In vitro initiation of transcription by RNA polymerase II on in vivo-assembled chromatin templates.

Abstract

We have studied the initiation of transcription in vitro by RNA polymerase II on simian virus 40 (SV40) minichromosomal templates isolated from infected cells. The efficiency and pattern of transcription from the chromatin templates were compared with those from viral DNA templates by using two in vitro transcription systems, either HeLa whole-cell extract or basal transcription factors, RNA polymerase II, and one of two SV40 promoter-binding transcription factors, LSF and Spl. Dramatic increases in numbers of transcripts upon addition of transcription extract and different patterns of usage of the multiple SV40 initiation sites upon addition of Spl versus LSF strongly suggested that transcripts were being initiated from the minichromosomal templates in vitro. That the majority of transcripts from the minichromosomes were due to initiation de novo was demonstrated by the efficient transcription observed in the presence of a-amanitin, which inhibited minichromosome-associated RNA polymerase II, and an a-amanitin-resistant RNA polymerase II, which initiated transcription in vitro. The pattern of transcription from the SV40 late and early promoters on the minichromosomal templates was similar to the in vivo pattern of transcription during the late stages of viral infection and was distinct from the pattern of transcription generated from viral DNA in vitro. In particular, the late promoter of the minichromosomal templates was transcribed with high efficiency, similar to viral DNA templates, while the early-early promoter of the minichromosomal templates was inhibited 10-to 15-fold. Finally, the number of minichromosomes competent to initiate transcription in vitro exceeded the amount actively being transcribed in vivo.Within eukaryotic cells, gene expression is regulated in the context of chromatin, a complex of genomic DNA, histones, and nonhistone proteins. Localized biochemical and structural changes of the chromatin have been correlated with the ability of individual genes to be actively transcribed. Such changes include higher concentrations of high-mobility-group proteins, modified histones, methylated cytosine residues (for reviews, see references 53 and 73), and the appearance of DNase I-hypersensitive sites, both 3' and 5' to the gene (for reviews, see references 20 and 29). Hypersensitivity to DNase I is believed to be due to the binding of specific transcription factors to promoter elements and to the displacement or the specific positioning of nucleosomes. Therefore, it is argued that chromatin plays a role in the regulation of gene expression.To investigate directly the role of chromatin in regulating transcription, in vitro assays have been developed to study the initiation and elongation of transcription on chromatin templates. Such in vitro transcription studies require substantial quantities of a defined DNA complexed into nucleosomes. Several previous studies have used as templates chromatin assembled in vitro by incubating DNA with either * Corresponding author.t Present address:

Alleviation of Histone H1-Mediated Transcriptional Repression and Chromatin Compaction by the Acidic Activation Region in Chromosomal Protein HMG-14

“…We have previously demonstrated the utility of SV40 MC promoters for in vitro transcription studies (7,8,19). Pol II initiates transcription in vitro from the late promoter on SV40 MCs at least as efficiently as it does on naked DNA (7). Only a small percentage of the SV40 MC templates or SV40 DNA templates are utilized for transcription in vitro.…”

“…SV40 MCs provide an exceptional system as a model for in vivo-assembled, transcriptionally competent chromatin. We have previously demonstrated the utility of SV40 MC promoters for in vitro transcription studies (7,8,19). Pol II initiates transcription in vitro from the late promoter on SV40 MCs at least as efficiently as it does on naked DNA (7).…”

“…Finally, transcriptional elongation was facilitated by incubation with high concentrations of unlabelled nucleotides (chase). The transcripts were analyzed by hybridization to single-stranded DNA encompassing the initiation region, followed by trimming of the selected transcripts by RNase T 1 to a maximal length (7,19). This protocol therefore both selects transcripts from a particular promoter and maps initiation sites for full-length transcripts (transcripts extending at least to the extent of the hybridizing single-stranded DNA).…”

“…For transcription assays involving T7 ⅐ HMG-14(1-73)/C24, which contains two cysteine residues, and T7 ⅐ HMG-14 (as a control), 1 mM DTT was also included. Quantitative analysis of RNA transcripts was essentially as described previously (7,19), involving hybridization of the radiolabelled SV40 late transcripts with complementary, unlabelled singlestranded recombinant DNA containing 1,488 bases of SV40 DNA sequence (from nucleotide 294 to 1782, where the major late promoter initiates at nucleotide 325), followed by cleavage with RNase T 1 . This results in the trimming of long transcripts from the circular SV40 MC templates to a maximal length with a unique 3Ј terminus.…”

Association of nucleosome-free regions and basal transcription factors within vivo-assembled chromatin templates activein vitro

“…Using isolated SV40 MC, we demonstrated previously that MC sedimenting throughout the 90S sucrose gradient peak, rather than just those sedimenting most rapidly, supported efficient transcription in vitro. In addition, the majority of transcription from all fractions of MC resulted from initiation de novo (30), rather than elongation of in vivo-initiated transcripts. Thus, a broader population of MC are competent to initiate transcription in vitro than are being actively transcribed in vivo (30).…”