de FACTo Nucleosome Dynamics*

The factors required for the delivery of RNA polymerase II to class II promoters using naked DNA were all identified by 1998, yet their exact mechanisms of action were not fully understood in all cases, and in some instances, their precise function still remains unknown. Nonetheless, a complete understanding of the complexity of the RNA polymerase II transcription cycle necessitated the development of assays that include chromatinized DNA templates. At this time, the field was actively searching for factors that allow transcription initiation on chromatinized templates. We began studies using chromatin templates in an attempt to identify factor(s) that permit RNA polymerase II to traverse nucleosomes, i.e. that allow elongation on chromatinized DNA templates. The challenge herein was to develop an assay that directly measured the ability of transcriptionally engaged RNA polymerase II to traverse nucleosomes. This approach resulted in the isolation of FACT, a heterodimer in humans comprised of Spt16 and SSRP1. Defined functional biochemical assays corroborated genetic studies in yeast that allowed the elucidation of FACT function in vivo. Collectively, these approaches demonstrate that FACT is a factor that allows RNA polymerase II to traverse nucleosomes in vitro and in vivo by removing one H2A/H2B dimer. More recent studies using a fully defined chromatin reconstitution/transcription assay revealed that FACT activity is greatly stimulated by post-translational modification of the histone polypeptides, specifically by monoubiquitination of lysine 120 of human histone H2B.

The factors required for the delivery of RNA polymerase II to class II promoters using naked DNA were all identified by 1998, yet their exact mechanisms of action were not fully understood in all cases, and in some instances, their precise function still remains unknown. Nonetheless, a complete understanding of the complexity of the RNA polymerase II transcription cycle necessitated the development of assays that include chromatinized DNA templates. At this time, the field was actively searching for factors that allow transcription initiation on chromatinized templates. We began studies using chromatin templates in an attempt to identify factor(s) that permit RNA polymerase II to traverse nucleosomes, i.e. that allow elongation on chromatinized DNA templates. The challenge herein was to develop an assay that directly measured the ability of transcriptionally engaged RNA polymerase II to traverse nucleosomes. This approach resulted in the isolation of FACT, a heterodimer in humans comprised of Spt16 and SSRP1. Defined functional biochemical assays corroborated genetic studies in yeast that allowed the elucidation of FACT function in vivo. Collectively, these approaches demonstrate that FACT is a factor that allows RNA polymerase II to traverse nucleosomes in vitro and in vivo by removing one H2A/H2B dimer. More recent studies using a fully defined chromatin reconstitution/transcription assay revealed that FACT activity is greatly stimulated by post-translational modification of the histone polypeptides, specifically by monoubiquitination of lysine 120 of human histone H2B.

The Discovery of FACT
A major challenge toward understanding the mechanistic aspects of transcriptional regulation is the recapitulation of this enzymatic process in vitro under physiological conditions. In the nucleus of cells, genomic DNA associates with numerous proteins to form chromatin. By its nature, DNA accessibility is highly restrictive when assembled into chromatin, which serves in a multifunctional capacity. At its most simple level, chromatin is a repeating unit of four histone proteins that holds 147 base pairs of DNA, termed the nucleosome. One histone H3/H4 tetramer and two H2A/H2B dimers comprise the core nucleosomal proteins. Chromatin serves to compact, protect, and regulate genomic DNA activities, in part by limiting auxiliary factors from contacting the DNA. This compaction and restriction has notable consequences for RNA transcription as well as other processes that necessitate access to DNA. RNA polymerase II (RNAPII) 3 transcribes protein-coding genes (into mRNA) in a highly regulated multienzymatic process. Early stages of gene activation in vivo require that promoters and other cis-elements are made available to their cognate transcriptional regulators. However, the cell must also deal with chromatin during all stages of mRNA biogenesis, including transcript elongation and mRNA processing events (1).
