Contribution of sequences downstream of the TATA element to a protein-DNA complex containing the TATA-binding protein.

A TATA complex that forms on the hsp70 promoter has been found to depend on sequence-specific interactions that occur at the transcription start and regions further downstream. The complex was detected with a gel shift assay and further characterized with interference assays. Antibodies reveal that the TATA-binding protein is in the complex. Interference assays localize specific contacts in the TATA element, the start site, and in a region approximately 25 bp downstream of the start site that contribute to either the assembly or the maintenance of the complex. Contact at the TATA element is made in the minor groove, as has been reported for the recombinant TATA-binding protein. Mutation in the TATA element or the start site of hsp70 causes complex formation to be more strongly dependent on contacts in the +25 region than in the normal core promoter. Examination of the hsp26 and histone H4 genes indicates that similar contacts contribute to the TATA complexes that form on these promoters. The results suggest that specific contacts downstream of the TATA element could play a key role in establishing the transcriptional potential of a gene by contributing to the interaction of the TATA-binding protein.

Protein-DNA cross-linking reveals dramatic variation in RNA polymerase II density on different histone repeats of Drosophila melanogaster.

In Drosophila melanogaster the five histone genes are within a 5-kilobase region which is repeated 100 times at a single chromosomal site. These 5-kilobase repeats are of two distinct classes, short and long, that differ by approximately 200 base pairs of DNA in the spacer region between the H1 and H3 genes. Since the mRNA-homologous regions of the repeats are highly conserved, one cannot examine differential expression of the repeats by classical hybridization methods. In this study, we assessed their transcriptional activity by measuring in vivo the relative amounts of RNA polymerase II that were cross-linked by UV irradiation to the two different histone repeats. The RNA polymerase II density on the long repeat in Schneider line 2 cells was strikingly lower (10-fold) than the density on the short repeat. The magnitude of this difference cannot be accounted for by reduced transcription of only one or two genes of the repeat. The density of topoisomerase I, an indicator of transcriptional activity, was also much higher on the short repeat than on the long repeat of line 2 cells. In contrast, the RNA polymerase II density was slightly higher on the long repeat than on the short repeat in a second cell line, KcH. The major difference between active (KcH) and inactive (S2) long repeats resides in the H1-H3 nontranscribed spacer. This portion of the spacer may contain a component necessary for expression that can act over a moderate distance and affect multiple genes of the repeat.

Localization of specific topoisomerase I interactions within the transcribed region of active heat shock genes by using the inhibitor camptothecin.

Camptothecin stabilizes the topoisomerase I-DNA covalent intermediate that forms during the relaxation of torsionally strained DNA. By mapping the position of the resultant DNA nicks, we analyzed the distribution of the covalent intermediates formed on heat shock genes in cultured Drosophila melanogaster cells. Topoisomerase I was found to interact with the transcriptionally active genes hsp22, hsp23, hsp26, and hsp28 after heat shock but not with the inactive genes before heat shock. The interaction occurred predominantly within the transcribed region, with specific sites occurring on both the transcribed and nontranscribed strands of the DNA. Little interaction was seen with nontranscribed flanking sequences. Camptothecin only partially inhibited transcription of the hsp28 gene during heat shock, causing a reduced level of transcripts which were nonetheless full length. Topoisomerase I also interacted with the DNA throughout the transcriptionally active hsp83 gene, including an intron, in both heat-shocked and non-heat-shocked cells. The results point to a dynamic set of interactions at the active locus.

In vivo interactions of RNA polymerase II with genes of Drosophila melanogaster.

We describe a method for examining the in vivo distribution of a protein on specific eucaryotic DNA sequences. In this method, proteins are cross-linked to DNA in intact cells, and the protein-DNA adducts are isolated by immunoprecipitation with antiserum against the protein. Characterization of the DNA cross-linked to the precipitated protein identifies the sequences with which the protein is associated in vivo. Here, we applied these methods to detect RNA polymerase II-DNA interactions in heat-shocked and untreated Drosophila melanogaster Schneider line 2 cells. The level of RNA polymerase II associated with several heat shock genes increased dramatically in response to heat shock, whereas the level associated with the copia genes decreased, indicating that both induction of heat shock gene expression and repression of the copia gene expression by heat shock occur at the transcriptional level. Low levels of RNA polymerase II were present on DNA outside of the transcription units, and for at least two genes, hsp83 and hsp26, RNA polymerase II initiated binding near the transcription start site. Moreover, for hsp70, the density of RNA polymerase II on sequences downstream of the polyadenylate addition site was much lower than that observed on the gene internal sequences. Examination of the amount of specific restriction fragments cross-linked to RNA polymerase II provides a means of detecting RNA polymerase II on individual members of multigene families. This analysis shows that RNA polymerase II is associated with only one of the two cytoplasmic actin genes.

