LeRoy G, Orphanides G, Lane WS, and Reinberg D.
Howard Hughes Medical Institute, Division of Nucleic Acid Enzymology,
Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway,
N.J. 08854, USA, and Harvard Microchemistry Facility, Harvard University,
16 Divinity Avenue, Cambridge MA 02138, USA.
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.
Intense biochemical efforts have resulted in the purification of a minimal set of factors necessary for transcription of class II genes in vitro. This minimal transcription system consisting of the general transcription factors (GTFs) TFIIB, TFIID, TFIIE, and TFIIH and RNA polymerase II (RNAPII) was established with assays that reconstituted accurate transcription from class II promoters on naked DNA templates (1, 2). In vivo, the DNA template is organized by histones into chromatin. The minimal RNAPII transcription system cannot transcribe DNA that is packaged into chromatin (3). However, transcription can be reconstituted on chromatin templates with nuclear extracts in the presence of an activator, suggesting that accessory factors are resent in crude nuclear extracts that facilitate RNAPII transcription from chromatin templates (4).
Different adenosine triphosphate (ATP)-dependent nucleosome remodeling
complexes have been isolated. The NURF (nucleosome-remodeling factor) and
ACF (ATP-utilizing chromatin assembly and remodeling factor) complexes
were purified from Drosophila nuclear extracts and have been shown to facilitate
activator-dependent transcription on chromatin templates (5, 6). Several
other ATP-dependent chromatin remodeling enzyme complexes have been purified
from different organisms: SWI/SNF from yeast, human, and Drosophila; RSC
(remodels the structure of chromatin) from yeast; and CHRAC (chromatin
accessibility complex) from Drosophila (7). Biochemical studies with the
human and yeast SWI/SNF complexes have suggested that it may participate
in transcription by assisting factor binding (8-10) and transcription elongation
(11) in the context of chromatin.
...
We have previously demonstrated that the failure of the reconstituted transcription system to elongate its transcripts on remodeled templates is due to nucleosome-induced stalling (26). Productive transcription through nucleosomes requires a factor termed FACT (facilitates chromatin transcription) (26). FACT is a heterodimeric factor of 140- and 80-kD polypeptides (Fig. 3), whose subunits have recently been identified (27). The addition of FACT in the presence of RSF and an activator facilitated the production of a 390-nucleotide (nt), full-length transcript (Fig. 4B, lane 7). However, FACT did not promote transcription in the absence of RSF, even when an activator was present (lane 4). Consistent with the results from our initiation assays, strong transcription was not observed in the absence of an activator (lanes 2 and 5) or in the presence of the GAL4 1-94 DNA-binding domain alone (lanes 3 and 6). The activator-dependent transcription observed in the presence of RSF and FACT was RNAPII-specific, because no product was detected when GTFs were omitted (Fig. 4B, lane 8).
This report describes a system that is capable of reconstituting
both the initiation and elongation phases of transcription from chromatin
templates in vitro. Previous studies that reconstituted transcription from
chromatin templates used crude systems with many unidentified proteins
(3). RSF is capable of directing activator-dependent transcription initiation
on chromatin templates. We believe that RSF facilitates transcription initiation
by remodeling nucleosomes in the promoter region to allow the formation
of preinitiation complexes. However, the activities of RSF are not sufficient
to allow polymerases to extend their transcripts. Elongation through nucleosomes
requires FACT, a chromatin-specific elongation factor (26).
...
In this study, we have defined the minimal factor requirements for
activator-dependent transcription on chromatin templates. It is likely
that there are other activities present in HeLa cells that will function
in reconstituted chromatin transcription assays. Additional coactivators
or mediator type complexes may be required for high levels of transcriptional
activation. Also, histone acetyltransferase complexes may stimulate transcription
from chromatin templates. The use of chromatin transcription assays will
provide a more natural environment to study transcription factors in vitro.
References and Notes:
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2. Roeder RG, Trends Biochem. Sci. 21, 327 (1996).
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403 (1994.
4. Kamakaka RT, Bulger M, Kadonaga JT, Genes Dev. 7, 1779 (1993).
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(1997).
6. Ito T, Bulger M, Pazin MJ, Kobayashi R, Kadonaga JT, Cell 90,
145 (1997).
7. Cairns BR, Trends Biochem. Sci. 23, 20 (1998).
8. Imbalzano AN, Kwon H, Green MR, Kingston RE, Nature 370, 481
(1994).
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370, 477 (1994).
10. Cote J, Quinn J, Workman JL, Peterson CL, Science 265 53 (1994).
11. Brown SA, Imbalzano AN, Kingston RE, Genes Dev. 10, 1479 (1996).
...
26. Orphanides G, LeRoy G, Chang C-H, Luse DS, Reinberg D, Cell
92, 105 (1998).
27. Orphanides G, Wu W, Lane WS, Hampsey M, Reinberg D, in preparation.
...
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