The FACT complex of vertebrate cells, comprising the Cdc68 (Spt16) and SSRP1 proteins, facilitates transcription elongation on a nucleosomal template and modulates the elongation-inhibitory effects of the DSIF complex in vitro. Genetic findings show that the related yeast (Saccharomyces cerevisiae) complex, termed CP, also mediates transcription. The CP components Cdc68 and Pob3 closely resemble the FACT components, except that the C-terminal high-mobility group (HMG) box domain of SSRP1 is not found in the yeast homolog Pob3. We show here that Nhp6a and Nhp6b, small HMG box proteins with overlapping functions in yeast, associate with the CP complex and mediate CP-related genetic effects on transcription. Absence of the Nhp6 proteins causes severe impairment in combination with mutations impairing the Swi-Snf chromatin-remodeling complex and the DSIF (Spt4 plus Spt5) elongation regulator, and sensitizes cells to 6-azauracil, characteristic of elongation effects. An artificial SSRP1-like protein, created by fusing the Pob3 and Nhp6a proteins, provides both Pob3 and Nhp6a functions for transcription, and competition experiments indicate that these functions are exerted in association with Cdc68. This particular Pob3-Nhp6a fusion protein was limited for certain Nhp6 activities, indicating that its Nhp6a function is compromised. These findings suggest that in yeast cells the Cdc68 partners may be both Pob3 and Nhp6, functioning as a bipartite analog of the vertebrate SSRP1 protein.Transcription of eukaryotic protein-coding genes takes place in the context of chromatin. In chromatin, DNA is associated with histone proteins in a nucleosomal configuration and with other proteins that have structural and/or regulatory roles. This intricate assembly of structural and regulatory proteins with DNA is generally inhibitory for transcription through the interference with transcription activator proteins and general transcription factors (reviewed in reference 57) and obstruction of the RNA polymerase II (RNAP II) elongation complex by nucleosomes (8; reviewed in reference 57). The processivity of elongation complexes is also inhibited by several nonnucleosomal chromatin components (19,50,60). Among the proteins regulating both nucleosomal and nonnucleosomal transcriptional inhibition is one named FACT (51).FACT is an abundant nuclear protein complex that has been purified and characterized based on its ability to allow transcription elongation along a nucleosomal template in vitro (27,33,34). In keeping with this activity, the FACT complex (also termed DUF [32]) has been shown to interact with nucleosomes and with histone H2A-H2B dimers and to have DNAuntwisting activity in vitro (32, 34). FACT has also been found to counteract the inhibitory effects on transcripton elongation imposed in vitro by the DSIF complex (51). In the budding yeast Saccharomyces cerevisiae, FACT has a structural, and possibly functional, homolog in the form of the CP complex, which has been implicated by genetic evidence in the interaction of chromati...

The Cdc68 protein of the yeast Saccharomyces cerevisiae is an essential nuclear protein that has been shown to be necessary for the trans-activation of many genes as well as for the maintenance of chromatin-mediated repression in the absence of trans-activation. These activities implicate the Cdc68 protein in the regulation of chromatin structure and/or function. Here we report that Cdc68 is found in association with another essential nuclear protein, Pob3, in what we term the CP complex. This dimer of Cdc68 with Pob3 is stable to partial purification, so that the functions of gene activation and repression that are assigned to Cdc68 are likely to be properties of the CP complex. The CP complex is highly abundant, suggesting that it may be widespread throughout chromatin.

No abstract

The abundant nuclear complex termed FACT affects several DNA transactions in a chromatin context, including transcription, replication, and repair. Earlier studies of yeast FACT, which indicated the apparent dispensability of conserved sequences at the N terminus of the FACT subunit Cdc68/Spt16, prompted genetic and biochemical studies reported here that suggest the domain organization for Spt16 and the other FACT subunit Pob3, the yeast homolog of the metazoan SSRP1 protein. Our findings suggest that each FACT subunit is a multidomain protein, and that FACT integrity depends on Pob3 interactions with the Spt16 Mid domain. The conserved Spt16 N-terminal domain (NTD) is shown to be without essential function during normal growth, but becomes important under conditions of replication stress. Genetic interactions suggest that some functions carried out by the Spt16 NTD may be partially redundant within FACT.

Transcription of nuclear genes usually involves trans-activators, whereas repression is exerted by chromatin. For several genes the transcription mediated by trans-activators and the repression mediated by chromatin depend on the CP complex, a recently described abundant yeast nuclear complex of the Pob3 and Cdc68/Spt16 proteins. We report that the N-terminal third of the Saccharomyces cerevisiae Cdc68 protein is dispensable for gene activation but necessary for the maintenance of chromatin repression. The absence of this 300-residue N-terminal domain also decreases the need for the Swi/Snf chromatin-remodeling complex in transcription and confers an Spt- effect characteristic of chromatin alterations. The repression domain, and indeed the entire Cdc68 protein, is highly conserved, as shown by the sequence of the Cdc68 functional homolog from the yeast Kluyveromyces lactis and by database searches. The repression-defective (truncated) form of Cdc68 is stable but less active at high temperatures, whereas the known point-mutant form of Cdc68, encoded by three independent mutant alleles, alters the N-terminal repression domain and destabilizes the mutant protein.