ABSTRACT
Proper chromatin regulation is central to genome function and maintenance. The group III chromodomain-helicase-DNA-binding (CHD) family of ATP-dependent chromatin remodeling enzymes, comprising CHD6, CHD7, CHD8, and CHD9, has well-documented roles in transcription regulation, impacting both organism development and disease etiology. These four enzymes are similar in their constituent domains, but they fill surprisingly non-redundant roles in the cell, with deficiencies in individual enzymes leading to dissimilar disease states such as CHARGE syndrome or autism spectrum disorders. The mechanisms explaining their divergent, non-overlapping functions are unclear. In this study, we performed an in-depth biochemical analysis of purified CHD6, CHD7, and CHD8 and discovered distinct differences in chromatin remodeling specificities and activities among them. We report that CHD6 and CHD7 both bind with high affinity to short linker DNA, whereas CHD8 requires longer DNA for binding. As a result, CHD8 slides nucleosomes into positions with more flanking linker DNA than CHD7. Moreover, we found that, although CHD7 and CHD8 slide nucleosomes, CHD6 disrupts nucleosomes in a distinct non-sliding manner. The different activities of these enzymes likely lead to differences in chromatin structure and, thereby, transcriptional control, at the enhancer and promoter loci where these enzymes bind. Overall, our work provides a mechanistic basis for both the non-redundant roles and the diverse mutant disease states of these enzymes in vivo.
Subject(s)
Adenosine Triphosphate/metabolism , Chromatin Assembly and Disassembly , DNA Helicases/metabolism , DNA-Binding Proteins/metabolism , DNA/metabolism , Nerve Tissue Proteins/metabolism , Nucleosomes/enzymology , Transcription Factors/metabolism , Animals , Biological Transport , DNA/chemistry , DNA Helicases/chemistry , DNA Helicases/genetics , DNA Helicases/isolation & purification , DNA, Recombinant/chemistry , DNA, Recombinant/metabolism , DNA, Viral/chemistry , DNA, Viral/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/isolation & purification , HeLa Cells , Humans , Hydrolysis , Kinetics , Molecular Weight , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/isolation & purification , Nucleosomes/metabolism , Phylogeny , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sf9 Cells , Spodoptera , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription Factors/isolation & purificationABSTRACT
Heterochromatin is a specialized chromatin structure that is central to eukaryotic transcriptional regulation and genome stability. Despite its globally repressive role, heterochromatin must also be dynamic, allowing for its repair and replication. In budding yeast, heterochromatin formation requires silent information regulators (Sirs) Sir2p, Sir3p, and Sir4p, and these Sir proteins create specialized chromatin structures at telomeres and silent mating-type loci. Previously, we found that the SWI/SNF chromatin remodeling enzyme can catalyze the ATP-dependent eviction of Sir3p from recombinant nucleosomal arrays, and this activity enhances early steps of recombinational repair in vitro. Here, we show that the ATPase subunit of SWI/SNF, Swi2p/Snf2p, interacts with the heterochromatin structural protein Sir3p. Two interaction surfaces are defined, including an interaction between the ATPase domain of Swi2p and the nucleosome binding, Bromo-Adjacent-Homology domain of Sir3p. A SWI/SNF complex harboring a Swi2p subunit that lacks this Sir3p interaction surface is unable to evict Sir3p from nucleosomes, even though its ATPase and remodeling activities are intact. In addition, we find that the interaction between Swi2p and Sir3p is key for SWI/SNF to promote resistance to replication stress in vivo and for establishment of heterochromatin at telomeres.
Subject(s)
Adenosine Triphosphatases/metabolism , Chromatin Assembly and Disassembly/physiology , Heterochromatin/metabolism , Histones/metabolism , Models, Molecular , Nucleosomes/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Silent Information Regulator Proteins, Saccharomyces cerevisiae/metabolism , Transcription Factors/metabolism , Animals , Blotting, Western , DNA Primers/genetics , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Oligonucleotides/genetics , Polymerase Chain Reaction , Rosaniline Dyes , Xenopus laevisABSTRACT
The optimal use of many biotherapeutics is restricted by Anti-drug antibodies (ADAs) and hypersensitivity responses which can affect potency and ability to administer a treatment. Here we demonstrate that Re-surfacing can be utilized as a generalizable approach to engineer proteins with extensive surface residue modifications in order to avoid binding by pre-existing ADAs. This technique was applied to E. coli Asparaginase (ASN) to produce functional mutants with up to 58 substitutions resulting in direct modification of 35% of surface residues. Re-surfaced ASNs exhibited significantly reduced binding to murine, rabbit and human polyclonal ADAs, with a negative correlation observed between binding and mutational distance from the native protein. Reductions in ADA binding correlated with diminished hypersensitivity responses in an in vivo mouse model. By using computational design approaches to traverse extended distances in mutational space while maintaining function, protein Re-surfacing may provide a means to generate novel or second line therapies for life-saving drugs with limited therapeutic alternatives.
Subject(s)
Asparaginase , Escherichia coli , Humans , Animals , Mice , Rabbits , Asparaginase/genetics , Asparaginase/therapeutic use , Escherichia coli/genetics , Antibodies , Membrane ProteinsABSTRACT
Heterochromatin is a repressive chromatin compartment essential for maintaining genomic integrity. A hallmark of heterochromatin is the presence of specialized nonhistone proteins that alter chromatin structure to inhibit transcription and recombination. It is generally assumed that heterochromatin is highly condensed. However, surprisingly little is known about the structure of heterochromatin or its dynamics in solution. In budding yeast, formation of heterochromatin at telomeres and the homothallic silent mating type loci require the Sir3 protein. Here, we use a combination of sedimentation velocity, atomic force microscopy and nucleosomal array capture to characterize the stoichiometry and conformation of Sir3 nucleosomal arrays. The results indicate that Sir3 interacts with nucleosomal arrays with a stoichiometry of two Sir3 monomers per nucleosome. We also find that Sir3 fibres are less compact than canonical magnesium-induced 30 nm fibres. We suggest that heterochromatin proteins promote silencing by 'coating' nucleosomal arrays, stabilizing interactions between nucleosomal histones and DNA.
Subject(s)
Heterochromatin/chemistry , Nucleosomes/metabolism , Silent Information Regulator Proteins, Saccharomyces cerevisiae/metabolism , Algorithms , Heterochromatin/metabolism , Microscopy, Atomic Force , Monte Carlo Method , Nucleosomes/chemistry , Nucleosomes/genetics , Protein Multimerization , Silent Information Regulator Proteins, Saccharomyces cerevisiae/chemistry , Silent Information Regulator Proteins, Saccharomyces cerevisiae/genetics , UltracentrifugationABSTRACT
Chromatin-remodeling enzymes use the energy from ATP hydrolysis to mobilize, disrupt or change the histone composition of nucleosomes, facilitating nearly every nuclear event. Two recent studies indicate that remodeling enzymes harness the power of an ancient constitutively active DNA translocase and that different remodeling enzymes may use specialized coupling domains that communicate the presence of nucleosomal epitopes to regulate translocase and remodeling activity.