The complete reconstitution of RNAPII transcription on naked DNA with purified, well defined factors remains a prized accomplishment toward establishing the basis for transcriptional regulation. However, it was established early on that transcription initiation and transcript elongation on chromatin templates in vitro are far less efficient in comparison with naked DNA (2,3). This discrepancy between naked and chromatinized templates suggested that specific factors are required for efficient transcription within the context of nucleosomes. In vivo, activators are required for efficient transcription although activator binding in vitro can be hampered by nucleosomes (4,5). To determine the minimal requirements for transcriptional activation on chromatin templates, a fully reconstituted transcription system was employed (6) (supplemental Fig. S1). Physiologically spaced nucleosomes were assembled using Drosophila embryo extract, and the resultant template was purified to remove contaminating proteins from the extract and templates that were not properly assembled. The GAL4-VP16 transcriptional activator, added to the DNA prior to chromatin assembly, was utilized to obtain remodeled chromatin templates at the promoter. The isolated chromatinized DNA was then incubated with the general transcription factors (IID, IIB, IIE, IIF, and IIH (7)), RNAPII, and the co-activators TFIIA and PC4. Proteins used in this reconstituted system were either bacterially produced or highly purified to complete the highly defined system.
Although transcription on naked DNA was observed in both the reconstituted system and in HeLa nuclear extracts, only nuclear extracts supported transcription on chromatinized templates, measured by the production of an ϳ400-nucleotide RNA. Therefore, a biochemical complementation assay was established to identify the soluble factor(s) required for chromatin-based transcription (supplemental Fig. S1A). Old-fashioned protein purification coupled with the biochemical complementation assay described above identified two polypeptides that allowed the production of an ϳ400-nucleotide RNA on chromatin templates (supplemental Fig. S1B). The purified heterodimer was named FACT (facilitates chromatin transcription) (6) and in humans consists of the highly conserved proteins Spt16 and the HMG-box containing SSRP1 (structure specific recognition protein-1) (8). Surprisingly, maximal FACT activity in vitro requires a near equimolar stoichiometry to nucleosomes (8). This observation foreshadowed the mechanistic activities of FACT as a histone chaperone (see below).
Before the biochemical isolation of FACT using the above complementation assay, genetic evidence in yeast suggested a functional role for SPT16 in transcriptional activation and chromatin modulation (9). A role for FACT in transcript elongation in vivo was established through genetic interactions with the known elongation factors TFIIS and Spt4 (6) (see below). Importantly, chromatin immunoprecipitation experiments in yeast revealed that FACT associates with active genes in vivo and co-localizes with RNAPII throughout the coding region (10). Moreover, FACT appears to localize just downstream of the promoter region, consistent with its necessary positioning for its function in assisting nucleosome-mediated transcript elongation (10). Similar results were observed in Drosophila, demonstrating that FACT is rapidly recruited to induced heat shock genes in a manner comparable with the elongation factors Spt5 and Spt6 (11) and to PAF, a multisubunit complex that modulates chromatin-related events downstream of transcription initiation including RNA processing (12)(13)(14)(15). Consistent with findings in yeast, FACT localizes most prominently downstream of promoter regions, appearing at the first nucleosome in Drosophila and mammalian cells (11,16). In the past, it was unclear how FACT is actively recruited to the correct location on transcribed genes in vivo, although recent findings suggest some mechanisms (see below).

Mechanistic Aspects of FACT Activity
Initially, FACT was observed to function differently from the canonical elongation factors, such as TFIIS and TFIIF (6). Several biochemical studies (described above) and genetic studies in yeast alluded to a role for FACT in histone dynamics. Early on, SPT16 was placed into the histone group of SPT genes along with DSIF (5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole sensitivity-inducing factor composed of Spt4 and Spt5) and Spt6, factors known to modulate chromatin structure (9,12). More importantly, mutational analyses in vivo of histones themselves revealed similar phenotypes as a yeast Spt16 mutant (Cdc68) (17); based on these findings, it was speculated that Spt16 directly regulates histone dimer-tetramer interactions (17). Mechanistic studies would put this hypothesis to the test.