RNA polymerase II interacts with the promoter region of the noninduced hsp70 gene in Drosophila melanogaster cells.

By using a protein-DNA cross-linking method (D. S. Gilmour and J. T. Lis, Mol. Cell. Biol. 5:2009-2018, 1985), we examined the in vivo distribution of RNA polymerase II on the hsp70 heat shock gene in Drosophila melanogaster Schneider line 2 cells. In heat shock-induced cells, a high level of RNA polymerase II was detected on the entire gene, while in noninduced cells, the RNA polymerase II was confined to the 5' end of the hsp70 gene, predominantly between nucleotides -12 and +65 relative to the start of transcription. This association of RNA polymerase II was apparent whether the cross-linking was performed by a 10-min UV irradiation of chilled cells with mercury vapor lamps or by a 40-microsecond irradiation of cells with a high-energy xenon flash lamp. We hypothesize that RNA polymerase II has access to, and a high affinity for, the promoter region of this gene before induction, and this poised RNA polymerase II may be critical in the mechanism of transcription activation.

TATA box-dependent protein-DNA interactions are detected on heat shock and histone gene promoters in nuclear extracts derived from Drosophila melanogaster embryos.

We monitored protein-DNA interactions that occur on the hsp26, hsp70, histone H3, and histone H4 promoters in nuclear extracts derived from frozen Drosophila melanogaster embryos. All four of these promoters were found to be transcribed in vitro at comparable levels by extracts from both heat-shocked and non-heat-shocked embryos. Factors were detected in both types of extracts that block exonuclease digestion from a downstream site at ca. +35 and -20 base pairs from the start of transcription of all four of these promoters. In addition, factors in extracts from heat-shocked embryos blocked exonuclease digestion at sites flanking the heat shock consensus sequences of hsp26 and hsp70. Competition experiments indicated that common factors cause the +35 and -20 barriers on all four promoters in both extracts. The formation of the barriers at +35 and -20 required a TATA box but did not appear to require specific sequences downstream of +7. We suggest that the factors responsible for the +35 and -20 barriers are components whose association with the promoter precedes transcriptional activation.

Promoter-proximal pausing on the hsp70 promoter in Drosophila melanogaster depends on the upstream regulator.

RNA polymerase II pauses in the promoter-proximal region of many genes during transcription. In the case of the hsp70 promoter from Drosophila melanogaster, this pause is long-lived and occurs even when the gene is not induced. Paused polymerase escapes during heat shock when the transcriptional activator heat shock factor associates with the promoter. However, pausing is still evident, especially when induction is at an intermediate level. Yeast Gal4 protein (Gal4p) will induce transcription of the hsp70 promoter in Drosophila when binding sites for Gal4p are positioned upstream from the hsp70 TATA element. To further our understanding of promoter-proximal pausing, we have analyzed the effect of Gal4p on promoter-proximal pausing in salivary glands of Drosophila larvae. Using permanganate genomic footprinting, we observed that various levels of Gal4p induction resulted in an even distribution of RNA polymerase throughout the first 76 nucleotides of the transcribed region. In contrast, promoter-proximal pausing still occurs on endogenous and transgenic hsp70 promoters in salivary glands when these promoters are induced by heat shock. We also determined that mutations introduced into the region where the polymerase pauses do not inhibit pausing in a cell-free system. Taken together, these results indicate that promoter-proximal pausing is dictated by the regulatory proteins interacting upstream from the core promoter region.

UV cross-linking identifies four polypeptides that require the TATA box to bind to the Drosophila hsp70 promoter.

