Nucleosome assembly in the wake of DNA replication is a key process that regulates cell identity and survival. Chromatin assembly factor 1 (CAF-1) is a H3-H4 histone chaperone that associates with the replisome and orchestrates chromatin assembly following DNA synthesis. Little is known about the mechanism and structure of this key complex. Here we investigate the CAF-1•H3-H4 binding mode and the mechanism of nucleosome assembly. We show that yeast CAF-1 binding to a H3-H4 dimer activates the Cac1 winged helix domain interaction with DNA. This drives the formation of a transient CAF-1•histone•DNA intermediate containing two CAF-1 complexes, each associated with one H3-H4 dimer. Here, the (H3-H4)2 tetramer is formed and deposited onto DNA. Our work elucidates the molecular mechanism for histone deposition by CAF-1, a reaction that has remained elusive for other histone chaperones, and it advances our understanding of how nucleosomes and their epigenetic information are maintained through DNA replication.DOI: http://dx.doi.org/10.7554/eLife.22799.001

The role of the Acanthamoeba casteUlanii TATA-binding protein (TBP) in transcription was examined. Specific antibodies against the nonconserved N-terminal domain of TBP were used to verify the presence of TBP in the fundamental transcription initiation factor for RNA polymerase I, TIF-IB, and to demonstrate that TBP is part of the committed initiation complex on the rRNA promoter. The same antibodies inhibit transcription in all three polymerase systems, but they do so differentially. Oligonucleotide competitors were used to evaluate the accessibility of the TATA-binding site in TIF-IB, TFIID, and TFIIIB. The results suggest that insertion of TBP into the polymerase II and III factors is more similar than insertion into the polymerase I factor.While the transcription systems of eukaryotic RNA polymerases I, II, and III obviously share some characteristics, initiation mechanisms for these transcription systems have been largely studied separately. The polymerases themselves have five subunits in common, seven in the case of the "odd pols," RNA polymerases I and III (45). Nevertheless, the general transcription factors involved with each polymerase have been examined in isolation, perhaps masking important generalizations about their functions. This approach began to change when it was discovered that some of the genes for small nuclear RNAs (snRNAs) are transcribed by polymerase III whereas most are transcribed by polymerase II (27,32,35,47 The full impact of this factor overlap was perhaps not realized because polymerase I still appeared to use dedicated factors. However, a flurry of studies (6,7,44,56) revealed that TBP was required for transcription of all genes. TBP is now known to be a subunit of TFIID (41), TFIIIB (17,24,48,55), and human TIF-IB (SL1) (6). TBP is associated with additional subunits (TAFs) to make up the functional factors (reviewed in reference 41). The TAFs appear to be different for each polymerase system (reviewed in reference 42), although overlap of TAFs has not been rigorously ruled out. All the TBP-containing factors are pivotal for their respective polymerases. Indeed, TFIIIB and TIF-IB are fundamental transcription factors; i.e., they appear to be responsible for the repetitive recruitment of RNA polymerase during successive rounds of initiation (16; reviewed in references 36 and 37).The manner by which the TBP-containing factor is recruited to the promoter differs from gene to gene. For RNA polymerase III genes with type I (SS RNA) or type II (tRNA, VAI, Alu, EBER, 7SL, 4.5S) internal control regions, additional general transcription factors are required to assemble TFIIIB onto the promoter (reviewed in references 10-12). Initiation complex formation on 5S and tRNA genes is an organized process in which the factors bind in an obligatory order, each relying on protein-DNA and protein-protein interactions with a previously bound factor(s) to join the complex. On some genes the DNA interaction site for TFIIIB is sequence specific, whereas on others specific sequence recognition is no...

The process of transcriptional elongation by RNA polymerase II (RNAPII) in a chromatin context involves a large number of crucial factors. Spn1 is a highly conserved protein encoded by an essential gene and is known to interact with RNAPII and the histone chaperone Spt6. Spn1 negatively regulates the ability of Spt6 to interact with nucleosomes, but the chromatin binding properties of Spn1 are largely unknown. Here, we demonstrate that full length Spn1 (amino acids 1–410) binds DNA, histones H3–H4, mononucleosomes and nucleosomal arrays, and has weak nucleosome assembly activity. The core domain of Spn1 (amino acids 141–305), which is necessary and sufficient in Saccharomyces cerevisiae for growth under ideal growth conditions, is unable to optimally interact with histones, nucleosomes and/or DNA and fails to assemble nucleosomes in vitro. Although competent for binding with Spt6 and RNAPII, the core domain derivative is not stably recruited to the CYC1 promoter, indicating chromatin interactions are an important aspect of normal Spn1 functions in vivo. Moreover, strong synthetic genetic interactions are observed with Spn1 mutants and deletions of histone chaperone genes. Taken together, these results indicate that Spn1 is a histone binding factor with histone chaperone functions.

