, TAF-I cannot stimulate transcript elongation when added after the other factors necessary for assembly of a preinitiation complex on naked DNA. Thus, TAF-I is required to facilitate transcription at a step after chromatin assembly but before transcript elongation.

Chromatin remodelling: it's a tracking business

Chromatin is a dynamic material consisting of DNA wrapped around nucleosomes. Chromatin assembly is very important for many processes, such as DNA replication and repair, and it is necessary for the compaction of the eukaryotic genome. One of the complexes involved in chromatin assembly is the ATP-dependent chromatin-assembly factor (ACF), which assembles nucleosomes onto DNA. It consists of two subunits: ISWI, which contains the ATP hydrolysis activity, and Acf1. ISWI has motifs in common with DNA helicases, which unwind the DNA helix. However, it is not known how ACF works, as no helicase activity has been detected for chromatin remodelling complexes. Fyodorov and Kadonaga have provided insight into the mechanism of ACF by showing that it operates as a processive DNA-tracking motor to assemble nucleosomes on DNA [1xDynamics of ATP-dependent chromatin assembly by ACF. Fyodorov, D.V. and Kadonaga, J.T. Nature. 2002; 418: 896–900CrossrefSee all References][1].If ACF is a DNA-tracking enzyme, a proportion of the ACF complexes will be in the process of tracking along DNA and, therefore, unavailable to assemble chromatin on a new DNA substrate. Using an in vitro chromatin assembly assay with two sequentially added substrates, the authors show that once chromatin assembly is initiated on the first DNA substrate, it is not initiated as efficiently on the second. This implies that the complex is involved in tracking along the DNA. The authors saw a lag of ∼15–30 minutes before nucleosome assembly on the second substrate, suggesting that this is the length of time ACF tracks along DNA. When the authors used a single template assay and sub-stoichiometric levels of ACF, the products consisted of two populations. One population had partially assembled chromatin, with about seven nucleosomes, and the other had none. This suggests that the complex loads about seven nucleosomes before dissociation. When these complexes were digested with a nuclease, the nucleosomes were shown to be in tandem arrays. Together, these data imply that ACF assembles nucleosomes by tracking processively along the DNA.Several other enzymes that track along DNA are ATP dependent. For example, helicases hydrolyze ∼1–3 ATP molecules per base pair. Fyodorov and Kadonaga examined the ATP requirement for chromatin assembly by ACF and found that the complex hydrolyzed 2–4 ATP molecules per base pair, comparable to the ATP consumption of helicases. Therefore, ACF is a DNA-tracking motor that requires large amounts of energy. ACF could require more energy than helicases if other steps in nucleosome assembly are ATP-dependent, but this is a subject for future research. It is likely that the tracking mechanism of ACF also applies to other chromatin remodelling complexes.

Dynamics of ATP-dependent chromatin assembly by ACF.

The assembly of DNA into chromatin is a critical step in the replication and repair of the eukaryotic genome. It has been known for nearly 20 years that chromatin assembly is an ATP-dependent process. 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. 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#150;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. 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.