A protein fraction that requires the TATA sequence to bind to the hsp70 promoter has been partially purified from nuclear extracts of Drosophila embryos. This TATA factor produces a large DNase I footprint that extends from -44 to +35 on the promoter. A mutation that changes TATA to TATG interferes both with the binding of this complex and with the transcription of the hsp70 promoter in vitro, indicating that this interaction is important for transcriptional activity. Using a highly specific protein-DNA cross-linking assay, we have identified four polypeptides that require the TATA sequence to bind to the hsp70 promoter. Polypeptides of 26 and 42 kilodaltons are in intimate contact with the TATA sequence. Polypeptides of 150 and 60 kilodaltons interact within the region from +24 to +47 in a TATA-dependent manner. Both the extended footprint and the polypeptides identified by UV cross-linking indicate that the Drosophila TATA factor is a multicomponent complex.

Molecular architecture of the hsp70 promoter after deletion of the TATA box or the upstream regulation region.

GAGA factor, TFIID, and paused polymerase are present on the hsp70 promoter in Drosophila melanogaster prior to transcriptional activation. In order to investigate the interplay between these components, mutant constructs were analyzed after they had been transformed into flies on P elements. One construct lacked the TATA box and the other lacked the upstream regulatory region where GAGA factor binds. Transcription of each mutant during heat shock was at least 50-fold less than that of a normal promoter construct. Before and after heat shock, both mutant promoters were found to adopt a DNase I hypersensitive state that included the region downstream from the transcription start site. High-resolution analysis of the DNase I cutting pattern identified proteins that could be contributing to the hypersensitivity. GAGA factor footprints were clearly evident in the upstream region of the TATA deletion construct, and a partial footprint possibly caused by TFIID was evident on the TATA box of the upstream deletion construct. Permanganate treatment of intact salivary glands was used to further characterize each promoter construct. Paused polymerase and TFIID were readily detected on the normal promoter construct, whereas both deletions exhibited reduced levels of each of these factors. Hence both the TATA box and the upstream region are required to efficiently recruit TFIID and a paused polymerase to the promoter prior to transcriptional activation. In contrast, GAGA factor appears to be capable of binding and establishing a DNase I hypersensitive region in the absence of TFIID and polymerase. Interestingly, purified GAGA factor was found to bind near the transcription start site, and the strength of this interaction was increased by the presence of the upstream region. GAGA factor alone might be capable of establishing an open chromatin structure that encompasses the upstream regulatory region as well as the core promoter region, thus facilitating the binding of TFIID.

Analyses of promoter-proximal pausing by RNA polymerase II on the hsp70 heat shock gene promoter in a Drosophila nuclear extract.

Analyses of Drosophila cells have revealed that RNA polymerase II is paused in a region 20 to 40 nucleotides downstream from the transcription start site of the hsp70 heat shock gene when the gene is not transcriptionally active. We have developed a cell-free system that reconstitutes this promoter-proximal pausing. The paused polymerase has been detected by monitoring the hyperreactivity of thymines in the transcription bubble toward potassium permanganate. The pattern of permanganate reactivity for the hsp70 promoter in the reconstituted system matches the pattern found on the promoter after it has been introduced back into files by P-element-mediated transposition. Matching patterns of permanganate reactivity are also observed for a non-heat shock promoter, the histone H3 promoter. Further analysis of the hsp70 promoter in the reconstituted system reveals that pausing does not depend on sequence-specific interactions located immediately downstream from the pause site. Sequences upstream from the TATA box influence the recruitment of polymerase rather than the efficiency of pausing. Kinetic analysis indicates that the polymerase rapidly enters the paused state and remains stably in this state for at least 25 min. Further analysis shows that the paused polymerase will initially resume elongation when Sarkosyl is added but loses this capacity within minutes of pausing. Using an alpha-amanitin-resistant polymerase, we provide evidence that promoter-proximal pausing does not require the carboxy-terminal domain of the polymerase.

Analysis of core promoter sequences located downstream from the TATA element in the hsp70 promoter from Drosophila melanogaster.

