Biomedical subjects

J T Lis

Publications and source records attributed to J T Lis.

At least 19 recordsLinked to original sources

PubMed1996

HSF recruitment and loss at most Drosophila heat shock loci is coordinated and depends on proximal promoter sequences.

The heat shock response in Drosophila is primarily dependent on the binding of the heat shock transcription factor, HSF, to conserved sequences in heat shock gene promoters, the heat shock elements (HSEs). Here we examine the kinetic relationship of HSF binding to chromosomal loci and heat shock gene transcription in vivo. The features of heat shock promoters that determine the kinetics of HSF binding are also examined. Analyses of HSF association by indirect immunofluorescence with an anti-HSF antibody reveal that fluorescent signals at many loci on polytene chromosomes rapidly increase and then gradually decrease as heat shock time progresses. While overall amounts of fluorescent signal vary from locus to locus, the patterns of acquisition and loss of HSF at most loci are coordinated with only one identified exception. Immunostaining with an anti-RNA polymerase II antibody indicates that the kinetics of RNA polymerase II accumulation on the heat shock loci are similar to those of HSF. Furthermore, nuclear run-on assays confirm that the major heat shock genes are coordinately transcribed during the attenuation period. In contrast, the kinetics of HSF association with HSE "polymers" in a transgenic fly strain are not coordinated with those of endogenous loci. The addition of core promoter sequences to one of the HSEs found in the polymer restores coordinate HSF binding, suggesting that the kinetic patterns of HSF binding depend on a core promoter located near the HSEs. Finally, the distribution of the heat shock protein HSP70 is examined for its role in regulating the attenuated response of HSF to heat shock.

Animals↗

PubMed1995

Binding of heat shock factor to and transcriptional activation of heat shock genes in Drosophila.

Heat shock factor (HSF) binds to heat shock elements (HSEs) and the binding can be highly cooperative. Here we report an analysis of binding of Drosophila HSF to both native and synthetic heat shock regulatory regions. We find that cooperative binding of HSF requires close proximity, rather than helical alignment, of HSEs. Two or more trimeric HSEs organized as contiguous 5 bp units show much higher levels of cooperativity than multiple but separated HSEs. We discuss these in vitro observations in the context of the in vivo status of heat shock genes under mild and full heat shock conditions. Finally, we show that the DNA binding and trimerization domains alone may be sufficient for the full level of binding cooperativity between HSF trimers. This last result suggests that close proximity of HSEs for cooperative binding of HSF is a result of protein-protein interactions near the point of DNA contact.

Animals↗

PubMed1995

HSF access to heat shock elements in vivo depends critically on promoter architecture defined by GAGA factor, TFIID, and RNA polymerase II binding sites.

Chromatin structure can modulate gene expression by limiting transcription factor access to gene promoters. We examined sequence elements of the Drosophila hsp70 promoter for their ability to facilitate the binding of the transcription factor, heat shock factor (HSF), to chromatin. We assayed HSF binding to various transgenic heat shock promoters in situ by measuring amounts of fluorescence at transgenic loci of polytene chromosomes that were stained with an HSF antibody. We found three promoter sequences that influence the access of HSF to its binding sites: the GAGA element, sequences surrounding the transcription start site, and a region in the leader of hsp70 where RNA polymerase II arrests during early elongation. The GAGA element has been shown previously to disrupt nucleosome structure. Because the two other critical regions include sequences that are required for stable binding of TFIID in vitro, we examined the in vivo occupancy of the TATA elements in the transgenic promoters. We found that TATA occupancy correlated with HSF binding for some promoters. However, in all cases HSF accessibility correlated with the presence of paused RNA polymerase II. We propose that a complex promoter architecture is established by multiple interdependent factors, including GAGA factor, TFIID, and RNA polymerase II, and that this structure is critical for HSF binding in vivo.

Animals↗

PubMed1995

Short transcripts of the ternary complex provide insight into RNA polymerase II elongational pausing.

