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.

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.

The DNA binding domain (DBD) of nuclear hormone receptors contains a highly conserved globular domain and a less conserved carboxyl-terminal extension (CTE). Despite previous observations that the CTEs of some classes of nuclear receptors are structured and interact with DNA outside of the hexanucleotide hormone response element (HRE), there has been no evidence for such a CTE among the steroid receptors. We have determined the structure of the progesterone receptor (PR)-DBD-CTE DNA complex at a resolution of 2.5 A, which revealed binding of the CTE to the minor groove flanking the HREs. Alanine substitutions of the interacting CTE residues reduced affinity for inverted repeat HREs separated by three nucleotides, and essentially abrogated binding to a single HRE. A highly compressed minor groove of the trinucleotide spacer and a novel dimerization interface were also observed. A PR binding site selection experiment revealed sequence preferences in the trinucleotide spacer and flanking DNA. These results, taken together, support the notion that sequences outside of the HREs influence the DNA binding affinity and specificity of steroid receptors.

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.

Previously, we and others reported that the high mobility group proteins, HMGB-1/-2, enhance DNA binding in vitro and transactivation in situ by the steroid hormone subgroup of nuclear receptors but did not influence these functions of class II receptors. We show here that the DNA binding domain (DBD) is sufficient to account for the selective influence of HMGB-1/-2 on the steroid class of receptors. Furthermore, the use of chimeric DBDs reveals that this selectivity is dependent on the C-terminal extension (CTE), amino acid sequences adjacent to the zinc finger core DBD. HMGB-1/-2 interact directly with the DBDs of steroid but not class II receptors, and this interaction requires the CTE. This in vitro interaction correlates with a requirement of the CTE for maximal HMGB-1/-2 enhancement of DNA binding in vitro and transcriptional activation in cells. Finally, class II receptor DBDs have a much higher intrinsic affinity for DNA than steroid receptor DBDs, and this affinity difference is also dependent on the CTE. These results reveal the importance of the steroid receptor CTE for DNA binding affinity and functional response to HMGB-1/-2.Nuclear hormone receptors comprise a superfamily of transcription factors that regulates diverse metabolic processes by binding to response elements in the enhancer regions of specific genes. This superfamily consists of three receptor subclasses: 1) the steroid hormone receptors for progesterone (PR) 1 , estrogen (ER), glucocorticoids (GR), androgens (AR), and mineralocorticoids (MR); 2) class II receptors for thyroid hormone (TR), retinoids (RAR and RXR), vitamin D3 (VDR), prostaglandins (PPAR), oxysterols, and bile acids; and 3) orphan receptors for which no endogenous ligand has been identified (1-4). Each of the receptor subclasses is characterized by a unique mechanism of action with respect to dimerization and DNA sequence recognition. Steroid receptors form homodimers that optimally recognize hexameric DNA elements arranged as inverted repeats separated by three unspecified base pairs. PR, GR, AR, and MR bind to the core hexamer AGAACA, whereas ER recognizes AGGTCA (2). Class II receptors preferentially function as heterodimers with RXR and recognize the AGGTCA hexamer arranged as direct repeats. Variable spacing between the direct repeats determines the RXR heterodimer binding specificity. Class II receptors, particularly TR, can also recognize an inverted repeat as homodimers, or half-sites as monomers. Orphan receptors can bind to the AGGTCA hexamer arranged either as a direct repeat, palindrome, or half-site as heterodimers with RXR, homodimers, or monomers (1, 5-7).DNA-bound nuclear receptors activate transcription through assembly of a coactivator protein complex (8 -10). Some of these coactivators possess enzyme activities that are thought to facilitate access of general transcription factors to chromatin templates (11,12). Additionally, the coactivator complex may serve as a protein bridge to facilitate assembly of the basal transcription apparatus (13, 14). We...