In a key experiment, covalent cross-linking of histone polypeptides within nucleosomes was observed to abrogate FACTdependent transcription, suggesting that the octamer must be disassembled during active transcription (8) (Fig. 1). This experiment suggested that FACT contained nucleosome-disassembly activity, a notion strongly supported by the physical association of Spt16 with H2A/H2B dimers (8,18) and histone H3/H4 interactions with SSRP1 (18). It was later discovered that FACT-mediated transcription through the nucleosome results in the loss of a single H2A/H2B dimer ( Fig. 2A and Ref. 18). Consistently, transcription in vitro is enhanced by the removal of H2A/H2B dimers (19), and specific loss of H2A/H2B dimers has been observed during active transcription mediated by RNAPII in vivo (20). Moreover, FACT was observed to contain histone chaperone activity (18), suggesting that FACT also functions to re-establish disassembled nucleosomes (Fig. 2B), an assertion previously hypothesized (21). Notably, inactivation of FACT results in transcription initiation from cryptic initiation sites within the coding region of genes in yeast, implying that FACT is required for maintaining chromatin structure in vivo (10,22). Taken collectively, a model can be formulated where FACT functionally exerts its effects on the nucleosome by removing one H2A/H2B dimer and facilitates nucleosome re-assembly after the wake of RNAPII transcription (Fig. 2B).

FACT Associates with Elongation Factors and Chromatin Modulators
Given the importance of FACT during transcription, it is not surprising that FACT displays physical and genetic interactions with many factors implicated in transcript elongation (12). Among these are the SPT proteins Spt4, Spt5, and Spt6, all established modulators of chromatin and transcript elongation (12). Similar to FACT, Spt6 regulates chromatin structure by interacting with histones, namely histone H3, and functions as a histone chaperone (23). Moreover, mutant strains of Spt6 allow spurious initiation from within the coding regions of genes comparable with FACT in yeast, suggesting that Spt6 is important for maintaining chromatin structure. Given that FACT and Spt6 selectively interact with histone H2A/H2B and H3, respectively, these two proteins may work collectively in the maintenance of chromatin structure during active transcription (Fig. 2B).
As discussed above, the nature of FACT spatial recruitment on active genes remains unclear. However, a number of recent studies have suggested a role for the chromatin-remodeling factor CHD1 in FACT localization. Several independent studies have demonstrated that FACT physically associates with CHD1 in higher organisms and yeast (24 -36). In yeast, chd1 mutant alleles are sensitive to 6-AU (27), and Chd1 displays genetic interactions with numerous elongation factors (26). Similar to FACT, CHD1 localizes throughout the coding region of genes (26) and can transfer core histones onto DNA (28), perhaps functioning in concert with FACT and Spt6 in the maintenance of chromatin structure. Recently, it was identified that human CHD1 specifically recognizes histone H3 trimethylated on lysine four (H3K4me3) (29,30). H3K4me3 correlates with the 5Ј-region of active genes and peaks near the start site of transcription in mammalian cells (31,32). Therefore, FACT may associate with CHD1 bound to H3K4me3 on active genes placing it in its proper position requisite for FACT function. Indeed, FACT can associate with CHD1 when bound to H3K4me3. 4 Quantitative chromatin immunoprecipitation studies have revealed a tight co-localization of FACT and H3K4me3 on active genes (16). Importantly, loss of H3K4me3 in vivo by small interfering RNA treatment resulted in reduced recruitment of FACT to active genes (34). Although CHD1/H3K4me3 may not be the exclusive mechanism for FACT recruitment during transcriptional activation, it is likely to be a major contributor to FACT localization on active genes.