Expression of the hsp70 gene of Drosophila melanogaster is controlled at the level of transcript elongation. In the uninduced state, hsp70 possesses an RNA polymerase II complex, elongationally engaged, but paused early in the transcription unit. In this study, we have used a powerful new selection-amplification technique to analyze the RNA transcripts associated with such "paused polymerases" under non-heat shock and heat shock-induced conditions. They reveal a region of pausing on the uninduced gene in vivo spanning from +21 to +35. This region is interrupted by an area of low polymerase density, centered at about +26. Upon induction, an accumulation of short transcripts, similar in size to those associated with the paused complex, was seen. Models for polymerase pausing and release are discussed in light of these data. The increased sensitivity of our new technique also allowed us to investigate ternary complexes associated with genes with much lower levels of engaged RNA polymerase. These included two of the small heat shock genes (hsp26 and hsp27) and two metabolic genes (Gapdh-1 and Gapdh-2), where paused polymerases were thought to be present, plus two other genes (Mtn and yp1), where polymerase pausing has not been detected in the past. In both of the small heat shock genes, we found evidence of previously unknown sites of transcriptional termination, residing immediately upstream of the regions of polymerase pausing.

Animals↗

PubMed1995

Sodium salicylate and yeast heat shock gene transcription.

The anti-inflammatory drug sodium salicylate modulates the activity of specific transcription factors in humans. Here we show that salicylate and sorbate, another organic acid, stimulate DNA binding by yeast heat shock transcription factor (HSF) in vivo. Surprisingly, salicylate inhibits heat shock gene transcription even in cells induced by a prior heat shock. This inhibition of transcription occurs at a step after HSF and transcription factor IID binding but before promoter melting by RNA polymerase. Salicylate appears to generate a tight binding but activation-impotent HSF by cytoplasmic acidification, since inhibiting proton efflux from cells triggers this same DNA binding and inhibition of heat shock gene expression.

Base Sequence↗

PubMed1995

Distribution of GAGA protein on Drosophila genes in vivo.

GAGA protein binds specific CT.GA-rich DNA sequences in vitro, and many of these sequences are required for transcription in vivo. GAGA protein has been implicated in the transcription of numerous Drosophila genes, including hsp70, hsp26, actin 5C, and Ubx. Here, we examine the in vivo distribution of GAGA protein on a number of Drosophila genes that do and do not have CT-rich sequences by use of a UV cross-linking technique. Prior to heat shock, GAGA protein is associated with the promoter regions of the uninduced hsp70 and hsp26 genes. Upon heat shock induction, GAGA protein is recruited to their transcription units with its distribution coincident with that of RNA polymerase II. The recruitment of GAGA protein to the hsp70 gene after an instantaneous heat shock occurs in a 5' to 3' manner with kinetics similar to RNA polymerase. GAGA protein has been shown to disrupt nucleosome both in vivo and in vitro. We propose that GAGA protein may function in vivo both by binding constitutively to its high-affinity binding sites and by spreading through the induced gene opening the chromatin structure allowing polymerase to elongate efficiently.

Actins↗

PubMed1995

Recruiting TATA-binding protein to a promoter: transcriptional activation without an upstream activator.

The binding of TATA-binding protein (TBP) to the TATA element is the first step in the initiation of RNA polymerase II transcription from many promoters in vitro. It has been proposed that upstream activator proteins stimulate transcription by recruiting TBP to the promoter, thus facilitating the assembly of a transcription complex. However, the role of activator proteins acting at this step to stimulate transcription in vivo remains largely speculative. To test whether recruitment of TBP to the promoter is sufficient for transcriptional activation in vivo, we constructed a hybrid protein containing TBP of the yeast Saccharomyces cerevisiae fused to the DNA-binding domain of GAL4. Our results show that TBP recruited by the GAL4 DNA-binding domain to promoters bearing a GAL4-binding site can interact with the TATA element and direct high levels of transcription. This finding indicates that binding of TBP to promoters in S. cerevisiae is a major rate-limiting step accelerated by upstream activator proteins.

Base Sequence↗

PubMed1995

Dynamic protein-DNA architecture of a yeast heat shock promoter.

