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The chromatin-specific transcription elongation factor FACT comprises human SPT16 and SSRP1 proteins

Abstract

The regulation of gene expression depends critically upon chromatin structure1. Transcription of protein-coding genes can be reconstituted on naked DNA with only the general transcription factors and RNA polymerase II (ref. 2). This minimal system cannot transcribe DNA packaged into chromatin, indicating thataccessory factors may facilitate access to DNA. Two classes of accessory factor, ATP-dependent chromatin-remodelling enzymes3 and histone acetyltransferases4, facilitate transcription initiation from chromatin templates. FACT (for facilitates chromatin transcription) is a chromatin-specific elongation factor required for transcription of chromatin templates in vitro5,6. 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.

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Figure 1: Recovery of FACT activity from renatured p140 and p80 subunits.
Figure 2: FACT activity co-elutes with FACTp140 and human SSRP1.
Figure 3: The spt16-197 and cdc68-11 alleles suppress the 6AUS phenotypes associated with the ppr2 Δ and spt4 Δ mutations.
Figure 4: FACT specifically binds to mono-nucleosomes and histone H2A/H2B dimers.
Figure 5: FACT activity requires disruption of the histone octamer.

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Acknowledgements

We thank D. Luse and R. Singer for helpful comments on the manuscript; E.Spooner, R. Robinson and K. Pierce for their technical expertise; T. Lagrange, X. Sun and R. Iratni for advice on cloning; J. Kurniawan, G. Johnston and R. Singer for communicating results before publication; G. LeRoy, D. Luse, G. Hartzog and F. Winston for useful suggestions; A. Loyola for purified histone dimers and tetramers; F. Winston and L. Breeden for strains; C. Kane for PPR2 constructs; S.Lippard for human SSRP1 antibodies; C. Crane-Robinson for HMG-1 protein; and G. Hartzog for oligonucleotides. This work was supported by grants from the NIH to D.R. and M.H. and from the Howard Hughes Medical Institute to D.R.

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Correspondence to Danny Reinberg.

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Orphanides, G., Wu, WH., Lane, W. et al. The chromatin-specific transcription elongation factor FACT comprises human SPT16 and SSRP1 proteins. Nature 400, 284–288 (1999). https://doi.org/10.1038/22350

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