In addition to the SPT group proteins and CHD1, FACT displays interactions with components of the PAF complex (25,35), which is comprised of Paf1, Cdc73, Ctr9, Leo1, and Rtf1 subunits in yeast (25,(35)(36)(37)(38). In human cells, the core PAF complex substitutes the Rtf1 subunit with the Ski8 protein, a factor that bridges the human PAF complex with RNA surveillance via the exosome (14). Genetic studies in yeast identified that components of the PAF complex functionally interact with FACT (35,39), and mutant alleles of PAF subunits display sensitivity to 6-AU. A physical association between FACT and the PAF complex supports these in vivo findings (25,35,40). Functionally, the PAF complex regulates distinct chromatin modifications associated with active transcription, including histone H3 methylation on lysines 4 and 79 (41)(42)(43), as well as monoubiquitination of H2B on lysine 120 (15) (H2B-123 in yeast (44,45)), a mark that facilitates H3K4me3. Given the physical and functional connections between FACT, PAF,  (20). In a control experiment, EC9 was converted into EC45, and only non-transcribed nucleosomes were released into solution (lane 1) (20). The positions of the polymerasefree nucleosomes N1 and N2, hexasomes, and DNA are indicated by dashes. Figure and legend taken from Ref. 18. B, the putative role of FACT, CHD1, and Spt6 in nucleosome reassembly.
Step 1, the removal of one H2A/H2B dimer by FACT facilitates transcript elongation.
Step 2, the chaperone activities of FACT, CHD1, and Spt6 may facilitate the re-assembly of nucleosomes following the wake of transcript elongation. See text for details. and H2B modification, it is likely that monoubiquitination of H2B facilitates FACT-mediated removal of H2A/H2B dimers during transcription. Consistently, a highly purified reconstituted chromatin system coupled to a highly reconstituted transcription system revealed that FACT-dependent transcript elongation is greatly enhanced by the PAF complex and the enzymes required for H2B monoubiquitination (Fig. 3A) (40). Moreover, the association between these three factors results in increased transcript elongation rates (Fig. 3B) (40). Additionally, the proteasomal ATPases Rpt4 and Sug1 are likely involved in the FACT/PAF/H2B monoubiquitination pathway. Rpt4 and Sug1 are subunits of the large 26 S proteasome, a cellular machine that degrades polyubiquitinated conjugated proteins (45). Sug1 and FACT subunits interact genetically in yeast (47), sug1 deletion strains display sensitivity to 6-AU (48), and FACT subunits (Cdc68) associate physically with the 19 S regulatory particle (47). More importantly, transcript elongation by RNAPII is stimulated in vitro by the 19 S proteasomal particle, although catalytic inhibition of the 26 S proteasome had no effect on elongation (48). Despite these findings, proteasome-mediated degradation has been shown to effect transcriptional regulation both at the level of activator turnover and RNAPII (49 -52). Interestingly, Rpt4 and Sug1 are recruited to active genes in a manner that requires H2B monoubiquitination, and mutant alleles of Rpt4 and Sug1 show defects in histone H3-K4 and H3-K79 methylation (53,54), similar to the PAF complex. Whether H2B monoubiquitination is functionally linked to Rpt4 and Sug1 remains to be determined, although a direct connection between these proteins seems likely. Mechanistically, it will be interesting to delineate how FACT, PAF, and Rpt4/Sug1 work together to facilitate RNA-PII passage through the nucleosome barrier via H2A/H2B dimer interactions and modifications (Fig. 4).

Additional FACT Activities
Much of the data on FACT have indicated a clear role during transcript elongation on chromatin templates. However, studies have indicated an expanded functional role for FACT in other aspects of DNA metabolism. Some of the more notable findings include FACT's recruitment of TATA-binding protein (TBP) to promoters (10,55,56) and its involvement in DNA damage and repair (33,63,64). DNA replication is also challenged with limited DNA accessibility due to chromatin compaction; therefore, it is not surprising that FACT participates in this process. How FACT mechanistically facilitates this process is unclear, but FACT is both physically (57,62) and functionally important for DNA replication (57)(58)(59)(60)(61)(62).
As FACT is intimately involved in the maintenance of chromatin structure, it is easy to see how FACT impacts many aspects of RNA and DNA polymerase function. New discoveries will not only enhance our understanding of FACT-specific activities but the overall understanding of the many processes it regulates.  4. An integrated model of FACT activity. Upon gene activation, histone H3 is trimethylated on lysine 4 (H3K4me3), which recruits CHD1. Through direct interations, CHD1 positions both FACT and PAF at the nucleosomal barrier. PAF association with RNF20/40 facilitates mono-ubiquitination of histone H2B on lysine 120, thus stimulating FACT activities.