Here we present an in vivo footprinting analysis of the Saccharomyces cerevisiae HSP82 promoter. Consistent with current models, we find that yeast heat shock factor (HSF) binds to strong heat shock elements (HSEs) in non-heat-shocked cells. Upon heat shock, however, additional binding of HSF becomes apparent at weak HSEs of the promoter as well. Recovery from heat shock results in a dramatic reduction in HSF binding at both strong and weak HSEs, consistent with a model in which HSF binding is subject to a negative feedback regulation by heat shock proteins. In vivo KMnO4 footprinting reveals that the interaction of the TATA-binding protein (TBP) with this promoter is also modulated: heat shock slightly increases TBP binding to the promoter and this binding is reduced upon recovery from heat shock. KMnO4 footprinting does not reveal a high density of polymerase at the promoter prior to heat shock, but a large open complex between the transcriptional start site and the TATA box is formed rapidly upon activation, similar to that observed in other yeast genes.

Base Sequence↗

PubMed1994

Phosphorylation of RNA polymerase II C-terminal domain and transcriptional elongation.

The carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II is essential in vivo, and is found in either an unphosphorylated (IIa) or hyperphosphorylated (IIo) form. The Drosophila uninduced hsp70 and hsp26 genes, and the constitutively expressed beta-1 tubulin and Gapdh-2 genes, contain an RNA polymerase II complex which pauses after synthesizing a short transcript. We report here that, using an in vivo ultraviolet crosslinking technique and antibodies directed against the IIa and IIo forms of the CTD, these paused polymerases have an unphosphorylated CTD. For genes containing a 5' paused polymerase, passage of the paused RNA polymerase into an elongationally competent mode in vivo coincides with phosphorylation of the CTD. Also, the level of phosphorylation of the CTD of elongating polymerases is shown not to be related to the level of transcription, but is promoter specific.

Animals↗

PubMed1994

The upstream activator CTF/NF1 and RNA polymerase II share a common element involved in transcriptional activation.

The carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II consists of tandem repeats of a heptapeptide with the consensus YSPTSPS. It has been shown that the heptapeptide repeat interacts directly with the general transcription factor TFIID. We report here that the CTD activates transcription when fused to the DNA-binding domain of GAL4. More importantly, we find that the proline-rich transcriptional activation domain of the CCAAT-box-binding factor CTF/NF1 contains a sequence with striking similarity to the heptapeptide repeats of the CTD. We show that this CTD-like motif is essential for the transcriptional activator function of the proline-rich domain of CTF/NF1. Deletion of and point mutations in this CTD-like motif abolish the transcriptional activator function of the proline-rich domain, while natural CTD repeats from RNA polymerase II are fully functional in place of the CTD-like motif. We further show that the proline-rich activation domain of CTF/NF1 interacts directly with the TATA-box-binding protein (TBP), and that a mutation in the CTD-like motif that abolishes transcriptional activation reduces the affinity of the proline-rich domain for TBP. These results demonstrate that a class of proline-rich activator proteins and RNA polymerase II possess a common structural and functional component which can interact with the same target in the general transcription machinery. We discuss the implications of these results for the mechanisms of transcriptional activation in eucaryotes.

Amino Acid Sequence↗

PubMed1994

Cooperative binding of heat shock transcription factor to the Hsp70 promoter in vivo and in vitro.

The minimal promoter of the Drosophila hsp70 gene contains a TATA box and two nonidentical HSE sequences, HSEI and HSEII, that synergistically activate the promoter. We have examined stereospecific alignment and spatial constraints in this promoter. Similar to deletion of HSEII, insertion in the spacer between the HSEs of 1 to 5 or 11 to 14 nucleotides (nt) reduced promoter activity to about 10%. In contrast, HSEII was capable of contributing to promoter activity when the spacer was either shortened by 2 or 4 nt or extended by 6 to 10 or 16 or 18 nt. Hence, half of the possible helical arrangements of HSEs are compatible, whereas the other half are essentially incompatible with efficient promoter function. HSEII was ineffective when its distance to HSEI was increased by more than 18 nt. In vitro, HSEII is a weak and HSEI a strong binding site for heat shock transcription factor HSF, and HSF binds to HSEII cooperatively. To find out whether the above periodicity reflects cooperative binding of HSF in vivo or represents the need of stereoalignment for synergistic activation of transcription, the weak HSF binding site HSEII was replaced with the strong binding site HSEI. This substitution greatly attenuated promoter periodicity, suggesting that the periodic effects are caused by cooperative binding of HSF to HSEII, and that stereoalignment of HSEs is not required for transcription activation. In agreement, in vitro assays using spacer mutants revealed cooperative binding of purified, recombinant HSF to HSEII with a similar periodicity as observed in vivo. Changing the distance between TATA and the HSEs did not produce promoter periodicity, indicating that stereoalignment of these elements is not important.

