Assays of nucleosome assembly and the inhibition of histone acetyltransferase activity
Lab/Group: Yokoyama Lab (RIKEN BRC), Nagata Lab (Tsukuba University)
Related Journal & Article Information
Journal: Nature Structural & Molecular Biology
Article Title: Regulation of histone acetylation and nucleosome assembly by transcription factor JDP2
Summary
Chromatin is a dynamic structure that can adopt markedly different conformations. During transcription, different conformations of chromatin act as important regulatory switches. A novel type of cellular complex, designated INHAT (inhibitor of histone acetyltransferases), was isolated recently and was shown to inhibit the HAT activity of p300/CBP and PCAF by binding to histones, preventing them from serving as substrates for acetyltransferases. This complex was demonstrated, initially, to have nucleosome-assembly activity. In order to examine whether other factors might have both HAT-inhibitory and nucleosome-assembly activities, similar to those of INHATs, we developed methods for measuring the inhibition of HAT activity and nucleosome-assembly activity in vitro and in vivo. Our methodology is particularly useful for measuring the histone-chaperone activity of specific proteins.
Introduction
The structure of chromatin, which influences numerous DNA-associated phenomena, such as transcription, replication, recombination and repair, is controlled by a complex combination of histone modifications, ATP-dependent chromatin-remodeling enzymes, and nucleosome-assembly factors1-3. The nucleosome is the basic unit of chromatin, consisting of a core of 147 bp of DNA that is wrapped around a histone octamer (two molecules each of H2A, H2B, H3 and H4). A stretch of linker DNA connects adjacent nucleosomes. The linker histone H1 promotes the packaging of strings of nucleosomes into 30-nm-diameter chromatin fibers4. Packaging of DNA into nucleosomes and into chromatin fibers greatly restricts the availability of the DNA for nuclear processes, such as transcription. Chromatin assembly occurs with the initial deposition of a histone (H3-H4)2 tetramer, prior to the addition of two heterodimers of histones H2A and H2B to form nucleosomes5,6. Transcriptional regulation is associated with rearrangements of chromatin structure that include histone modifications and changes in nucleosome structures7-11. Compaction of chromatin and arrangement of the nucleosome itself represent barriers that have to be overcome for the activation of transcription. The amino-terminal (N-terminal) histone tails protruding from the nucleosome do not participate in nucleosome formation to any significant extent but, rather, they provide docking sites for other proteins and protein complexes to regulate chromatin compaction9. Among the identified N-terminal modifications of histones, acetylation occurs at highest frequency and has been studied the most extensively, with the resultant identification of a number of histone acetyltransferases (HATs) and histone deacetylases (HDACs), as well as their target lysine residues within histones in the chromatin. As components of larger complexes, many HATs have been identified independently as regulators of transcription, for example, as activators, coactivators, elongators and components of general transcription factors. These various observations indicate that HATs are involved in almost all aspects of transcription12-16. An enzyme responsible for histone acetylation, designated HATA, was identified first in Tetrahymena17. HATA is strongly homologous, at the amino acid level, to the yeast protein GCN5, which also catalyzes the acetylation of histones. To date, close to 30 proteins have been shown to have HAT activity. It is possible that each HAT might be specific for a particular histone substrate and that the various HATs act specifically with regard to the amino acid(s) within the histone(s) that is acetylated. The human MYST family of acetyltransferases includes hMOF, TIP60, HBO1, MOZ and MORF18-22. Although these proteins are not homologous, at the amino acid level, to CBP/p300, PCAF and GCN523-25, they have been implicated in the regulation of several critical steps in the response to DNA damage.
The activities of HATs are not only antagonized by HDACs but can also be regulated by certain cellular and viral regulatory factors and by post-translational modification26. Thus, for example, the basic helix-loop-helix protein (bHLH protein) Twist and the adenoviral oncoprotein E1A inhibit the acetyltransferase activities of p300/CBP and PCAF27,28, while TAT, the transactivator protein of HIV1, represses acetylation that involves Tip60 and TAFII25029,30. One potential mechanism for such inhibition includes the direct binding of acetyltransferases to these regulatory factors. Alternatively, for example, the activity of GCN5-HAT might be inhibited via phosphorylation by the Ku-DNA-dependent protein kinase31, while the HAT activities of CBP/p300 and ATF-2 are stimulated by phosphorylation32-34.
The structure of chromatin changes, to allow greater accessibility by transcription factors, during gene activation9. It has been suggested that a more open chromatin state, associated with gene activation but separate from histone modification and alterations in nucleosomal arrays, also results from changes in nucleosome integrity due to displacement of histones8. Moreover, it has been demonstrated that histone chaperones play important roles in these processes35-37.
Both the deposition and arrangement of nucleosomes can represent barriers to transcriptional activation. Thus, it is tempting to speculate that histone chaperones are also involved in the compaction of chromatin. It is also important to determine whether a transcriptional corepressors influence nucleosome depostition and assembly through the regulation of histone chaperone activity.
