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Eukaryotic cells store their genetic information in the form of a highly organized nucleoprotein complex termed chromatin. The high degree of compaction of DNA within chromatin places severe constraints on proteins that require access to the DNA template to facilitate gene transcription, DNA replication, and DNA repair. As a consequence, eukaryotic cells have developed sophisticated mechanisms to allow chromatin to be rapidly decompacted locally for access by DNA-binding proteins. Once thought to play only a structural role, it now appears that chromatin plays a key regulatory role by marshalling access to the DNA template. We have reviewed the role played by chromatin in the cellular response to physiological and toxicological stimuli and described how changes in chromatin structure may in the future be used as markers of toxicity. We also review the evidence that chromatin itself is the direct target of certain toxicants and that toxicant-induced perturbations in chromatin structure may precipitate adverse effects.

The machinery that transcribes protein-coding genes in eukaryotic cells must contend with repressive chromatin structures in order to find its target DNA sequences. Diverse arrays of proteins modify the structure of chromatin at gene promoters to help transcriptional regulatory proteins access their DNA recognition sites. The way in which disruption of chromatin structure at a promoter is transmitted through a whole gene has not been defined. Recent breakthroughs suggest that the passage of an RNA polymerase through a gene is coupled to mechanisms that propagate the breakdown of chromatin.

The regulation of gene expression depends critically upon chromatin structure. Transcription of protein-coding genes can be reconstituted on naked DNA with only the general transcription factors and RNA polymerase II. This minimal system cannot transcribe DNA packaged into chromatin, indicating that accessory factors may facilitate access to DNA. Two classes of accessory factor, ATP-dependent chromatin-remodelling enzymes and histone acetyltransferases, facilitate transcription initiation from chromatin templates. FACT (for facilitates chromatin transcription) is a chromatin-specific elongation factor required for transcription of chromatin templates in vitro. Here we show that FACT comprises a new human homologue of the Saccharomyces cerevisiae Spt16/Cdc68 protein and the high-mobility group-1-like protein structure-specific recognition protein-1. Yeast SPT16/CDC68 is an essential gene that has been implicated in transcription and cell-cycle regulation. Consistent with our biochemical analysis of FACT, we provide evidence that Spt16/Cdc68 is involved in transcript elongation in vivo. Moreover, FACT specifically interacts with nucleosomes and histone H2A/H2B dimers, indicating that it may work by promoting nucleosome disassembly upon transcription. In support of this model, we show that FACT activity is abrogated by covalently crosslinking nucleosomal histones.

Transcription of naked DNA in vitro requires the general transcription factors and RNA polymerase II. However, this minimal set of factors is not sufficient for transcription when the DNA template is packaged into chromatin. Here, a factor that facilitates activator-dependent transcription initiation on chromatin templates was purified. This factor, remodeling and spacing factor (RSF), has adenosine triphosphate-dependent nucleosome-remodeling and spacing activities. Polymerases that initiate transcription with RSF can only extend their transcripts in the presence of FACT (facilitates chromatin transcription). Thus, the minimal factor requirements for activator-dependent transcription on chromatin templates in vitro have been defined.

We have isolated a human RNA polymerase II complex that contains chromatin structure remodeling activity and histone acetyltransferase activity. This complex contains the Srb proteins, the Swi-Snf complex, and the histone acetyltransferases CBP and PCAF in addition to RNA polymerase II. Notably, the general transcription factors are absent from this complex. The complex was purified by two different methods: conventional chromatography and affinity chromatography using antibodies directed against CDK8, the human homolog of the yeast Srb10 protein. Protein interaction studies demonstrate a direct interaction between RNA polymerase II and the histone acetyltransferases p300 and PCAF. Importantly, p300 interacts specifically with the nonphosphorylated, initiation-competent form of RNA polymerase II. In contrast, PCAF interacts with the elongation-competent, phosphorylated form of RNA polymerase II.