Animals↗

PubMed1994

Fine structure analyses of the Drosophila and Saccharomyces heat shock factor--heat shock element interactions.

Heat shock genes are activated by the binding of the heat shock transcription factor (HSF) to heat shock elements (HSEs), consisting of arrays of the 5-bp unit NGAAN arranged as inverted repeats. Here, we have investigated the interaction of the 5-bp unit with HSFs of Drosophila and Saccharomyces. Mutations within the conserved, central trinucleotide GAA reduce the relative binding affinity of both HSFs. In addition, the base at position 1 (N1) also influences binding, with a strong preference for an A at this position. Methylation interference initially indicated that HSF contacts A1 in the minor groove, but interacts with the immediately adjacent base G2 in the major groove. Further characterization of this apparently abrupt minor to major groove transition by substitution of A1 with an inosine, shows that HSF contacts A1 in the major groove. We offer an explanation for this apparent contradiction and propose that HSF recognizes the HSE primarily through contacts within the major groove of the DNA helix. Finally, based on these observations and a re-evaluation of the base frequencies and criteria for consensus sequence assignment, we propose that the sequence AGAAN more accurately represents the consensus HSE motif.

Animals↗

PubMed1994

Distribution of B52 within a chromosomal locus depends on the level of transcription.

Drosophila B52 protein is a homologue of human ASF/SF2 that functions in vitro as an essential pre-mRNA splicing factor. Immunofluorescence analysis of polytene chromosomes has shown that B52 generally colocalizes with RNA polymerase II; however, in contrast to other splicing factors, B52 brackets RNA polymerase II at highly active heat-shock puffs. Also, UV cross-linking in nonpolytene cells has shown that B52 cross-links in vivo to DNA flanking the highly active transcription units. Here, we find that the distribution of cross-linked B52 at heat-shock loci depends on transcription levels. Heat shocks at low and moderate temperatures, which induce corresponding levels of transcription, recruit B52 both to transcribed DNA and to flanking DNA, whereas a full heat-shock induction concentrates B52 on the DNA that brackets the entire activated region. We have also identified a 46-kDa protein from Chironomus tentans that binds Drosophila B52 antibodies and has a distribution on chromosomes analogous to B52. This protein is found throughout the moderately transcribed Balbiani rings. However, when transcription at these rings is hyperinduced to levels comparable to fully induced Drosophila heat-shock genes, the protein is restricted to the boundaries of highly decondensed chromatin. We suggest that B52 tracks to chromatin fibers that are folding or unfolding, and we discuss this in light of B52's proposed roles in pre-mRNA splicing and control.

Animals↗

PubMed1994

The SR protein B52/SRp55 is essential for Drosophila development.

B52, also called SRp55, is a 52-kDa member of the Drosophila SR protein family of general splicing factors. Escherichia coli-produced B52 is capable of both activating splicing and affecting the alternative splice site choice in human in vitro splicing reactions. Here we report the isolation of a B52 null mutant generated by remobilizing a P element residing near the B52 gene. The resulting deletion, B52(28), is confined to the B52 gene and its neighbor the Hrb87F gene. Second-instar larvae homozygous for the deletion are deficient in both B52 mRNA and protein. The B52 null mutant is lethal at the first- and second-instar larval stages. Germ line transformation of Drosophila flies with B52 genomic DNA rescues this lethality. Thus, B52 is an essential gene and has a critical role in Drosophila development. Larvae deficient in B52 are still capable of splicing the five endogenous pre-mRNAs tested here, including both constitutively and alternatively spliced genes. Therefore, B52 is not required for all splicing in vivo. This is the first in vivo deficiency analysis of a member of the SR protein family.

Animals↗

PubMed1994

A highly conserved domain of RNA polymerase II shares a functional element with acidic activation domains of upstream transcription factors.