A novel complex, designated INHAT (inhibitor of histone acetyltransferases), was isolated recently from human cells and shown to inhibit the HAT activities of p300/CBP and PCAF by binding to histones, thereby preventing them from serving as substrates for acetyltransferases35. One type of INHAT complex is known as template-activating factor-1β (TAF-1β; also known as the myeloid leukemia-associated oncoprotein Set), and this complex was shown initially to have nucleosome-assembly activity36-38. The nuclear protein pp32 is another type of INHAT39,40.
In view of their potentially important roles, it is of interest to determine whether other factors might have both HAT-inhibitory and nucleosome-assembly activities that are similar to those of INHAT.
We have developed a method for measuring the histone-chaperone activity that is associated with inhibition of histone acetylation and nucleosome assembly in vitro and in vitro. We describe this method here and demonstrate its power using transcription factor JDP2 as a model protein with HAT-inhibitory and nucleosome-assembly activity.
Discussion
Histone acetyl transferases (HATs) are a diverse set of enzymes that can be classified on the basis of their catalytic domains. Different HAT complexes, such as members of the Gcn5 N-acetyltransferase (GNAT) family, MYST HATs and the Orphan class of HATs, are composed of various unique subunits. Combinations of these subunits participate in the formation of HAT complexes. For example, some subunits have domains that cooperate to recruit the HAT to the appropriate location on the genome. These domains include bromodomains, chromodomains, WD40 repeats, Tudor domains and PHD fingers2 and they are all chromatin-binding domains that recognize histone tails. Moreover, the inhibitory activities of the HAT-like histone deacetylase complex (HDAC complex) and inhibitor of histone acetyltransferase (INHAT) are also important for regulation of chromatin structure via binding to histones, thereby preventing them from serving as substrates for HAT.
Assays of inhibition of HAT activity and nucleosome assembly, as well as of histone displacement, are frequently used to demonstrate the activity of histone chaperones. We have described, here, methods for examining inhibition of HAT and nucleosome-assembly activities using the JDP2 transcription factor as a model protein.
Some histone chaperones have HAT-inhibitory activity for regulation of the modification of histones. Such modifications probably have dramatic effects on protein assembly on the chromatin. Although the molecular relationships between the activities of histone chaperones and of HAT inhibitors remain to be defined, the procedures and assays described here might help to resolve these issues in the future.
Materials
Reagents
Equipment
Procedure
There are 9 protocols in this series. Please follow the links below to find each protocol.
Inhibition of Histone Actyltransferase (HAT) Activity
http://www.natureprotocols.com/2007/07/30/assays_of_nucleosome_assembly_1.php
Isolation and labeling of DNA fragments (includes information on Assembly of Chromatin in vitro, Experiments with Mononucleosomes, Isolation of 5'-End-Radiolabeled Fragments of pB100-Uless/strider DNA, and DRE and CRE Elements)
http://www.natureprotocols.com/2007/07/30/assays_of_nucleosome_assembly_2.php
Preparation of Nuclei from HeLa Cells
http://www.natureprotocols.com/2007/07/30/assays_of_nucleosome_assembly_3.php
Preparation of Histone H1-Depleted Chromatin
http://www.natureprotocols.com/2007/07/30/assays_of_nucleosome_assembly_4.php
Preparation of Core Histones (Includes Preparation of Core Histones by FPLC and Further Purification and Concentration of Core Histones)
http://www.natureprotocols.com/2007/07/30/assays_of_nucleosome_assembly_5.php
Reconstitution of Chromatin, Salt Dialysis Using Purified Core Histones, Octamer Transfer from Donor Chromatin, Analysis of Nucleoproteins on an Agarose Gel, Purification of Reconstituted Chromatin on a Sucrose Gradient, and Binding of Linker Histones to Reconstituted Chromatin
http://www.natureprotocols.com/2007/07/30/assays_of_nucleosome_assembly_6.php
Plasmid Super-Coiling Assay and Nucleosome Assembly on a Fragment of 5S DNA
http://www.natureprotocols.com/2007/07/30/assays_of_nucleosome_assembly_7.php
Digestion of Chromatin in Permeabilized Cells with Micrococcal Nuclease (MNase), Permeabilization of cells and digestion with MNase, Purification and Characterization of DNA after Digestion of Chromatin, and Nuclease Cleavage and Mapping Strategies
http://www.natureprotocols.com/2007/07/30/assays_of_nucleosome_assembly_8.php
Ligation-Mediated Single-Sided PCR (LMPCR)
(including: First-strand Synthesis, Ligation-Mediated PCR for Nucleosome Mapping in vivo, and Ligation-Mediated Polymerase Chain Reaction (LM-PCR))
http://www.natureprotocols.com/2007/07/30/assays_of_nucleosome_assembly_9.php
Troubleshooting
Critical Steps
Anticipated Results
References
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Acknowledgements
Keywords
Histone Chaperone, Nucleosome assembly, Inhibition of HAT, Transcription factor, AP-1