Sarah C. Roemer

CAF-1-induced oligomerization of histones H3/H4 and mutually exclusive interactions with Asf1 guide H3/H4 transitions among histone chaperones and DNA

Anti-silencing function 1 (Asf1) and Chromatin Assembly Factor 1 (CAF-1) chaperone histones H3/H4 during the assembly of nucleosomes on newly replicated DNA. To understand the mechanism of histone H3/H4 transfer among Asf1, CAF-1 and DNA from a thermodynamic perspective, we developed and employed biophysical approaches using full-length proteins in the budding yeast system. We find that the C-terminal tail of Asf1 enhances the interaction of Asf1 with CAF-1. Surprisingly, although H3/H4 also enhances the interaction of Asf1 with the CAF-1 subunit Cac2, H3/H4 forms a tight complex with CAF-1 exclusive of Asf1, with an affinity weaker than Asf1–H3/H4 or H3/H4–DNA interactions. Unlike Asf1, monomeric CAF-1 binds to multiple H3/H4 dimers, which ultimately promotes the formation of (H3/H4)2 tetramers on DNA. Thus, transition of H3/H4 from the Asf1-associated dimer to the DNA-associated tetramer is promoted by CAF-1-induced H3/H4 oligomerization.

Structural and Functional Analysis of Domains of the Progesterone Receptor

Steroid hormone receptors are multi-domain proteins composed of conserved well-structured regions, such as ligand (LBD) and DNA binding domains (DBD), plus other naturally unstructured regions including the amino-terminal domain (NTD) and the hinge region between the LBD and DBD. The hinge is more than just a flexible region between the DBD and LBD and is capable of binding co-regulatory proteins and the minor groove of DNA flanking hormone response elements. Because the hinge can directly participate in DNA binding it has also been termed the carboxyl terminal extension (CTE) of the DNA binding domain. The CTE and NTD are dynamic regions of the receptor that can adopt multiple conformations depending on the environment of interacting proteins and DNA. Both regions have important regulatory roles for multiple receptor functions that are related to the ability of the CTE and NTD to form multiple active conformations. This review focuses on studies of the CTE and NTD of progesterone receptor (PR), as well as related work with other steroid/nuclear receptors.

Mechanism of high-mobility group protein B enhancement of progesterone receptor sequence-specific DNA binding

The DNA-binding domain (DBD) of progesterone receptor (PR) is bipartite containing a zinc module core that interacts with progesterone response elements (PRE), and a short flexible carboxyl terminal extension (CTE) that interacts with the minor groove flanking the PRE. The chromosomal high-mobility group B proteins (HMGB), defined as DNA architectural proteins capable of bending DNA, also function as auxiliary factors that increase the DNA-binding affinity of PR and other steroid receptors by mechanisms that are not well defined. Here we show that the CTE of PR contains a specific binding site for HMGB that is required for stimulation of PR-PRE binding, whereas the DNA architectural properties of HMGB are dispensable. Specific PRE DNA inhibited HMGB binding to the CTE, indicating that DNA and HMGB–CTE interactions are mutually exclusive. Exogenous CTE peptide increased PR-binding affinity for PRE as did deletion of the CTE. In a PR-binding site selection assay, A/T sequences flanking the PRE were enriched by HMGB, indicating that PR DNA-binding specificity is also altered by HMGB. We conclude that a transient HMGB–CTE interaction alters a repressive conformation of the flexible CTE enabling it to bind to preferred sequences flanking the PRE.

A Progesterone Receptor Co-activator (JDP2) Mediates Activity through Interaction with Residues in the Carboxyl-terminal Extension of the DNA Binding Domain

Progesterone receptor (PR) belongs to the nuclear receptor family of ligand-dependent transcription factors and mediates the major biological effects of progesterone. Transcriptional co-activators that are recruited by PR through the carboxyl-terminal ligand binding domain have been studied extensively. Much less is known about co-activators that interact with other regions of receptors. Jun dimerization protein 2 (JDP2) is a PR co-activator that enhances the transcriptional activity of the amino-terminal domain by increasing the α-helical content and stability of the intrinsically disordered amino-terminal domain. To gain insights into the mechanism of JDP2 co-activation of PR, the structural basis of JDP2-PR interaction was analyzed using NMR. The smallest regions of each protein needed for efficient protein interaction were used for NMR and included the basic region plus leucine zipper (bZIP) domain of JDP2 and the core zinc modules of the PR DNA binding domain plus the intrinsically disordered carboxyl-terminal extension (CTE) of the DNA binding domain. Chemical shift changes in PR upon titration with JDP2 revealed that most of the residues involved in binding of JDP2 reside within the CTE. The importance of the CTE for binding JDP2 was confirmed by peptide competition and mutational analyses. Point mutations within CTE sites identified by NMR and a CTE domain swapping experiment also confirmed the functional importance of JDP2 interaction with the CTE for enhancement of PR transcriptional activity. These studies provide insights into the role and functional importance of the CTE for co-activator interactions.