We report here that the largest subunit of yeast RNA polymerase II contains an acidic domain that is similar to acidic activators of transcription. This domain includes the highly conserved homology box H. A hybrid protein containing this acidic domain fused to the DNA-binding domain of GAL4 is a potent activator of transcription in the yeast Saccharomyces cerevisiae. Interestingly, mutations that reduce the upstream activating activity of this acidic domain also abolish the normal function of RNA polymerase II. Such functional defects can be rescued by the acidic activation domains of VP16 and GAL4 when inserted into the mutant derivatives of RNA polymerase II. We further show that this acidic domain of RNA polymerase II interacts directly with two general transcription factors, the TATA-binding protein and TFIIB, and that the acidic activation domain of VP16 can compete specifically with the acidic domain of the RNA polymerase for these interactions. We discuss the implications of this finding for the mechanisms of transcriptional activation in eucaryotes.

Amino Acid Sequence↗

PubMed1994

The concentration of B52, an essential splicing factor and regulator of splice site choice in vitro, is critical for Drosophila development.

B52 is a Drosophila melanogaster protein that plays a role in general and alternative splicing in vitro. It is homologous to the human splicing factor ASF/SF2 which is essential for an early step(s) in spliceosome assembly in vitro and also regulates 5' and 3' alternative splice site choice in a concentration-dependent manner. In vitro, B52 can function as both a general splicing factor and a regulator of 5' alternative splice site choice. Its activity in vivo, however, is largely uncharacterized. In this study, we have further characterized B52 in vivo. Using Western blot (immunoblot) analysis and whole-mount immunofluorescence, we demonstrate that B52 is widely expressed throughout development, although some developmental stages and tissues appear to have higher B52 levels than others do. In particular, B52 accumulates in ovaries, where it is packaged into the developing egg and is localized to nuclei by the late blastoderm stage of embryonic development. We also overexpressed this protein in transgenic flies in a variety of developmental and tissue-specific patterns to examine the effects of altering the concentration of this splicing factor in vivo. We show that, in many cell types, changing the concentration of B52 adversely affects the development of the organism. We discuss the significance of these observations with regard to previous in vitro results.

Animals↗

PubMed1993

Polymerase processivity and termination on Drosophila heat shock genes.

5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) limits RNA polymerase II transcription to a gene's 5'-end. Transcription of the uninduced Drosophila hsp70 gene is likewise restricted to the 5'-end, where the polymerase resides in a paused state. Furthermore, paused elongation complexes formed on the uninduced hsp70 gene and in DRB-inhibited reactions can both be restarted by Sarkosyl or high salt. These similarities prompted us to explore whether these complexes were generated by a block at the same polymerase modification step. In vivo UV cross-linking and KMnO4 hyperreactive site mapping show that while the naturally paused polymerase is restricted to the first approximately 42 base pairs of hsp70, DRB treatment of heat-induced cells allows the polymerase to transcribe past this site. Therefore, the DRB-sensitive step is probably not rate-limiting for hsp70 transcription under uninduced conditions. DRB treatment did, however, lead to the reduction of KMnO4 hyper-reactivity on hsp70 and hsp26 in a region correlating with open polymerase and/or early elongation complexes, suggesting a site for the DRB-sensitive polymerase modification step. Finally, we used the techniques of polymerase-DNA cross-linking and KMnO4 hyper-reactive site mapping to analyze the natural polymerase termination process at the 3'-end of the hsp26 gene. The data obtained are consistent with polymerases terminating at multiple sites downstream of the polyadenylation site.

Animals↗

PubMed1993

In vivo transcriptional pausing and cap formation on three Drosophila heat shock genes.

The regulation of many eukaryotic genes occurs at the level of transcriptional elongation. On the uninduced hsp70 gene of Drosophila melanogaster, for example, an RNA polymerase II complex has initiated transcription but has paused early in elongation. In this study, we examine pausing on hsp70 and two of the small heat shock genes (hsp27 and hsp26) at high resolution, using a technique that utilizes paramagnetic particle-mediated selection of terminated run-on transcripts. This technique provides precise information on the distribution of RNA polymerase within each transcription unit. It also details the progression of 5' cap formation on the elongating transcripts. For each gene, we find polymerases paused over a relatively narrow promoter-proximal region. The regions are generally around 20 nucleotides wide, with two preferred pausing positions spaced roughly 10 nucleotides apart or about one turn of the helix. The bulk of capping occurs as transcripts pass between 20 and 30 nucleotides in length. Interestingly, in the three genes examined here, elongational pausing and 5' cap formation appear largely coincident.

Animals↗