Site-specific photo-cross-linking of the rRNA committed transcription complex was carried out by using 5-[N-(p-azidobenzoyl)-3-aminoallyl]-dUMP-derivatized promoter DNA. Putative TAFIs of 145, 99, 96, and 91 kDa, as well as TATA-binding protein (TBP), were found to specifically photo-cross-link to different positions along the promoter. These had been identified as potential subunits of the fundamental transcription initiation factor TIF-IB (also known as SL1, factor D, and TFID) from Acanthamoeba castellanii by purification to apparent homogeneity. No other polypeptides attributable to the rRNA architectural transcription factor UBF were identified, suggesting that this protein is not part of the committed complex. Scanning transmission electron microscopy of the complexes was used to estimate the mass of the complex and the contour length of the DNA in the complex. This showed that a single molecule of TIF-IB is in each committed complex and that the DNA is not looped around the protein, as would be expected if UBF were in the complex. A circular permutation analysis of DNA bending resulting from TIF-IB binding revealed a 45 +/- 3.1 degrees (n = 14) bend centered 23 bp upstream of the transcription initiation site. This degree of bending and the position of the bend relative to the site of TBP photo-cross-linking are consistent with earlier data showing that the TBP TATA box-binding domain is not utilized in the assembly of the rRNA committed complex (C. A. Radebaugh, J. L. Mathews, G. K. Geiss, F. Liu, J. Wong, E. Bateman, S. Camier, A. Sentenac, and M. R. Paule, Mol. Cell. Biol. 14:597-605, 1994).

Spn1 plays essential roles in the regulation of gene expression by RNA Polymerase II (RNAPII), and it is highly conserved in organisms ranging from yeast to humans. Spn1 physically and/or genetically interacts with RNAPII, TBP, TFIIS and a number of chromatin remodeling factors (Swi/Snf and Spt6). The central domain of Spn1 (residues 141-305 out of 410) is necessary and sufficient for performing the essential functions of SPN1 in yeast cells. Here we report the high-resolution (1.85Å) crystal structure of the conserved central domain of Saccharomyces cerevisiae Spn1. The central domain is comprised of eight alpha-helices in a right handed super helical arrangement, and exhibits structural similarity to domain I of TFIIS. A unique structural feature of Spn1 is a highly conserved loop, which defines one side of a pronounced cavity. The loop and the other residues forming the cavity are highly conserved at the amino acid level among all Spn1 family members, suggesting that this is a signature motif for Spn1 orthologs. The locations and the molecular characterization of temperature-sensitive mutations in Spn1 indicate that the cavity is a key attribute of Spn1 that is critical for its regulatory functions during RNAPII-mediated transcriptional activity.

Acanthamoeba castellanii transcription initiation factor-IB (TIF-IB) is the TATA-binding protein-containing transcription factor that binds the rRNA promoter to form the committed complex. Minor groove-specific drugs inhibit TIF-IB binding, with higher concentrations needed to disrupt preformed complexes because of drug exclusion by bound TIF-IB. TIF-IB/DNA interactions were mapped by hydroxyl radical and uranyl nitrate footprinting. TIF-IB contacts four minor grooves in its binding site. TIF-IB and DNA wrap around each other in a right-handed superhelix of high pitch, so the upstream and downstream contacts are on opposite faces of the helix. Dimethyl sulfate protection assays revealed limited contact with a few guanines in the major groove. This detailed analysis suggests significant DNA conformation dependence of the interaction.Extensive in vitro and in vivo analyses have shown that efficient transcription of the rRNA gene by RNA polymerase I requires formation of a stable transcription factor-promoter DNA complex, the committed complex (for review, see Refs. 1 and 2). In most species, two regions of the rRNA promoter, the core promoter element (CPE) 1 and upstream promoter element (UPE), participate in expression of the rRNA gene and formation of the committed complex. The CPE is located between Ϫ50 and ϩ10 relative to the transcription start site (position ϩ1), is absolutely required in all organisms, and is sufficient in some for initiation. It is the primary binding site for the TBPcontaining transcription factor. The UPE is located farther upstream, from approximately Ϫ150 to Ϫ110, and acts primarily to stimulate transcription. It has a variable requirement between species; at one end of the spectrum, Acanthamoeba castellanii has no UPE discernible in vitro (3). We (4) and others (5) have shown recently that the CPE can be subdivided into an element that interacts intimately with TIF-IB and an element that functions like the initiator element (Inr) of some RNA polymerase II promoters, interacting with a specific TAF I .The UPE and CPE are important because they serve as the binding sites for two RNA polymerase I transcription factors, UBF and TIF-IB (also known as SL1, factor D, Rib1, or corebinding factor) respectively. These two proteins interact, by mechanisms that are still unclear but apparently involve DNA looping (6), to form a stable initiation complex capable of directing specific RNA polymerase I recruitment through multiple rounds of transcription. The need for UBF is variable between species; UBF is required in human and Xenopus (7-9), but is dispensable in rat (10), mouse (11), yeast (12-14), and A. castellanii (15). Therefore, TIF-IB is, by elimination, the fundamental transcription factor for rRNA genes, recruiting RNA polymerase I in the next step of the initiation process (16).TIF-IB has recently been purified to homogeneity from several eukaryotic species, including human (17), mouse (18), yeast (12,14), and A. castellanii.2 Biochemical analysis has shown that TIF-IB consists of TBP an...