TFIID recognizes multiple sequence elements in the hsp70 promoter of Drosophila. Here, we investigate the function of sequences downstream from the TATA element. A mutation in the initiator was identified that caused an eightfold reduction in binding of TFIID and a fourfold reduction in transcription in vitro. Another mutation in the +24 to +29 region was somewhat less inhibitory, but a mutation in the +14 to +19 region had essentially no effect. The normal promoter and the mutants in the initiator and the +24 to +29 region were transformed into flies by P element-mediated transformation. The initiator mutation reduced expression an average of twofold in adult flies, whereas the mutation in the +24 to +29 region had essentially no effect. In contrast, a promoter combining the two mutations was expressed an average of sixfold less than the wild type. The results suggest that the initiator and the +24 to +29 region could serve overlapping functions in vivo. Protein-DNA cross-linking was used to identify which subunits of TFIID contact the +24 to +29 region and the initiator. No specific subunits were found to cross-link to the +24 to +29 region. In contrast, the initiator cross-linked exclusively to dTAF230. Remarkably, dTAF230 cross-links approximately 10 times more efficiently to the nontranscribed strand than to the transcribed strand at the initiator.

Heterochromatic silencing of Drosophila heat shock genes acts at the level of promoter potentiation.

In a variety of organisms, genes placed near heterochromatin are transcriptionally silenced. In order to understand the molecular mechanisms responsible for this block in transcription, high resolution in vivo chromatin structure analysis was performed on two heat shock genes, hsp26 and hsp70. These genes normally reside in euchromatin where GAGA factor and RNA Pol II are present on the promoter prior to heat shock induction. P-element transformation experiments led to the identification of stocks in which these two genes were inserted within heterochromatin of the chromosome 4 telomeric region. These transgenes exhibit silencing that is partially suppressed by mutations in the gene encoding HP1. Micrococcal nuclease analysis revealed that the heterochromatic transgenes were packaged in a more regular nucleosome array than when located in euchromatin. High resolution DNase I analysis demonstrated that GAGA factor and TFIID were not associated with these promoters in heterochromatin; potassium permanganate experiments showed a loss of Pol II association. Taken together, these data suggest that occlusion of trans-acting factors from their cis- acting regulatory elements leading to a block in promoter potentiation is a mechanism for heterochromatin gene silencing.

Association of topoisomerase I with transcriptionally active loci in Drosophila.

Immunofluorescence staining of the polytene chromosomes of Drosophila shows high levels of topoisomerase I associated with transcriptionally active regions. A photocrosslinking technique demonstrates the presence of topoisomerase I in the region of transcription of the active heat-shock genes. Camptothecin stabilizes the topoisomerase I-DNA covalent intermediate that forms during the relaxation of torsionally strained DNA. By mapping the position of the resultant DNA nicks, topoisomerase I is found to interact with the transcriptionally active genes hsp23, hsp26, and hsp28 after heat shock but not with the inactive genes prior to heat shock. The interaction occurs predominantly within the transcribed region, with specific sites observed on both the transcribed and nontranscribed strands of the DNA. Little interaction is seen with nontranscribed flanking sequences. Camptothecin only partially inhibits transcription of the hsp28 gene during heat shock, causing a reduced level of transcripts which are nonetheless full length. The results point to a dynamic set of interactions at the active locus.

Transcription factor TFIID recognizes DNA sequences downstream of the TATA element in the Hsp70 heat shock gene.

The interaction between the Hsp70 heat shock gene promoter and a Drosophila protein complex which contains the TATA-binding protein depends on sequence-specific interactions located in the region downstream of the transcription start site. Immunopurification of the complex through the use of antibodies against the TATA-binding protein reveals that the complex is transcription factor TFIID. Binding assays with the immunopurified TFIID confirm that sequence-specific contacts are made in the region between nucleotides +18 and +33 relative to the transcription start site. These sequence-specific interactions could play key roles in recognition of TATA-containing and TATA-less promoters.

Genomic footprinting of the hsp70 and histone H3 promoters in Drosophila embryos reveals novel protein-DNA interactions.