The Cac1 subunit of histone chaperone CAF-1 organizes CAF-1-H3/H4 architecture and tetramerizes histones

The histone chaperone Chromatin Assembly Factor 1 (CAF-1) deposits tetrameric (H3/H4)2 histones onto newly-synthesized DNA during DNA replication. To understand the mechanism of the tri-subunit CAF-1 complex in this process, we investigated the protein-protein interactions within the CAF-1-H3/H4 architecture using biophysical and biochemical approaches. Hydrogen/deuterium exchange and chemical cross-linking coupled to mass spectrometry reveal interactions that are essential for CAF-1 function in budding yeast, and importantly indicate that the Cac1 subunit functions as a scaffold within the CAF-1-H3/H4 complex. Cac1 alone not only binds H3/H4 with high affinity, but also promotes histone tetramerization independent of the other subunits. Moreover, we identify a minimal region in the C-terminus of Cac1, including the structured winged helix domain and glutamate/aspartate-rich domain, which is sufficient to induce (H3/H4)2 tetramerization. These findings reveal a key role of Cac1 in histone tetramerization, providing a new model for CAF-1-H3/H4 architecture and function during eukaryotic replication. DOI: http://dx.doi.org/10.7554/eLife.18023.001

Reversed-phase ion-pair liquid chromatography method for purification of duplex DNA with single base pair resolution

Obtaining quantities of highly pure duplex DNA is a bottleneck in the biophysical analysis of protein–DNA complexes. In traditional DNA purification methods, the individual cognate DNA strands are purified separately before annealing to form DNA duplexes. This approach works well for palindromic sequences, in which top and bottom strands are identical and duplex formation is typically complete. However, in cases where the DNA is non-palindromic, excess of single-stranded DNA must be removed through additional purification steps to prevent it from interfering in further experiments. Here we describe and apply a novel reversed-phase ion-pair liquid chromatography purification method for double-stranded DNA ranging in lengths from 17 to 51 bp. Both palindromic and non-palindromic DNA can be readily purified. This method has the unique ability to separate blunt double-stranded DNA from pre-attenuated (n-1, n-2, etc) synthesis products, and from DNA duplexes with single base pair overhangs. Additionally, palindromic DNA sequences with only minor differences in the central spacer sequence of the DNA can be separated, and the purified DNA is suitable for co-crystallization of protein–DNA complexes. Thus, double-stranded ion-pair liquid chromatography is a useful approach for duplex DNA purification for many applications.

Thermodynamic insights into histone transfer among chaperones

Background Together with non-histone proteins nucleosomes assemble the eukaryotic genome into higher order structures known as chromatin. Chromatin structure is dynamic, as it is continually assembled and disassembled for factors that carry out the processes of transcription, replication, DNA repair, and recombination to gain access to the DNA. The deposition of the histones H3/H4 onto DNA to give the tetrasome intermediate and the displacement of H3/H4 from DNA are thought to be first and last steps in nucleosome assembly and disassembly, respectively. Anti-silencing function 1 (Asf1) and Chromatin Assembly Factor (CAF-1) are chaperones of histones H3/ H4 that function together in the replication dependent chromatin assembly pathway. Materials and methods In order to investigate the molecular basis of the activity of Asf1 and CAF-1 in chromatin assembly and disassembly, we employed a variety of methods, including Xray crystallography, biophysical and biochemical methods using full-length proteins in the budding yeast system. Results To understand the mechanism of histone H3/H4 transfer among Asf1, CAF-1, and DNA from a thermodynamic perspective, we measured the binding affinities for their various complexes. Asf1 has the ability to directly deposit H3/H4 dimers onto the DNA, but additional factors are required for their removal from DNA. The C-terminal tail of Asf1 greatly enhances the interaction of Asf1 with H3/H4 and with CAF-1. Surprisingly, although H3/H4 also enhances the interaction of Asf1 with the CAF-1 subunit Cac2, H3/H4 forms a tight complex with CAF-1 exclusive of Asf1, with an affinity weaker than Asf1-H3/ H4 or H3/ H4-DNA interactions. Unlike Asf1, monomeric CAF-1 binds to multiple H3/H4 dimers, which ultimately promotes the formation of (H3/H4)2 tetramers on DNA. Conclusions The H3/H4-Asf1 complex is a thermodynamic histone sink. The transition of H3/H4 from the Asf1-associated dimer to the DNA-associated tetramer is thermodynamically favored, but occurs through a less stable intermediate complex with CAF-1.