The fundamental transcription initiation factor (TIF) for ribosomal RNA expression by eukaryotic RNA polymerase I, TIF-IB, has been purified to near homogeneity from Acanthamoeba castellanii using standard techniques. The purified factor consists of the TATA-binding protein and four TATA-binding protein-associated factors with relative molecular weights of 145,000, 99,000, 96,000, and 91,000. This yields a calculated native molecular weight of 460,000, which compares well with its mass determined by scanning transmission electron microscopy (493,000) and its sedimentation rate, which is close to RNA polymerase I (515,000). Both impure and nearly homogeneous TIF-IB exhibit an apparent equilibrium dissociation constant of 56 ؎ 3 pM. However, although impure TIF-IB can form a promoter-DNA complex resistant to challenge by other promotercontaining DNAs, near homogeneous TIF-IB cannot do so. An additional transcription factor, dubbed TIF-IE, restores the ability of near homogeneous TIF-IB to sequester DNA into a committed complex.

c Histone chaperones, like nucleosome assembly protein 1 (Nap1), play a critical role in the maintenance of chromatin architecture. Here, we use the GAL locus in Saccharomyces cerevisiae to investigate the influence of Nap1 on chromatin structure and histone dynamics during distinct transcriptional states. When the GAL locus is not expressed, cells lacking Nap1 show an accumulation of histone H2A-H2B but not histone H3-H4 at this locus. Excess H2A-H2B interacts with the linker DNA between nucleosomes, and the interaction is independent of the inherent DNA-binding affinity of H2A-H2B for these particular sequences as measured in vitro. When the GAL locus is transcribed, excess H2A-H2B is reversed, and levels of all chromatinbound histones are depleted in cells lacking Nap1. We developed an in vivo system to measure histone exchange at the GAL locus and observed considerable variability in the rate of exchange across the locus in wild-type cells. We recapitulate this variability with in vitro nucleosome reconstitutions, which suggests a contribution of DNA sequence to histone dynamics. We also find that Nap1 is required for transcription-dependent H2A-H2B exchange. Altogether, these results indicate that Nap1 is essential for maintaining proper chromatin composition and modulating the exchange of H2A-H2B in vivo.T he basic unit of chromatin is the nucleosome, which forms when DNA is wrapped around two copies each of the four core histones arranged as two histone H2A-H2B dimers and a histone H3-H4 tetramer (1). Nucleosomes are highly dynamic, capable of multiple structural transitions between completely assembled and entirely disassembled structures (2). Indeed, H2A-H2B and H3-H4 are actively exchanged during both DNA replication-dependent and -independent events (3-9). Chromatin transitions have the potential to profoundly affect gene expression, and a diverse spectrum of factors, including histone chaperones, participate in this process (10).Histone chaperones are histone-binding proteins that facilitate nucleosome assembly and/or disassembly in an ATP-independent fashion (11-13). The histone chaperone nucleosome assembly protein 1 (Nap1) is a highly conserved chaperone that binds H2A-H2B in vitro with nanomolar affinity (12, 14) in a conformation that shields interfaces required for nucleosome assembly (15). Although functional in the assembly of nucleosomes in vitro (16), a number of studies support a role for Nap1 in transcription-dependent processes of disassembly of nucleosomes. Nap1 is critical for the eviction of histones during transcription in a mammalian in vitro system (17), and Nap1 (with the ATP-dependent chromatin remodeler RSC [remodels the structure of chromatin]) can facilitate the elongation of RNA polymerase II (RNAPII) on chromatin templates using yeast in vitro systems (18,19). Our previous in vivo studies indicated that Nap1 prevents excess H2A-H2B accumulation on chromatin (20), and here, we expand our analysis to investigate the role of Nap1 in histone exchange and occupancy. As a model sy...