The transcriptional potential of the hsp70 heat shock gene promoter is established prior to induction by stress. It has been shown previously that the TBP subunit of TFIID is associated with the TATA element and that RNA polymerase II is paused downstream from the transcription start site. In order to identify new interactions involved in establishing this potentiated state, a detailed analysis of the molecular architecture of a single copy of the hsp70 promoter was performed. A suitably marked promoter was stably integrated using P-element-mediated transformation so as to overcome any ambiguity that might be associated with analyzing the five copies of the endogenous gene. Genomic footprinting using DNase I revealed two previously unidentified interactions. First, the GAGA element located at -120 is protected by protein. Secondly, the pattern of DNase I cleavage in the vicinity of the transcription start is found to bear significant similarity to the pattern associated with binding of purified TFIID. Noting that purified GAGA factor and TFIID interact similarly with the hsp70 and H3 promoters, the architecture of the endogenous H3 promoter was analyzed to determine what interactions might be needed to establish a potentiated state containing a paused polymerase. Despite the detection of TFIID and GAGA on the H3 promoter, no paused polymerase is evident. In addition, no proteins appear to interact with the transcription start. These results suggest that the GAGA factor and TFIID are not sufficient to establish a potentiated state containing paused polymerase and that TFIID interactions downstream from the TATA element could be important for pausing.

Nucleosomes are not necessary for promoter-proximal pausing in vitro on the Drosophila hsp70 promoter.

RNA polymerase II has been found to pause stably on several metazoan genes in a promoter-proximal region located 20-40 nt downstream from the start site of transcription. Escape of polymerase from this paused state has been proposed to be a rate limiting step in transcription of some genes. A study of the human hsp70 promoter showed that a nucleosome positioned downstream from the transcription start was a key component in establishing a stably paused polymerase in one cell-free system. We tested whether these results could be extended to the Drosophila hsp70 promoter in a Drosophila cell-free system and found that polymerase paused stably on the promoter even when the length of DNA downstream from the transcription start was not sufficient for assembly of a nucleosome. Our results indicate that a downstream nucleosome is not a universal requirement for stably pausing RNA polymerase in the promoter-proximal region.

Detecting protein-DNA interactions in vivo: distribution of RNA polymerase on specific bacterial genes.

We present an approach for determining the in vivo distribution of a protein on specific segments of chromosomal DNA. First, proteins are joined covalently to DNA by irradiating intact cells with UV light. Second, these cells are disrupted in detergent, and a specific protein is immunoprecipitated from the lysate. Third, the DNA that is covalently attached to the protein in the precipitate is purified and assayed by hybridization. To test this approach, we examine the cross-linking in Escherichia coli of RNA polymerase to a constitutively expressed, lambda cI gene, and to the uninduced and isopropyl beta-D-thiogalactoside (IPTG)-induced lac operon. As expected, the recovery of the constitutively expressed gene in the immunoprecipitate is dependent on the irradiation of cells and on the addition of RNA polymerase antiserum. The recovery of the lac operon DNA also requires transcriptional activation with IPTG prior to the cross-linking step. After these initial tests, we examine the distribution of RNA polymerase on the leucine operon of Salmonella in wild-type, attenuator mutant, and promoter mutant strains. Our in vivo data are in complete agreement with the predictions of the attenuation model of regulation. From these and other experiments, we discuss the resolution, sensitivity, and generality of these methods.

Protein/DNA crosslinking of a TFIID complex reveals novel interactions downstream of the transcription start.

A protein--DNA complex containing TFIID has been analyzed by crosslinking. The TBP subunit of TFIID crosslinked to the TATA element but not to any of the regions further downstream which were tested. A 150 kd polypeptide, which corresponds in size to one of the TBP-associated factors (TAFs), crosslinked to a region between +10 and +15 and a second region between +35 and +47. Another polypeptide of greater than 205 kd (also a potential TAF) crosslinked preferentially to the region between +35 and +42. The +10 to +15 region has been recently implicated in hsp70 promoter recognition by TFIID, and the most downstream contacts overlap with the region where RNA polymerase II pauses on the hsp70 promoter in noninduced cells. Crosslinking revealed that as the salt concentration was increased, the TBP interaction was largely unaffected whereas the protein/DNA interactions downstream of the TATA element were disrupted. We propose that during the formation of a transcription complex, TATA-dependent interactions could be disrupted in the vicinity of the start site and the region immediately downstream. A protein contact downstream of +35 might function in pausing polymerase.

Genome-wide analysis of protein-DNA interactions in living cells.

Understanding the regulation of gene expression requires an analysis of gene-specific transcription factors. This review highlights recent work that uses protein-DNA crosslinking, immunoprecipitation and DNA microarrays to determine the binding sites for specific transcription factors throughout the yeast genome.